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ORF-A/2 Deficient Molecular Clone of Feline Immunodeficiency Virus (FIV) Demonstrates Diminished Viral Gene Expression i...

Permanent Link: http://ufdc.ufl.edu/UFE0021204/00001

Material Information

Title: ORF-A/2 Deficient Molecular Clone of Feline Immunodeficiency Virus (FIV) Demonstrates Diminished Viral Gene Expression in Lymphoid Tissues of Neonatal Cats during Acute and Latent Infection
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Novak, Janelle M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acute, chronic, feline, fiv, immunodeficiency, lymphoid, neonatal, orf, virus
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the present study, neonatal cats were infected at birth with either JSY3 (WT) or JSY3?ORF-A/2 for comparison of viral gene expression, proviral load, pathogenicity in thymus, lymph nodes, peripheral blood mononuclear cells (PBMCs) and sorted lymphocyte subpopulations of the blood, during the acute (8 weeks) and chronic (16 weeks) stages of infection. The objective of this study was to further examine the impact of the ORF-A/2-deletion in relation to proviral load during acute and chronic FIV infection, viral gene expression during acute and chronic FIV infection, as well as track individual changes in proviral load and viral gene expression in the blood lymphocyte populations during acute and chronic infection. The hypothesis is that proviral load and viral gene transcription will be lower in cats infected with the ORF-A/2 deletion-mutant during acute and chronic FIV infection. Relative proviral load and expression of viral gag and rev genes was determined using real-time quantitative PCR. Compared to cats infected with JSY3 (WT), there was lower gag and rev gene transcription in thymus, lymph nodes and PBMCs at 8 and 16 weeks in cats infected with JSY3?ORF-A/2. Proviral load in all three lymphoid tissues was lower in JSY3?ORF-A/2 infected cats at 8 weeks only. For peripheral blood lymphocytes, relative proviral load and transcription of viral gag and rev genes was reduced in cats infected with JSY3?ORF-A/2. In particular, relative proviral load and viral transcription in JSY3?ORF-A/2 sorted B cells of the PBMCs was statistically reduced during chronic FIV infection. In addition to reduced viral integration and transcription, there was delayed CD4+/CD8+ T cell inversion and less B cell expansion in PBMCs of JSY3?ORF-A/2 infected cats. Immunohistochemical detection of FIV p24 Gag protein revealed distribution of infected cells to the lymphoid follicles and medullary areas in the thymus of JSY3 (WT) and JSY3?ORF-A/2 infected cats. This type of distribution pattern was not observed previously (Norway et al., 2001) but has been observed in HIV/AIDS patients (Prevot et al., 1992) (Burke et al., 1995) and SIV-infected monkeys (Li et al., 1995). Immunohistochemical analyses of the thymus with the Ki67 antibody demonstrated primarily cortical labeling, indiscriminant medullary staining and exclusion from the follicles. The labeling of the cortical cells represents active cell cycling of non-infected thymocytes. JSY3?ORF-A/2 infected cats had a higher mean number of Ki67-positive cells compared to uninfected or JSY3 (WT) infected cats at either 8 or 16 weeks.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Janelle M Novak.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Mergia, Ayalew.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021204:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021204/00001

Material Information

Title: ORF-A/2 Deficient Molecular Clone of Feline Immunodeficiency Virus (FIV) Demonstrates Diminished Viral Gene Expression in Lymphoid Tissues of Neonatal Cats during Acute and Latent Infection
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Novak, Janelle M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acute, chronic, feline, fiv, immunodeficiency, lymphoid, neonatal, orf, virus
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the present study, neonatal cats were infected at birth with either JSY3 (WT) or JSY3?ORF-A/2 for comparison of viral gene expression, proviral load, pathogenicity in thymus, lymph nodes, peripheral blood mononuclear cells (PBMCs) and sorted lymphocyte subpopulations of the blood, during the acute (8 weeks) and chronic (16 weeks) stages of infection. The objective of this study was to further examine the impact of the ORF-A/2-deletion in relation to proviral load during acute and chronic FIV infection, viral gene expression during acute and chronic FIV infection, as well as track individual changes in proviral load and viral gene expression in the blood lymphocyte populations during acute and chronic infection. The hypothesis is that proviral load and viral gene transcription will be lower in cats infected with the ORF-A/2 deletion-mutant during acute and chronic FIV infection. Relative proviral load and expression of viral gag and rev genes was determined using real-time quantitative PCR. Compared to cats infected with JSY3 (WT), there was lower gag and rev gene transcription in thymus, lymph nodes and PBMCs at 8 and 16 weeks in cats infected with JSY3?ORF-A/2. Proviral load in all three lymphoid tissues was lower in JSY3?ORF-A/2 infected cats at 8 weeks only. For peripheral blood lymphocytes, relative proviral load and transcription of viral gag and rev genes was reduced in cats infected with JSY3?ORF-A/2. In particular, relative proviral load and viral transcription in JSY3?ORF-A/2 sorted B cells of the PBMCs was statistically reduced during chronic FIV infection. In addition to reduced viral integration and transcription, there was delayed CD4+/CD8+ T cell inversion and less B cell expansion in PBMCs of JSY3?ORF-A/2 infected cats. Immunohistochemical detection of FIV p24 Gag protein revealed distribution of infected cells to the lymphoid follicles and medullary areas in the thymus of JSY3 (WT) and JSY3?ORF-A/2 infected cats. This type of distribution pattern was not observed previously (Norway et al., 2001) but has been observed in HIV/AIDS patients (Prevot et al., 1992) (Burke et al., 1995) and SIV-infected monkeys (Li et al., 1995). Immunohistochemical analyses of the thymus with the Ki67 antibody demonstrated primarily cortical labeling, indiscriminant medullary staining and exclusion from the follicles. The labeling of the cortical cells represents active cell cycling of non-infected thymocytes. JSY3?ORF-A/2 infected cats had a higher mean number of Ki67-positive cells compared to uninfected or JSY3 (WT) infected cats at either 8 or 16 weeks.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Janelle M Novak.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Mergia, Ayalew.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021204:00001


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1 ORF-A/2 DEFICIENT MOLECULAR CLONE OF FELINE IMMUNODEFICIENCY VIRUS (FIV) DEMONSTRATES DIMINISHED VI RAL GENE EXPRESSION IN LYMPHOID TISSUES OF NEONATAL CATS DU RING ACUTE AND LATENT INFECTION By JANELLE MARISA NOVAK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Janelle Marisa Novak

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3 To my beautiful research kittens. Without them, this work would not have been possible.

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4 ACKNOWLEDGMENTS I would like to thank Dr. Ayalew Mergia for s upervising my research, for his belief in my abilities, for his wisdom, and hi s patience. I thank Dr. Calvin Johnson for his mentoring and assistance with my research animals. I especi ally thank Dr. Cynda Crawford for agreeing to assist with my project; her a dvice, leadership, enthusiasm, and support greatly enhanced this research endeavor. I sincerely thank Dr. Goodenow, Dr. Levy, and Dr. Romero, for presiding on my thesis committee. Their professionalism, expertise, su pport, and guidance we re essential to the completion of this work. I would like to tha nk George Papadi and Peter Nadeau for their countless support in the lab, for their technical assistance, and advice. I thank Sally OConnell, for her expertise, pr oficiency, helpfulness, and positive spirit. Without her assistance, I coul d not have stayed on track with the Ph.D. requirements. I thank all of my friends who believed in me, stood by me through difficult times, and never let me give up. Specifically, I thank Dr. Holly Kolenda-Roberts for her friendship, support, collaboration an d assistance in the lab. I would like to especially thank my family fo r their support. To my father, Dr. Raymond Novak, who has inspired me with his own researc h, and mentored me in my research. To my mother, Frances Novak, who has motivated me with her patience and steadfast dedication. To my sisters Jennifer, Jessica, and Joanna, each of whom have dedicated countless hours of advice, graphics support, and encouragement. Lastly, I thank Matthew Jensen, for his endless supply of enthusiasm, support, advice, and love. He has been a source of strength th at has seen me through these challenging, yet wonderful years.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............13 CHAPTER 1 INTRODUCTION..................................................................................................................15 Human Immunodeficiency Virus (HIV).................................................................................15 Animal Model for HIV....................................................................................................16 Neonatal FIV Infection....................................................................................................16 Significance of the FIV ORF-A/2 Gene..................................................................................17 In Vitro ORF -A/2 Studies................................................................................................17 In Vivo ORF-A/2 Studies.................................................................................................18 Use of ORF-A/2 in FIV/HIV Vaccine Development......................................................19 Role of ORF-A/2 in Sorted Cell Populations..................................................................20 Goals of the Study............................................................................................................. .....21 2 LITERATURE REVIEW.......................................................................................................24 Retroviridae................................................................................................................... .........24 Lentiviruses................................................................................................................... ..25 Feline Immunodeficiency Virus (FIV)............................................................................26 Physical properties of FIV........................................................................................26 Molecular properties of FIV.....................................................................................27 FIV genome..............................................................................................................27 FIV infection: cellular tropism.................................................................................30 FIV infection: tissue tropism....................................................................................31 Pathogenesis in natural host.....................................................................................32 FIV diagnosis and control........................................................................................32 3 VIRAL GENE EXPRESSION IN LYMPHOCYTE SUBPOPULATIONS OF NEONTAL CATS INFECTED WITH THE MOLECULAR CLONE FIV OR ORF-A/2 DEFECTIVE FIV DURING ACUTE AND CHRONIC STAGES OF INFECTION...........35 Introduction................................................................................................................... ..........35 Materials and Methods.......................................................................................................... .36 Cell Lines..................................................................................................................... ....36 Construction of CMVFIV Wild-Type a nd CMVFIVORF-A/2-Deficient Molecular Clones......................................................................................................................... .36 Virus Preparation.............................................................................................................39

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6 Animals and Inoculation..................................................................................................40 Blood Samples.................................................................................................................40 Tissue Collection and Processing....................................................................................41 Immunomagnetic Selection of Peri pheral Blood Lymphocyte Populations...................41 Flow Cytometry...............................................................................................................43 Quantitative Real-Time PCR for FIV Provirus...............................................................44 Quantitative Real-Time PCR for FIV Transcription.......................................................45 Statistical Analysis..........................................................................................................46 Results........................................................................................................................ .............46 Blood Lymphocyte Subpopulations................................................................................46 Thymus and Lymph Node Lymphocyte Subpopulations................................................48 Proviral Load in Lymphoid Tissues and Lymphocyte Subpopulations..........................49 Transcription of gag and rev in Lymphoid Tissues.........................................................50 Transcription of gag and rev in Blood Lymphocyte Subpopulations.............................52 Discussion..................................................................................................................... ..........53 4 VISUALIZATION OF VIRUS-INFE CTED CELLS AND ACTIVELY REPLICATING THYMOCYTES AND LYMP HOCYTES OF JSY3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACUTE AND CHRONIC STAGES OF INFECTION..................................................................................80 Introduction................................................................................................................... ..........80 Materials and Methods.......................................................................................................... .81 Immunohistochemistry Assay for FI V p24 Protein and Ki67 Protein............................81 Statistical Analysis..........................................................................................................82 Results........................................................................................................................ .............82 Discussion..................................................................................................................... ..........84 5 MEASUREMENT OF CYTOKINES IL-4, IL-7, IL-15, INTERFERON-ALPHA AND INTERFERON-GAMMA IN JSY3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACUTE AND CHRONIC FIV INFECTION..................94 Introduction................................................................................................................... ..........94 Materials and Methods.......................................................................................................... .95 Cytokine Primers and Probes..........................................................................................95 Quantitative Real Time PCR for Cytokine Transcription...............................................95 Statistical Analysis..........................................................................................................96 Results........................................................................................................................ .............97 IL-4........................................................................................................................... .......97 IL-7........................................................................................................................... .......98 IL-15.......................................................................................................................... ......98 Interferon Gamma (IFN)..............................................................................................99 Interferon Alpha (IFN)...............................................................................................100 Discussion..................................................................................................................... ........101

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7 6 VISUALIZATION OF PLASMACYTOID DE NDRITIC CELLS (PDC) WITHIN THE THYMUS AND LYMPH NODES OF FIV JSY3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACUTE AND CHRONIC INFECTION...................................................................................................................... ...113 Introduction................................................................................................................... ........113 Materials and Methods.........................................................................................................114 Immunohistochemistry Assay for DLEC and IFN....................................................114 Statistical Analysis........................................................................................................115 Results........................................................................................................................ ...........115 Discussion..................................................................................................................... ........117 7 CONCLUSIONS..................................................................................................................126 LIST OF REFERENCES.............................................................................................................131 BIOGRAPHICAL SKETCH.......................................................................................................144

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8 LIST OF TABLES Table page 3-1 The sequences of the primers used for conventional PCR analyses for FIV.....................63 3-2 The sequences of the primers and pr obes used for quantitative real time PCR analyses for FIV............................................................................................................... ..64 3-3 The FIV proviral load in the thym us, lymph nodes, and peripheral blood lymphocytes of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2)......................................................71 3-4 The FIV proviral load in CD4+ T cells, CD8+ T cells, and B cells in the peripheral blood of cats neonatally infected with JS Y3 (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2).....................................................................................73 3-5 Transcription of the FIV gag gene in the thymus, lymph nodes, and peripheral blood lymphocytes of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ).....................................................75 3-6 Transcription of the FIV rev gene in the thymus, lym ph nodes, and peripheral blood lymphocytes of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ).....................................................76 3-7 Transcription of the FIV gag gene in CD4+ T cells, CD8+ T cells, and B cells in the peripheral blood of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ).....................................................78 3-8 Transcription of the FIV rev gene in CD4+ T cells, CD8+ T cells, and B cells in the peripheral blood of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ).....................................................79 4-1 Mean number of p24+ cells per thymic area.....................................................................87 4-2 Mean number of p24+ cells per lymph node area..............................................................87 4-3 Mean number of Ki67+ cells per 200 counted thymic cells..............................................88 4-4 Mean number of Ki67+ cells per 200 counted lymph node cells......................................88 5-1 Primers and probes utilized for the cytokine real time quantitative PCR........................105 5-2 Transcription of cytokine genes (IL-4, IL-7, IL-15, Interferon-gamma, Interferonalpha) in the thymus of cats neonatally inf ected with JSY3 wild type FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 )...................................................111

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9 5-3 Transcription of cytokine genes (IL-4, IL-7, IL-15, Interferon-gamma, Interferonalpha) in the lymph nodes of cats neonatally infected with JSY3 wild type FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ).............................................112 6-1 Mean number of IFNpositive cells as measured per total thymus area.......................120 6-2 Mean number of IFNpositive cells as measured per total lymph node area...............120 6-3 Mean number of DLEC-positive cells as measured per total thymus area......................121 6-4 Mean number of DLEC-positive cells as measured per total lymph node area...............121

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10 LIST OF FIGURES Figure page 2-1 Genomic structure of Feline Immunodeficiency Virus (FIV)...........................................34 3-1 Construction of the CMV-driv en JSY3 (WT) molecular clone.........................................58 3-2 Construction of the CMV-driven JSY3 ORF-A/2 molecular clone.................................59 3-3 Schematic of viral events depicting what transpired utilizing the CMV driven plasmids....................................................................................................................... ......60 3-4 ORF-A/2 gene DNA sequence data from the PCR product of JSY3 ORF-A/2 infected CD4E cells...........................................................................................................61 3-5 ORF-A/2 gene DNA sequence data from the PCR product of JSY3 (WT) infected CD4E cells..................................................................................................................... ....62 3-6 Absolute CD4:CD8 T cell ratios from birth to 16 weeks..................................................65 3-7 Absolute CD4+ T cells in PBMC from birth to 16 weeks.................................................65 3-8 Absolute CD8+ T cells in PBMC from birth to 16 weeks.................................................66 3-9 Absolute B cells in PBMC from birth to 16 weeks...........................................................66 3-10 Absolute thymocyte subpopulations at 8 and 16 weeks as determined by flow cytometry...................................................................................................................... .....67 3-11 Absolute lymphocyte subpopulations at 8 and 16 weeks as determined by flow cytometry...................................................................................................................... .....68 3-12 Relative proviral load of the FIV gag gene in thymus (TH), lymph node (LN) and blood (peripheral blood mononuclear cells (PBM Cs)) of 8-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus......................................................................69 3-13 Relative proviral load in thymus (T H), lymph node (LN) and peripheral blood mononuclear cells (PBMCs) 16-week old ki ttens infected with JSY3 (WT) and JSY3 ORF-A/2 virus........................................................................................................69 3-14 Relative proviral load per gram thymus weight at 8 and 16 weeks. The relative proviral load of FIV JSY3 gag gene in thymocytes was determined by real-time PCR assay.......................................................................................................................... .........70 3-15 Relative proviral load per gram lymph node weight at 8 and 16 weeks. The relative proviral load of FIV JSY3 gag gene in lymph node cells was determined by real-time PCR assay...................................................................................................................... ....70

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11 3-16 Relative proviral load of the FIV gag gene in CD4+, CD8+, and B cells of the blood of 8-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus.....................72 3-17 Relative proviral load of the FIV gag gene in CD4+, CD8+, and B cells of the blood of 16-week old kittens infect ed with JSY3 (WT) and JSY3 ORF-A/2 virus...................72 3-18 Relative gene expression of FIV gag and rev in the thymus (TH), lymph node (LN) and blood (PBMC) of 8-week and 16-week ol d kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus........................................................................................................74 3-19 Relative gene expression of the FIV gag and rev gene in CD4+, CD8+ and B lymphocytes in the blood of 8-week old a nd 16-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus.......................................................................................77 4-1 Histologic section of the JSY3 (WT) infected Week 8 thymus demonstrating p24 antibody-stained cells (black) within the follicles (F) and the medullary (M) areas of the lobule..................................................................................................................... .......89 4-2 Histologic section of the JSY3 ORF-A/2 infected Week 8 lymph node demonstrating p24 antibody-stained cells (bl ack) localized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C)...............................................90 4-3 Histologic section of a JSY3 ORF-A/2 infected Week 16 thymus demonstrating Ki67 antibody-stained cells (black)...................................................................................91 4-4 Histologic section of JSY3 ORF-A/2 infected Week 16 thymus demonstrating Ki67 antibody-stained cells (black) (arrows) within the cortex..................................................91 4-5 Histologic section of the JSY3 (WT) infected Week 8 lymph node demonstrating Ki67 antibody-stained cells (black) (arrows).....................................................................92 4-6 Histologic section of the thymus dem onstrating p24 antibody-st ained cells (black) within the follicles (F ) and the medullary (M) areas of the lobule....................................93 5-1 IL-4 gene expression in the thymus from kittens at 8 weeks and 16 weeks....................106 5-2 IL-4 gene expression in the lymph node from kittens at 8 weeks and 16 weeks.............106 5-3 IL-7 gene expression in the thymus from kittens at 8 weeks and 16 weeks....................107 5-4 IL-7 gene expression in the lymph node from kittens at 8 weeks and 16 weeks.............107 5-5 IL-15 gene expression in the thymus from kittens at 8 weeks and 16 weeks..................108 5-6 IL-15 gene expression in the lymph node from kittens at 8 weeks and 16 weeks...........108 5-7 IFNgene expression in the thymus from kittens at 8 weeks and 16 weeks.................109 5-8 IFNgene expression in the lymph node from kittens at 8 weeks and 16 weeks..........109

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12 5-9 IFNgene expression in the thymus from kittens at 8 weeks and 16 weeks.................110 5-10 IFNgene expression in the lymph node fr om kittens at 8 weeks and 16 weeks..........110 6-1 Histologic section of an uninfected thymus at 16 weeks stained with anti-IFNantibody....................................................................................................................... .....122 6-2 Histologic section of a JSY3 (WT)-infect ed thymus at 8 weeks demonstrating IFNantibody-stained cells (black) within the follicles (F) and the medullary (M) areas of the lobule..................................................................................................................... .....122 6-3 Histologic section of a JSY3 ORF-A/2-infected thymus at 16 weeks demonstrating IFNantibody-stained cells (black) within th e follicles (F) and the medullary (M) areas of the lobule............................................................................................................123 6-4 Histologic section of a JSY3 ORF-A/2-infected lymph node at 8 weeks demonstrating IFNantibody-stained cells (black) lo calized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C).............................................123 6-5 Histologic section of a JSY3 ORF-A/2-infected thymus at 16 weeks demonstrating DLEC-antibody-stained cells (black) within the lymphoid follicles (F) of the lobule. Note the lack of DLEC-positive cells within the cortex (C)............................................124 6-6 Histologic section of a JSY3 (WT)-inf ected thymus at 8 weeks demonstrating DLEC-antibody-stained cells (black) within the lymphoid follicles (F) of the lobule....124 6-7 Histologic section of a JSY3 ORF-A/2-infected lymph node at 8 weeks demonstrating DLEC antibody-stained cells (b lack) localized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C).............................................125

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ORF-A/2 DEFICIENT MOLECULAR CLONE OF FELINE IMMUNODEFICIENCY VIRUS (FIV) DEMONSTRATES DIMINISHED VI RAL GENE EXPRESSION IN LYMPHOID TISSUES OF NEONATAL CATS DU RING ACUTE AND LATENT INFECTION By Janelle Marisa Novak December 2007 Chair: Ayalew Mergia Major: Veterinary Medical Sciences In the present study, neonatal cat s were infected at birth with either JSY3 (WT) or JSY3 ORF-A/2 for comparison of viral gene expressi on, proviral load, pathogenicity in thymus, lymph nodes, peripheral blood mononuclear cells (PBMCs) and sorted lymphocyte subpopulations of the blood, during the acute (8 weeks) and chronic ( 16 weeks) stages of infection. The objective of this study was to further examine the impact of the ORF-A/2-deletion in relation to proviral load duri ng acute and chronic FIV infecti on, viral gene expression during acute and chronic FIV infection, as well as track individual change s in proviral load and viral gene expression in the blood lymphocyte populati ons during acute and ch ronic infection. The hypothesis is that proviral load and viral gene transcription will be lo wer in cats infected with the ORF-A/2 deletion-mutant during acute and chronic FIV infecti on. Relative proviral load and expression of viral gag and rev genes was determined using real-time quantitative PCR. Compared to cats infected with JSY3 (WT), there was lower gag and rev gene transcription in thymus, lymph nodes and PBMCs at 8 and 16 weeks in cats infected with JSY3 ORF-A/2. Proviral load in all three lymphoid tissues was lower in JSY3 ORF-A/2 infected cats at 8 weeks only. For peripheral blood lymphocytes, relativ e proviral load and transcription of viral gag and

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14 rev genes was reduced in cats infected with JSY3 ORF-A/2. In particular relative proviral load and viral transcription in JSY3 ORF-A/2 sorted B cells of the PBMCs was statis tically reduced during chronic FIV infection. In addition to re duced viral integration and transcription, there was delayed CD4+/CD8+ T cell inversion and less B cell expansion in PBMCs of JSY3 ORFA/2 infected cats. Immunohistochemical detectio n of FIV p24 Gag protei n revealed distribution of infected cells to the lymphoi d follicles and medullary areas in the thymus of JSY3 (WT) and JSY3 ORF-A/2 infected cats. This type of dist ribution pattern was not observed previously (Norway et al., 2001) but has been observed in HI V/AIDS patients (Prevot et al., 1992) (Burke et al., 1995) and SIV-infected monkeys (Li et al., 1995). Immunohistochemi cal analyses of the thymus with the Ki67 antibody demonstrated pr imarily cortical labeling, indiscriminant medullary staining and exclusion from the follicles. The labeling of the cortical cells represents active cell cycling of non-in fected thymocytes. JSY3 ORF-A/2 infected cats had a higher mean number of Ki67-positive cells compar ed to uninfected or JSY3 (WT) infected cats at either 8 or 16 weeks.

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15 CHAPTER 1 INTRODUCTION Human Immunodeficiency Virus (HIV) As of the year 2006, approximately 40 million people worldwide have contracted human immunodeficiency virus (HIV), of which 18 m illion were women and 2.3 million were children (UNAIDS/WHO; http://www.unaids.org/en/HIV_data /epi2006/). In this same year, 530,000 children were newly infected. The predom inance of infection in women and children emphasizes the need for further HIV research in an effort to decrease the propagation of the virus, by developing additional anti-retrovira l drugs, and potentially an HIV vaccine. The focus on pediatric HIV is important as thes e patients acquire the virus from infected mothers through vertical tran smission which can include in utero peripartum or post-partum exposure. In utero transmission of HIV is increasingly important as human infants infected in utero lose thymic competency and progress to an AI DS state at an accelerated rate compared to infants infected peripartum (C hakraborty, 2005). The same outcome occurs in fetal cats inoculated in utero with feline immunodeficiency virus (FIV), and represents a significant difference in viral pathogenesis based on immune competency of the host (Johnson et al., 2001). At the same time, FIV-infected neonatal cats and older adult cats are more susceptible to infection than young cats, exemplifying that age at infection influences the severity of the disease (George et al., 1993). The numbers of children contracting HIV a nd dying from AIDS annually, mainly from a lack of immune competency at the time of exposur e to the virus, will conti nue to increase. This difference in immune competency is critical to emulate in an animal model if we are to better understand pediatric lentiviral pathogenesis.

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16 Animal Model for HIV A suitable animal model is necessary for pediatric HIV research, since controlled infections of humans is not possible and access to valuable tissues is invasive and limited. HIV, feline immunodeficiency virus (FIV), and simian immunodeficiency virus (SIV), all belong to the lentivirus genus of retroviruses (Knipe et al., 2001). Th e close phylogenetic relationship and clinical course of HIV, to feline immunodefici ency virus (FIV), and simian immunodeficiency virus (SIV), allows the explorati on and use animal models of lentiv irus infection with the goal of understanding HIV (Overbaugh et al., 1997) (Le vy, 1996) (Coffin et al., 1997). All three of these viruses cause an initial acu te infection (fever, malaise, neutropenia) with a resulting clinically latent infection (low plasma viremi a, lymphadenopathy) where the subject displays no overt clinical illness. These viruses have cellular tropisms for CD4+ T cells and the progressive depletion of these cells results in the inability to mount an ef fective immune response against invading pathogens (Pedersen et al., 1987) (Yamamoto et al., 1988) (Chakrabarti et al., 1987). The host experiences a gradual lo ss of CD4+ T cells, inversion of the CD4+:CD8+ T cell ratio, and eventually immunosuppression with a gradual wasting syndrome that results in an acquired immunodeficiency syndrome (AIDS) (Coffin et al ., 1997) (Knipe et al., 2001) (Pedersen et al., 1987) (Chakrabarti et al., 1987). Neonatal FIV Infection In pediatric FIV and HIV, the thymus is th e primary target for virus replication, which causes atrophy and loss of T cell production, whil e the peripheral lymph nodes and blood may function as secondary storage sites for virusinfected cells (Johnson et al., 2001). In the neonatally-infected cat model, acu te infection occurs at arou nd ~8 weeks and asymptomatic latency around ~16 weeks. Utilization of the neonatally-FIV-infected cat model provides many

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17 benefits due to the availabili ty of tissues, control in timi ng of infection and method of inoculation, as well as decreased expense and larger litter size compared to the primate model. Significance of the FIV ORF-A/2 Gene In Vitro ORF -A/2 Studies From the beginning of FIV research, the ORF-A/2 gene has been a target of investigation in order to better understand its role in vi ral replication and pa thogenesis. Early in vitro work demonstrated that frameshift mutations in the ORF-A/2 gene resulted in decreased viral replication, reduced viral reverse transcri ptase (RT) activity, a nd reduced FIV p24 Gag expression in primary peripheral blood lymphocyt es and established T ce ll lines (Tomonaga et al., 1993). Subsequent in vitro work demonstrated that the OR F-A/2-mutated San Diego isolate of FIV (pPR-strain) was growth restricted in primary feline blood leukocytes and a T lymphocyte line (MCH5-4), but not in a felin e astrocyte cell line (G355-5) or Crandell feline kidney cells (CrFK). Another ORF-A/2-mutated FIV strain 34 TF10, demonstrated the ab ility to replicate in the G355-5 cell line and the CrFK cell line, but not in primary fe line blood leukocytes or in the MCH5-4 T cell line (Waters et al., 1 996). This data suggested that ORF-A/2 mutations had an effect on the ability of the virus to replicate in certain cell types, specifically drawing attention to a lymphocyte-restriction. Their data went on to suggest that th e ORF-A/2 protein could behave as a transactivator on the FIV long terminal re peat (LTR), to promote viral gene expression, somewhat like the tat transactivator gene of HIV-1 (Wat ers et al., 1996). In 1999, this same research group demonstrated that the San Die go isolate of FIV (pPR-s train) ORF-A/2 protein transactivated the FIV LTR, resulting in a 14 to 20-fold increase in expression above the basal level (de Parseval et al., 1999). Additionally, th e removal of three site s in the LTR totally abrogated ORF-A/2-mediated transactivation in tr ansient transfection assays (de Parseval et al., 1999).

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18 However, other ORF-A/2 mutations of the pPR-strain of FIV demonstrated little-to-no effect on LTR-driven viral expression in PBMCs or IL-2 dependent T cell lines (Gemeniano et al., 2003). These investigators did find that virions derived from the mutants had decreased infectivity in PBMC and these mutants could be rescued by co-transfection with a wild-type ORF-A/2 expression vector. Taken together, their resear ch suggests that ORFA/2 is involved in virion infectivity and virion formati on potentially signifying that the ORF-A/2 gene is analogous to other accessory genes of primate lentiviruses ( vpr, vpu, nef ). In 2004, this same group demonstrated that the ORF-A/2 gene encoded a nuclear loca lization signal and the ORF-A/2 protein was detected in the nuc leus of CrFK and African gr een monkey kidney (COS-7) cells (Gemeniano et al., 2004). They also f ound that ORF-A/2 protein could induce a G2 cell cycle arrest of transfected cells, thus lending support to their previous statement about the ORF-A/2 gene functionally resembling an accessory gene, vpr, of HIV-1. In Vivo ORF-A/2 Studies There is less work in vivo that has focused on the FIV ORF-A/2 gene. Use of the FIV TM2 strain with ORF-A/2 deletions, in specific pathogen-free (SPF) cats infected between 4-9 weeks of age, demonstrated clinically normal cats at 16 weeks post-infection, wi th varying levels of FIV provirus in lymphoid tissues (Inoshima et al ., 1998). The data from this study suggested that ORF-A/2 was neither required for the viral life cycle nor needed for viral in fection to occur. In 2001, Norway et. al. utilized a highly pat hogenic molecular clone JSY3-point-mutated ORFA/2 and the neonatally-infected SPF kitten mode l (Norway et al., 2001). The study revealed reduced viral replication in JSY3 ORF-A/2 infected cats by lymphoc yte co-culture, quantitative PCR, and immunohistochemistry at 14 weeks post-infection. In addition, JSY3 ORF-A/2 infected thymus demonstrated equivalent ly mphoid follicular hyperplasia with JSY3 (WT) infected thymus (Norway et al., 2001). In 2002, Pistello et. al. infect ed 10 month old SPF cats

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19 with p34TF10-derived mutations of the ORF-A/2 gene. Their data demonstrated reduced plasma viremia, reduced proviral loads in PBMC, and reduced infectious cells in circulation compared to animals with an intact ORF-A/2 gene. It appeared that the inability of ORF-A/2 mutants to replicate in lymphocytes in vitro, as reported previously (Wat ers et al., 1996), was not as absolute in vivo (Pistello et al., 2002). Use of ORF-A/2 in FIV/HIV Vaccine Development The significance of reduced viral replication in lymphocytes and a re duced plasma viremia has propelled an interest in the use of a mutated ORF-A/2 gene in a live attenuated FIV vaccine (Pistello et al., 2005). Vaccination of 10 month old SPF cats with a defective ORF-A/2 gene demonstrated normal peripheral blood lymphocyte numbers and subsets, with no reversion to wild type infection over 22 months of observation. However, 6 of 9 vaccine recipients displayed evidence of challenge virus over 15-months of observation with an increased proviral burden. The results of this work could not be applied to HIV-1 vaccine development, as there is no HIV1 accessory gene that has been implicated in the restriction of virus repl ication in lymphocytes and the use of live attenuated vaccines is not favo red due to safety concerns (Girard et al., 2006). In recent work, the use of a recombinant OR F-A/2 protein to vacc inate six month old SPF cats, showed enhancement of the acute-phase FIV infection post-challenge, but overall the vaccinated cats exhibited reduced plasma FIV an d a slower decline in peripheral CD4+ T cells compared to unvaccinated-FIV-challenged cats (Pis tello et al., 2006). The enhancement of the acute-phase infection was thought to be attributable to an increas ed expression of CD134, a cell surface receptor implicated in FIV cellular tropism, on ORF-A/2-immunized PBMC. Thus, these data support the use of lentiviral accessory proteins, such as ORF-A/2, to improve the ability of the host to control vira l replication and disease progression ; at the same time, the use of accessory proteins in vaccine development is precarious and may exacerbate subsequent

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20 lentiviral infections as seen in cases of SIV (Staprans et al., 2004) (Amara et al., 2005). The enhanced expression of CD134 on the surface of ORF-A/2-immunized PBMC suggests that the ORF-A/2 protein may play an important role in vi ral entry into the cell, and it may not be a safe candidate in the development of a FIV vaccine. Role of ORF-A/2 in Sorted Cell Populations Several laboratories have examined the role of select lymphocyte cell populations in the pathogenesis of FIV, but none of these studies have includ ed the use of an ORF-A/2 deletion mutant virus. In 1993, English et. al. looked at FIV in vivo tropism utilizing the NCSU1 isolate in 16 adult cats. The experiment involved the extraction of lymphocytes and their subsequent maintenance in culture with IL-2 and Con-A. In 2004, Shimojima et. al. (Shimojima et al., 2004) published a report that indicated that CD134 ( OX40) acts as a primary receptor for FIV. Additionally, CD134 is up-regulated on CD4+ T cells activated by treatment with IL-2 and Con A (de Parseval et al., 2004). This study was an ex vivo analysis as opposed to in vivo analysis of cellular tropism with the modification by IL-2 an d Con-A. Proviral load was then examined by Southern blot and infectivity was measure by reve rse transcriptase (RT) assay. It was found that all cats had provirus in CD4+, CD 8+ and B cells and that CD4+ T cells had the highest level of provirus 2 to 4 weeks afte r infection, while B cells had the highes t proviral load in chronic status ( 1 yr). The study also looked at monocyte popul ation and found that only one out of ten cats had detectable FIV. While this study re ported initial FIV cellu lar tropism, albeit ex vivo there was no quantification of viral gene expression from these sorted cells or the use of an ORF-A/2 mutant at acute and chroni c stages of FIV infection. Yang et. al. (Yang et al., 1996) characteri zed the molecular clone JSY3 (parent NCSU1) in adult cats. As previously repor ted, FIV provirus was first detected in CD4+ T cells during acute infection with JSY3 (2-4 weeks post infecti on.), from 14 wks on, provirus was found in CD8+, B

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21 and CD4+ T cells. Their data suggested FIV infec tion of CD8+ T cells and B cells to be latent, and non-cytolytic. On the contrary, the reductio n in the number of CD4+ T cells into chronic infection was the result of activ e, cytolytic, infection. The authors did not use a neonatally infected kitten model, examine viral gene expr ession, quantitate provirus by Real-Time PCR, or examine proviral load in the absence of the ORF-A/2 gene in sorted populations at acute and chronic phases of infection. In 1996, Dean et. al. (Dean et al., 1996) examined proviral burden and viral kinetics of FIV in lymphocyte subsets of the blood and lymph node Using the Petaluma strain and adolescent cats, they looked at acute inf ection (2-12 months) and chroni c infection (6-10 months) and subsequently sorted for CD4+, CD8+, and CD21 cel ls. They examined proviral levels by whole cell, endpoint dilution and semi-qua ntitative PCR and RT-PCR. Thr ough this they were able to demonstrate that CD4+, CD8+ and B cells contai ned provirus in acute and chronic states of infection. They also reported that there was a greater increase in proviral levels in acute infection as opposed to chronic infection, in pe ripheral blood mononuclear cells (PBMCs). At acute stages of infection the pr oviral load was comparable in lymph node and PBMCs. At 20 weeks they measured RNA in the lymph node of 5 cats and demonstrated that they were RTPCR positive (i.e. presence of viral RNA). There was no quantification of viral gene expression from the lymph node or the sorted cells of the PBMCs, or the use of an ORF-A/2 mutant at acute and chronic stages of FIV inf ection. Thus, the use of an ORF-A/2 -deleted mutant virus, as examined in this research, will help elucidate the role of the ORF-A/2 gene in viral gene expression, in different cellular environments. Goals of the Study Previous in vivo work with the FIV ORF-A/2 gene has distinctions which include animal age differences, viral strain differences, level of sensitivity in assays performed, lack of viral

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22 gene expression, and the absence of information regarding ORF-A/2s involvement in sorted cell populations. The goal of this research project is to more fully characte rize the role of the ORFA/2 gene expression in vivo and to understand the mechanism of FIV pathogenesis by analyzing proviral load and gene expression from the acute to the chronic phase of infection, in the presence or absence of a functional ORF-A/2 gene, utilizing the high ly pathogenic molecular clone JSY3. To accomplish this objective, the neonatally-infected cat was selected as the infection model. In this study, neonatal cats we re infected with a highly pathogenic wild type FIV (JSY3) or its ORF-A/2 deletion mutant. The major lymphoid tissue compartments the thymus, lymph nodes, and periphe ral blood mononuclear cells (P BMCs) were harvested during the productive and chronic phases of infection. These compartments were analyzed for proviral load, viral gene transcription, a nd the localiza tion of virus-infected and actively replicating cells (in the thymus and lymph node) of cats infected with the JSY3 wild type virus or an ORF-A/2 mutant. The hypothesis is that proviral load and viral gene e xpression will be lower in the thymus, lymph node, and PBMCs of cats infected with the ORF-A/2 deletion-mutant during acute and chronic FIV infection. In the following chapters, feline i mmunodeficiency virus (FIV) and the ORF-A/2 gene will be discussed in full (Chapter 2) The methods, experimental desi gn, and results of this project will be discussed in detail in the subsequent chapters: Chapter 3: The quantification of proviral load and the quantification of viral gene expression ( gag and rev ) in thymocytes, lymph node cells, peripheral blood lymphoc ytes, and blood lymphocyte subs ets during the productive and chronic phase of virus infection; Chapter 4: The localizat ion and quantifica tion of p24-virusinfected-and actively re plicating thymocytes and lymphocytes during the productive and chronic phase of virus infection u tilizing immunohistochemistry.

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23 Supplemental information on the charac terization and analysis of the FIV ORF-A/2 gene will be detailed in Chapters 5, and 6: Chapter 5 presents data on the cytokine(s) (IL-4, IL-7, IL15, IFN, and IFN) gene expression in JSY3 (WT) and JSY3 ORF-A/2 infected cats during acute and chronic infection. A nd, Chapter 6 describes the visualization of the plasmacytoid dendritic cell type (PDC) util izing immunohistochemistry in th ymus and lymph node samples of JSY3 (WT) and JSY3 ORF-A/2 infected cats during acute and chronic infection.

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24 CHAPTER 2 LITERATURE REVIEW Retroviridae The retroviridae comprise a large family of vi ruses found in all verteb rates (Knipe et al., 2001). Retroviruses are termed because they have a single-stranded RNA genome which is reverse-transcribed into double stranded DNA, by its own viri on-associated RNA-dependent DNA-polymerase (reverse transcript ase) (Coffin et al., 1997). There are seven genera of retrovirus classification and they are categorized as either simp le or complex retroviruses. Simple viruses encode Gag, Pol, and Env gene products while complex viruses encode additional regulatory proteins. The al pharetroviruses, betaretroviru ses and gammaretroviruses are considered simple viruses, whereas the deltav iruses, epsilonretroviruses, lentiviruses, and spumaviruses are complex viruses (Knipe et al., 2001). Feline immunode ficiency virus (FIV) belongs in the lentivirinae group and is a complex virus (Pedersen N.C., 1993a). The viral genome consists of two identical RNA molecules about 7-10 kilobases in length and they are modified similar to cellular mRNAs including capping at the 5 end and polyadenylation at the 3 end. The order of the structural genes is gag-pro-pol-env All lentiviruses have at least thr ee auxiliary genes in addition to gag-pro-pol-env (Knipe et al., 2001). FIV has three auxiliary genes: vif, rev and ORF-A/2 (Tomonaga et al., 1996). Retroviruses have an enveloped virion, wh ich is approximately 100 nm in diameter, derived from the env gene (Knipe et al., 2001) (Pedersen N.C., 1993a). The internal core is made up of 3-4 products of the gag gene, as well as catalytic proteins prot ease, reverse transcriptase and integrase (Coffin et al., 1997) Reverse transcriptas e is responsible for converting single-stranded RNA into double-st randed DNA, and integrase is necessary for inserting virus into cellular DNA to form provirus (Coffin et al., 1997).

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25 Viral replication occurs in two stages. The fi rst stage includes entry of the virion core into the cytoplasm, synthesis of double-stranded DNA, (utilizing the single-stranded genome as template), transfer of the pre-integration complex to the nucleus and integration of DNA into the host genome (Coffin et al., 1997). This integrati on takes place in the absence of viral gene expression and is regulated by proteins found w ithin the virion (Pedersen N.C., 1993a). The second stage of replication in cludes synthesis and processing of viral genomes, mRNAs and proteins using the host cells RNA polymerase (C offin et al., 1997). Virion assembly occurs by encapsidation of the genome by unprocessed precursors of gag, pro and pol genes, association of the nucleocapsids with the cell membrane release of the virion by budding and final processing of the precursors to finished produc ts (Coffin et al., 1997) (Pedersen N.C., 1993a) (Knipe et al., 2001). Lentiviruses Lentiviruses are characterized by a unique cyli ndrical or conical viri on core (Knipe et al., 2001). Examples of lentiviruses include HIV-1, visna virus, capri ne arthritis encephalitis virus (CAEV), equine infectious anemia virus (E IAV), bovine immunodefici ency virus, simian immunodeficiency virus (SIV), and feline immun odeficiency virus (FIV) (Knipe et al., 2001) (Coffin et al., 1997). All lentiviruses bud from the plasma membrane without a preformed nucleoid (Knipe et al., 2001). All lentiviruses have a specif ic tropism for macrophages, are cytopathic and persist in cells (Brown et al., 2006) (Dean et al., 1999) (Brunner et al., 1989) (Dow et al., 1990) (Coffin et al., 1997). They ca use chronic degeneration and a slow progressive disease in its host (Michael et al., 1991). The lentivirus genus includes viruses responsible for a variety of neurological and immunologi cal diseases (Coffin et al., 1997).

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26 Feline Immunodeficiency Virus (FIV) The etiologic agent of a chronic, progre ssive disease found in felines is feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus (HIV-1) that causes AIDS in humans (Pedersen et al., 1987). This virus was first is olated in Petaluma, California in 1986, from a multiple cat household. FIV was reported after the discovery of HIV1 in 1983 and HIV-2 in 1986 (Barre-Sinoussi et al., 1983;Clavel et al., 1986). Analogous to HIV, at 4 to 6 weeks post-FIV infection, a generali zed immune suppression is prominent, with an acute infection that begins with mild fever, lymph node enlargement a nd a gradual decline in CD4+ T cells (Pedersen et al., 1987). This decl ine continues into a latent asymptomatic infectious stage marked with an inverted CD4: CD8 T cell ratio (CD8+ T cell population increases; B cells may increase and will infiltr ate the thymus). Generally, other opportunist infections mark the remaining co urse of life with gradual wast ing syndrome such as seen in human acquired immunodeficiency syndrome (AIDS) patients (Yam amoto et al., 1988) (Ackley et al., 1990). As a result of its ability to induce immunodeficiency, FIV is an important small animal model for use in HIV/AIDS research (Siebelink et al., 1990). Physical properties of FIV The complete virion of FIV is 105-125 nanometers in diameter, is spherical to ellipsoid in shape and contains short envelope projections (Pedersen N.C., 1993a). The virus buds from the plasma membrane of infected cells much like other retroviruses crea ting a crescent-shaped appearance (Pedersen N.C., 1993a). FIV has a typical buoyant density in sucrose of 1.15-1.17 g/cm3, much like other retroviruses a nd it is readily inactivated by disinfecting agents such as alcohol, chlorine, and phenolic compounds (Pedersen N.C., 1993b).

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27 Molecular properties of FIV The genomic organization of FIV (Figure 2-1) is similar to other lentiviral genomes in that it contains several open reading frames which contain genes enc oding for structural proteins (Gag, Pro, Pol, Env) and the genomic size is anal ogous to the genomic size of lentiviruses about 9.4 kilobase pairs (Miyazawa, 2002). Additional ope n reading frames (ORFs) exist which code for vif, rev and orf-a/2 genes (Miyazawa et al., 1994) (Tomon aga et al., 1992) (Tomonaga et al., 1996) (Kiyomasu et al., 1991). At the 3 end of the env gene is a 243-nucleotide region that is the rev -response-element (RRE) where the Rev protei n binds (Phillips et al., 1992) The provirus genome (the integrated viral genome) contains long termin al repeat (LTR) sequences, at both ends which are comprised of three regions designated as U3, R and U5 (Figure 2-1) (Miyazawa, 2002). FIV genome The length of the FIV LTR is similar to ot her lentiviruses (CAEV, EIAV, V-MV) but shorter than SIV or HIV (Pedersen N.C., 1993a). The FIV LTR, like the LTR of CAEV and VMV, is capable of a high basal level of transcription in establ ished cell lines (Sparger et al., 1992) (Neuveut et al., 1993) (Saltarelli et al., 1993). The U3 promoter region of the FIV LTR c ontains binding sites for enhancer/promoter proteins such as AP-1, C/EBP, AP-4, ATF, a nd NF-1 (Inoshima et al., 1998) (Miyazawa et al., 1991) (Phillips et al., 1990) (Thompson et al., 1994) Nuclear proteins can specifically bind to the AP-1, ATF, AP-4, and C/EBP elements (Thom pson et al., 1994) (Ikeda et al., 1998). T-cell activation takes place via the AP-1 site by protein kinase C or c-Fo s; the ATF site is required for protein kinase A-mediated cyclic AMP (cAMP) response (Ikeda et al., 1996) (Miyazawa et al., 1993) (Sparger et al., 1992). The jun/fos family of proteins regulates the transcription of genes containing the AP-1 sites; and the CAAT enha ncing binding proteins (C/EBP) regulate the

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28 transcription of genes the C/EBP sites (LekstromHimes et al., 1998) (Rahmsdorf, 1996). Cyclic AMP response element (CRE) sequences serve as the binding site for members of the ATG/CREB (CRE binding proteins) family of proteins (M ontminy, 1997) (Meyer et al., 1993). The four FIV structural genes gag-pro-pol-env have different functions. Gag codes for internal structural proteins of the virion, while pro which is located at the 5 end of the pol gene, encodes for the virion protease (Knipe et al., 200 1) (Coffin et al., 1997) (Pedersen N.C., 1993a). Pol codes for reverse transcriptase and env codes for envelope glycoprot eins of the virion (Knipe et al., 2001) (Coffin et al., 1997) (Pedersen N.C., 1993a). Vif has been shown to encode for an accesso ry protein, which is necessary for viral infectivity and efficient viral replication (Shackle tt et al., 1994) (Lockridge et al., 1999). This gene is similar in size and position to the vif of primate lentiviruses but no significant genetic homology exists. Vif protein lo calizes to the nucleus (Chatterji et al., 200 0), and may enhance infectivity in non-permissive cells by blocking a ce ll specific inhibitor as s een in HIV-1 (Yang et al., 2007) (Russell et al., 2007) (Opi et al., 2007). The Rev gene encodes a post-transcriptional re gulatory protein that binds to a rev response-element (RRE) (Phillips et al., 1992). The rev gene product is critical for viral replication (Kiyomasu et al., 1991) (Tomonaga et al., 1995) (Phillips et al., 1992). Rev is an essential viral protein encoded by a small, mu ltiply spliced mRNA synthesized at early times after infection (Coffin et al., 1997). Multiply spli ced events include Rev, and in the case of HIV1, Tat and Nef. Multiply spliced RNA first appear s in the cytoplasm early after transcription. When a level of Rev is reached, unspliced and singly spliced RNAs begin to accumulate in the cytoplasm, allowing productive infection to pr oceed (Michael et al., 1991) (Kiyomasu et al., 1991) (Tomonaga et al., 1995) (Phi llips et al., 1992) (Tomonaga et al., 1996). Full length or

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29 singly spliced mRNA code for viri on structural proteins Gag, Pro, Pol and Env and thus leads to virion formation and a productive infection. The functions of the ORF-A/2 gene product have not been fully elucidated. Similarities in the genomic placement of ORF-A/2 in FIV, to the tat transactivator gene of HIV, have provoked further studies into the role of ORF-A/2 protein as a putative tr ansactivator that regulates FIV gene expression (de Parseval et al., 1999). In HIV-1, the Tat protein behaves as a Type I Tat protein that up-regulates viral gene expression via interaction with a Tat transacting response region (TAR), an RNA stem-loop structure encode d in the long terminal repeat (Roy et al., 1990). In FIV however, the LTR does not contai n a hairpin RNA loop st ructure like the HIV-1 TAR, and the FIV LTR has a str ong basal activity (Sparger et al ., 1992) (Thompson et al., 1994). ORF-A/2 encodes a protein of 79 amino acids with a C-terminal cysteine-rich region like other tat genes of lentiviruses (Tomonaga et al., 1996). The ORF-A/2 pr otein also contains a leucinerich region in the middle of the sequence, an N-terminal acidic/hydrophobic region but does not have the core and basiccoding regions present in tat genes of primate lentiviruses (Tomonaga et al., 1996) (Gemeniano et al., 2003). Based on the amino acid sequence of ORF-A/2 protein and the strong basal promoter activity of the FIV LTR the FIV ORF-A/2 protein has been postulated to regulate transactivation in a transacting re sponse (TAR)-independent manner (as Type 2 Tat protein) (Tomonaga et al., 1996). In vitro the ORF-A/2 gene product has demonstrated slight transactivation of the FIV LTR (de Parseval et al., 1999). However, other studies demonstrate only a small or no effect created by the ORFA/2 protein on the FIV LTR gene expression (Thompson et al., 1994) (Miyazawa et al., 1993) (Sparger et al ., 1992) (Waters et al., 1996). In 2001, Norway et. al. demonstrated a decreased rate of viral replicat ion and pathogenesis in 14 week old-JSY3 ORF-A/2 infected cats, relative to th e JSY3 (WT), with reduced proviral

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30 load, although the extent of thymic damage betw een both groups was equivalent (Norway et al., 2001). In 2003, Gemeniano et. al, (Gemen iano et al., 2003), demonstrated that ORF-A/2 may function at multiple steps of the FIV life cycle to include both virion formation infectivity, and may have properties with the HIV-1 vpr gene, including nuclear local ization and the ability to induce G2 cell-cycle arrest (Gemeniano et al., 2004) In summary, the ORF-A/2 protein may have a variety of functions and its complete role in FIV pathoge nesis remains somewhat elusive. FIV infection: cellular tropism In HIV-1 infection, the virus binds to th e CD4 cell surface molecule, in addition to chemokine receptors CXCR4 or CCR5 (Coffin et al., 1997). These binding events determine virus cellular tropism and whether or not the infection is productiv e. In FIV infection, it has been demonstrated that FIV utilizes chemokine receptor CXCR4 (Garg et al., 2004) (Frey et al., 2001). In 2004, Shimojima et. al. (Shimojima et al., 2004) published a re port indicating that CD134 (OX40) acts as a primary receptor for FIV; CD134 is up-regulated on CD4+ T cells activated by treatment w ith IL-2 and Con A (de Parseval et al., 2004a). Soluble CD134 also facilitates FIV entry into CX CR4 positive cells (de Parseval et al., 2006). Although FIV does not use CD4 as the primary recep tor, it does share similarities with HIV-1 in the use of CXCR4, DC-SIGN, and cell-surface heparans for viral entr y (Geijtenbeek et al., 2000) (Saphire et al., 2001;de et al., 2001) (de Pa rseval et al., 2004b). In vitro Petaluma and PPR isolates of FIV have been reported to target the CD4+ and CD8+ T lymphocytes during infection (Brown et al., 1991). In vivo FIV has also been detected in lymphocyt es (Pedersen et al., 1987), in peritoneal macrophages (Brunner et al., 1989), and in central nervous system tissue (Dow et al., 1990). English et al. (English et al., 1993) subsequently published that bot h T-cell subsets are infected in vivo as well as immunoglobulin-positive (Ig+) cells. Dean et. al. (D ean et al., 1996), reported that CD4+, CD8+ and B cells cont ained provirus in acute and chroni c states of infection. They

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31 also reported that there was an increase in proviral levels in the peripheral blood mononuclear cells (PBMCs) in acute FIV infec tion rather than chronic FIV in fection. The proviral load was the same in the acute stages of in fection in the ly mph node and PBMC. FIV infection: tissue tropism Upon infection with FIV, the ea rliest places to detect the virus are the thymus, the lymph nodes, notably the mesenteric lymph node, as well as the central nervous system (Beebe et al., 1994) (Dua et al., 1994) (Obert et al., 2002) (Rogers et al., 1998) (Toyosaki et al., 1993). At later stages, FIV is detectable in the intest ines, kidney, liver, bone marrow and salivary gland (Beebe et al., 1994) (Rogers et al ., 1998) (Bishop et al., 1996) (Obert et al., 2000b) (Obert et al., 2000a) (Park et al., 1995). The route of exposure to the virus does not seem to be important, rather virus strain, and the type of inoculum are the determining factors (Bishop et al., 1996) (Obert et al., 2000b). The thymus is particularly sensitive to FIV infection and can become atrophic (Johnson et al., 2001) (Beebe et al., 1994). Fe tal FIV infection cause s a temporary depletion of thymocytes, which is followed by a rebound in thymocytes to promote further viral re plication (Johnson et al., 2001). However, in neonatal kittens, a progressi ve atrophy occurs with a decreased ability to support replication over time (Johnson et al., 2001) Prominent B-cell foll icles form within the thymus during the acute stage of infection, and increase during the latent or asymptomatic phase (Liang et al., 2000) (Orandle et al., 2000). This formation of follicles does not appear to be directly related to virus re plication as seen in previous work (Norway et al., 2001). In the lymph node, follicular and paracortical hyperplasia occur. Polyclonal B cell activation and expansion of the B cell population seems to be correlated with the development of lymphoid follicles in other ti ssues including the kidney, thymus eye, bone marrow and salivary gland (Flynn et al., 1994) (Walker et al., 1994).

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32 Pathogenesis in natural host Isolates of FIV have come from domestic cats, but research has reve aled that FIV-related viruses have also been found in a number of wild cat species, including African lions, leopards, and cougars (Barr et al., 1995) (V andeWoude et al., 1997) (Brown et al., 1994). Studies have shown that each of these wild cat species is infected with its own particular form of FIV. While domestic cats infected with FIV often suffer fr om AIDS-like symptoms (B ull et al., 2003), wild cat species infected with their form of FIV appear to show no signs of disease (HofmannLehmann et al., 1996). It is thought that this lack of disease c ould stem from a long history of co-evolution between virus and its wild cat ho sts (Olmsted et al., 1992) (Pedersen N.C., 1993b). FIV had been identified in many parts of th e world including the United States, Canada, South Africa, Europe, China, Japan, New Zeala nd and Australia (Pedersen N.C., 1993b). The virus has been recovered from the bodily fluids of infected cats, such as blood, saliva, plasma and serum (Yamamoto et al., 1988) (Dow et al ., 1990). Contact, as a means for horizontal transmission, can occur but it is not as efficient as bite wounds. It has been shown that a single experimentally administered bite from a naturally or experimentally infected cat will transmit the infection to another cat (Yamamoto et al., 1988). Subsequent studies have show that it can also be transmitted by mucosal exposure, blood transfer and vertically from mother to offspring (Pedersen et al., 1987) (Callanan et al., 1991) (O'Neil et al., 1995) (O'Neil et al., 1996) (Sellon et al., 1994). FIV diagnosis and control In the feral and pet cat popul ation, routine screening for FI V antibodies is performed via patient-side immunochromatic lateral flow antibo dy tests. Confirmation of FIV infection is made by Western Blot, immunofluorescent antibody test (IFA), P CR or virus culture. Since FIV is typically contracted through bite wounds, the trap, neuter, and release programs (TNR) serve

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33 to decrease the population of feral cats, especially intact male cat s, which are over-represented in FIV infection. In 2002, Fort Dodge Animal Hea lth released Fel-O-Vax, the first vaccine licensed for the preven tion of FIV. This vaccine is dual-subtype cont aining subtype A (Petaluma) and subtype D (Shiz uoka) which is in the inactivated form (Pu et al., 2001). The vaccine is licensed for use in cats 8 weeks and older and consists of the administration of 3 doses, 2 to 3 weeks apart. Annual boosters are recommended thereafter. The vaccine has shown substantial protection against FIV challenge duri ng vaccine trials but it remains uncertain how the vaccine will protect against other strains outside of subtypes A and D. The vaccine contains whole virus and as a result cats develop antibodi es that are indistingu ishable from antibodies produced during natural inf ection. This currently presents a challenge in the diagnosis of FIV infection as vaccinated cats may be false-po sitive on all methods of testing (ELISA, IFA, Western blot) (Crawford et al., 2007). Treatment of FIV-infected cats consists mostly of supportive care and addressing secondary conditions (stomatitis, anemia, a nd lymphoma). The use of 3'-azido-2',3'dideoxythymidine (AZT) in cats has been more thoroughly researched and appears to reduce the plasma viral load and increase the CD4+ cell co unt. Unfortunately, most of the other antiretroviral pharmaceuticals available for humans are t oo toxic or have not yet been studied in cats. The goal of this research project is to more fully characterize the role of the ORF-A/2 gene expression in vivo and to understand the mechanism of FIV pathogenesis by analyzing proviral load and gene expression from the acute to the chronic phase of infecti on, in the presence or absence of a functional ORF-A/2 gene, utilizing the highly pa thogenic molecular clone JSY3. The methods, experimental design, and results of th is project will be discussed in detail in the subsequent chapters: Chapter 3: The quantification of proviral load and the quantification of

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34 viral gene expression ( gag and rev ) in thymocytes, lymph node cells, peripheral blood lymphocytes, and blood lymphocyte subsets durin g the productive and chronic phase of virus infection; Chapter 4: The localization and qua ntification of p24-viru s-infected-and actively replicating thymocytes and lymphocytes duri ng the productive and chronic phase of virus infection utilizing immunohistochemistry. Supplemental information on the charac terization and analysis of the FIV ORF-A/2 gene will be detailed in Chapters 5, and 6: Chapter 5 presents data on the cytokine(s) (IL-4, IL-7, IL15, IFN, and IFN) gene expression in JSY3 (WT) and JSY3 ORF-A/2 infected cats during acute and chronic infection. A nd, Chapter 6 describes the visualization of the plasmacytoid dendritic cell type (PDC) util izing immunohistochemistry in th ymus and lymph node samples of JSY3 (WT) and JSY3 ORF-A/2 infected cats during acute and chronic infection. Figure 2-1. Genomic struct ure of Feline Immunode ficiency Virus (FIV).

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35 CHAPTER 3 VIRAL GENE EXPRESSION IN LYMPHOCYTE SUBPOPULATIONS OF NEONTAL CATS INFECTED WITH THE MOLECULAR CLONE FIV OR ORF-A/2 DEFECTIVE FIV DURING ACUTE AND CHRONIC STAGES OF INFECTION Introduction ORF-A/2 mutations have shown various effect s on the ability of FIV to replicate in vitro including the preservation of replication in fibr oblastoid cell culture line s (Crandell feline kidney cells (CrFK)) and monocyte-derived macrophage cultu res, but the restricti on of virus replication in feline peripheral blood leukocytes (PBLs) (T omonaga et al., 1993a) (W aters et al., 1996). Increased viral gene expr ession, as a result of ORF-A/2 gene product transact ivation of the FIV long terminal repeat (LTR), has also been repo rted CrFK and HeLa cells (Waters et al., 1996) (de Parseval et al., 1999). In vivo ORF-A/2 mutations restrict virus replicati on in peripheral bl ood mononuclear cells (PBMC) of infected specific-pathogen-free (SPF) cats (Dean et al., 1999) (Norway et al., 2001). Norway et al. also demonstrated th at neonatal cats infected with an ORF-A/2 deletion mutant of FIV had a delayed onset of CD4+:CD8+ T cell i nversion, reduced proviral load in lymphocytes, reduced p24 Gag-positive-thymocytes, lower plasma viremia, and reduced expansion of CD8+ T cells compared to cats infected with the wild type parent viruses (Norway et al., 2001). However, whether the ORF-A/2 mutant deletion affects virus ge ne expression and replication in different lymphoid compartments during acute and chronic FIV inf ection is unknown. The hypothesis is that proviral load a nd viral gene expression will be lo wer in cats infected with the ORF-A/2 deletion-mutant during acute and chronic FIV infection. To accomplish this objective, the neonatally-i nfected cat was selected as the infection model. Neonatal cats were infected with a hi ghly pathogenic wild type FIV (JSY3) or its ORFA/2 deletion mutant. The major lymphoid tissue co mpartments the thymus, lymph nodes, and

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36 peripheral blood mononuclear cells (PBMCs) were harvested during the productive and chronic phases of infection for the comp arison of provirus load, and vira l gene expression between cats infected with the wild type virus or an ORF-A/2 mutant. Materials and Methods Cell Lines Human fibroblast 293T cells were cultured in Dulbeccos minimal essential medium (DMEM), supplemented with 200nM L-glutamin e, 10% fetal bovine serum (Serologicals Corporation, Norcross, GA), 20 units/ml penic illin, and 120 g/ml streptomycin. The human 293 T cells were maintained at 37C in 5% CO2. Feline CD4+ T cells (CD4E), kindly provided by Wayne and Mary Tompkins (North Carolina Stat e University, Raleigh, NC ), were cultured in RPMI 1640 (cRPMI) supplemented with 10% feta l bovine serum, 2mM L-glutamine, 10mM HEPES, 0.075% sodium bicarbonate, 2mM sodium pyruvate, 20 units/ml penicillin, 120g/ml streptomycin, 0.0004% 2-mercapto ethanol, and 100 U/mL of recombinant human interleukin 2 (rIL-2; provided by the NIH AIDS Research and Reference Reagent Program, Rockville, MD) (complete RPMI, cRPMI). The feline CD4E cells were maintained at 37C in 7% CO2. Construction of CMVFIV Wild-Type and CM VFIVORF-A/2-Deficient Molecular Clones Molecularly cloned JSY3 FIV provirus (Ya ng et al., 1996) was ki ndly provided by Wayne Tompkins (North Carolina State Un iversity, Raleigh, NC.). The JSY3 ORF-A/2-deficient molecular clone was constructed by site-speci fic-mutagenesis involvi ng two doublet nucleotide deletions in regions [6013/6014] and [6028/6029] of JSY3 as desc ribed previously (Norway et al., 2001) (Figure 3-4). To allo w for one round of replication of these feline viruses in the human 293 T cell line, a cytomegalovirus (CMV) prom oter was inserted in place of the FIV U3 promoter in the JSY3 and the JSY3 ORF-A/2 provirus (Figure 3-1; Figure 3-2). Insertion of the

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37 CMV promoter was conducted using standard te chniques and plasmids containing JSY3 and the JSY3 ORF-A/2 provirus (Sambrook J., 1989). The following narrative describes the details of cloning the CMV promoter to the JSY3 ORF-A/2 construct. Briefly, PSP72 ORF-A/2 (Norway et al., 2001) and PSP72CMVFIV were digested wi th restriction endonucleases Sph I and Eco RI for 1.5 hours at 37C, to reveal an approximate 9 Kb and a ~2 Kb fragment for each plasmid. PSP72 ORF-A/2 was phosphatase (calf intestine) tr eated for 30 min at 37C then placed on ice till gel loading. After enzyme digestion was complete, a 1% ag arose gel was run to visualize both digested products. The 9 Kb PSP72 ORF-A/2 plasmid backbone was extracted from the agarose gel as well as the ~2 Kb insert from the PSP72CMVFI V, which contained the CMV promoter. This was done utilizing the QIAEX II agarose gel extr action protocol (Qiagen Inc., Valencia, CA). From the gel extraction, DNA was isolated for li gation purposes. Overni ght ligation protocol included 1L T4 DNA ligase (New England Bi olabs, Ipswich, MA.), 2L vector (PSP72 ORFA/2) and insert (CMV) on ice. The following da y, ligation reaction was tr ansformed with E. coli cells (DH5 ). Briefly, 100L bacterial cells were adde d to the ligation mix and incubated for 30 min on ice. Cells were then heat shocked for ex actly 90 seconds at 42C. Afterwards, cells were placed on ice for 1-2 minutes. Liquid broth (LB) media was then added to snap-top polystyrene tubes and transformation mix was added to tubes then allowed to incubate at 37C for 45 to 60 minutes. The mix was pelleted at room temperat ure for 30 seconds. All supernatant was then removed except the last 50-100L, which was used to mix pelleted cells by repeated pipetting up and down. Cells were then streaked onto an agar plate, coating evenly. The plate was incubated for 16 to 18 hours at 37C, till the following day for colony picking.

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38 After overnight plate growth, multiple col onies were selected and grown in 3 mL LB/ampicillin (100g/mL final concentration of ampicillin) in snap-top polystyrene tubes. Samples were placed in a rotary shaker at 37C for overnight growth. The following day, DNA was isolated from the samples. In brief, the bacterial culture was pelle ted and re-suspended in GTE buffer (50mM glucose, 10mM EDTA pH 8.0, 25 mM Tris CL pH 8.0) containing 2mg/mL lysozyme. The suspension was vortexed and then 200 L of 0.2 N NaOH, 1% SDS was added to each tube, and tube was inverte d. 150L of 3M sodium acetate pH 4.8 was added, tube inverted and incubated at -20C for 10 minutes. The suspension wa s centrifuged at maximum speed for 15 minutes and then supernatant was decanted into a fresh tube containing 900L of absolute ethanol. The suspension was inverted to mix and centrifuged at maximum speed for 5 minutes. The supernatant was then aspirated off from the pellet, and 750L of abso lute ethanol was added to the tube. The suspension was i nverted to mix, and incubated at -70C for 10 minutes. The suspension was then centrifuged for 5 minutes at maximum speed, and the supernatant was aspirated. The sample was then speed-vac until dry. The DNA sample was then re-dissolved in 25-50L of distilled water. DNA mini-preparations were then screened by restriction endonucleas es to determine if they contained the proper construct. Once th e proper construct was identified, the clone was grown in 250 L of ampicillin and 250 mL of LB media for amplification. Isolation of the plasmid was accomplished using the Qiagen pl asmid purification protocol (Qiagen Inc., Valencia, CA) according to manufacturers instructions. The sequence of the pCMVFIV wild-type a nd pCMVFIVORF-A/2-deficient constructs was confirmed by the University of Florida DNA Se quencing Core Laboratory (Gainesville, FL). The CMV promoter facilitated strong FIV expre ssion in the human 293 T cells to amplify virus

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39 production. During subsequent repl ication of these constructs in feline CD4E cells, the CMV promoter was deleted during reverse transcripti on and replaced by the FIV U3 promoter in the progeny virions (Figure 3-3). Virus Preparation Human 293 T cells (4x105 cells/mL) were transfected with 1.5 g of plasmids containing the pCMVFIV wild-type or pCMVFIVORF-A/2deficient constructs using the reagent LipofectAMINE according to manufacturers instruc tions (Life Technologies, Gaithersburg, MD). After 8 hours, the transf ection solution was replaced with minimal essential media (MEM; Invitrogen Corporation, Carlsbad, California). After 24 hours, 106 feline CD4E cells in cRPMI were added to the transfected 293 T cells. Foll owing 4 days of incubation at 37C in 7% CO2, suspended CD4E cells were carefully removed fr om the adherent 293 T cells and placed into new culture plates for furthe r incubation at 37C in 7% CO2. A 25L aliquot of culture supernatant was removed every other day for measurement of Mg++-dependent viral reverse transcriptase (RT) activity as previously descri bed (Johnson et al., 1990). Culture supernatants were harvested and then utilized to infect 2x106 CD4E cells. The supernatant of cultures containing JSY3 wild-type virus was harveste d on day 7, while supernatant containing JSY3 ORF-A/2 deficient virus was harvested on day 13. The culture supernatants were passed through a 45-m filter and viral genomi c DNA was isolated using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA). The DNA was am plified by PCR using primers that flank the ORF-A/2 gene. These primers were designed for dete ction of point mutations introduced into the ORF-A/2 gene (primers RMN 3 and RMN 4, Table 3-1) The presence of JSY3 wild-type and JSY3 ORF-A/2 FIV viruses in DNA extracted from culture supernatants was determined by conventional PCR performed on a reaction mixture containing 2 L of RMN 3 and RMN 4

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40 primers (20pmol/L) (Table 3-1), 2 L of D NA (100-200 ng), 1L of 10 mM dNTP mixture, 1 L of PFU, and 10 L of 10x PFU buffer (Stratag ene, La Jolla, CA). PCR cycling conditions were: 94C for 1min, 58C for 2 min, and 72C for 1 min for 30 cycles. The PCR products were sequen ced to confirm that the ORF-A/2 deletions were still present (University of Florida DNA Sequencing Core Labor atory; Gainesville, FL) (Figures 3-4, 3-5). The TCID50 for each virus was determined by 4-fold titration in CD4E cells as previously described (Johnson et al., 1990). The ti ter of JSY3 wild-type virus was 7.4x106 TCID50/mL and the titer of the JSY3ORFA/2 deletion mutant was 2.9x104 TCID50/mL. Animals and Inoculation One-day-old specific pathogen-free kittens we re injected intraperitoneally with 200 L cRPMI 1640 media containing 104 TCID50 of either the JSY3 wild-typ e virus (WT) (n=6) or the JSY3ORF-A/2 deletion virus (n=10). Following i noculation, the kittens were returned to their respective queens for the duration of the study. Infection was confirmed by PCR analysis of blood samples as described below. The kittens were euthanatized by intravenous administration of a barbiturate (Beuthansia, Schering-Plough Animal Health Corp, Union, NJ ) at 8 weeks (n=3 for JSY3 wild type (WT), n=4 for JSY3 ORF-A/2 deletion virus) and 16 weeks (n=3 for JSY3 wild type (WT), n=6 for JSY3ORF-A/2 de letion virus) post-infec tion for collection of tissues. The kittens and their queens were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The study was approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). Blood Samples Blood samples from each kitten were collect ed via jugular venipuncture into tubes containing EDTA prior to inoculation (0.5 mL) a nd at biweekly interval s thereafter (1-2 mL)

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41 until the time of euthanasia. In addition to a complete blood cell count with differential, lymphocyte subsets were quantified in each samp le by flow cytometry as described below. Genomic DNA was extracted from the blood samples (QIAamp DNA Mini Kit; Qiagen Inc., Valencia, CA) and the presence of JSY3 wild-type and JSY3 ORF-A/2 FIV viruses in genomic DNA was determined by conventional PCR using gagspecific primers FIV-1(-2) and FIV-5(-4) (Table 3-1). The reaction mixt ure contained 0.3uL of 50M FI V-1(-2) and FIV-5 (-4), 2L of genomic DNA (100-200 ng), 1 L of 10mM dNTP mixture, 11 L of 25mM MgCl2, 1 L of Taq polymerase, and 10 L of 10x Taq buffer (Roche Applied Science, Indi anapolis, IN.). PCR cycling conditions were: 94C for 1min, 58C for 2 min, and 72C for 1 min for 30 cycles. Tissue Collection and Processing At the time of euthanasia, each kitten was anes thetized by intraperitone al administration of ketamine (2 mg/kg, Ketaset, Wyeth, Madison, NJ) and acep romazine (0.1 mg/kg, PromAce, Fort Dodge Animal Health, Overland Park, KS) for collection of blood by cardiac puncture into tubes containing EDTA anticoagulant. Periph eral blood mononuclear cells (PBMC) were isolated from the blood by a disconti nuous density gradient of Percoll (Sigma-Aldrich Corp. St. Louis, MO) as previously descri bed (Tompkins et al., 1989). The thymus and axillary, prescapular and popliteal lymph nodes were aseptica lly collected from each kitten and weighed. Portions of the tissues were fixed in OCT tissue matrix (BD Biosciences, San Jose, CA) for immunohistochemistry, and other portions were processed into single cell suspensions as previously described (Orandle et al., 2000). Immunomagnetic Selection of Periph eral Blood Lymphocyte Populations The PBMC were fractionated into T cell subsets and B cells using the MiniMACS (Miltenyi Biotech, Auburn, CA) immunomagnetic bead-sorting protocols according to the manufacturers instruct ions. The monoclonal antibodies used for selection of CD4+ T cells

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42 included anti-feline CD4 anti body conjugated to phycoerythrin (PE) (MCA 1346 PE, Serotec Inc., Raleigh, NC) and anti-feline CD4 antibody conjugated to biotin (clone CAT30A, Wayne and Mary Tompkins, North Carolina State Univer sity, Raleigh, NC). The monoclonal antibodies used for selection of CD8+ T cells in cluded anti-feline CD8 antibody conjugated to phycoerythrin (PE) (MCA 1347PE, Serotec Inc. Raleigh, NC) and anti-feline CD8 antibody conjugated to fluorescein isothiocyanate (FIT C) (clone CAT357, Wayne and Mary Tompkins, North Carolina State University, Raleigh, NC). The monoclonal antibody used for selection of B cells was anti-CD79a conjugated to allophycocyanin (APC) (C7252; clone HM57, DakoCytomation California Inc., Carpinteria, CA). PBMC aliquots were incubated with a fluorochrome-conjugated CD4 antibody, CD 8 antibody, or B cell antibody followed by incubation with magnetic beads conjugated with antibody to the fluorochrome (anti-PE beads, anti-FITC beads, anti-biotin beads, antiAPC beads, Miltenyi Biotech, Auburn, CA). Briefly, PBMC were fractionated into T ce ll subsets and B cells using the MiniMACS (Miltenyi Biotech, Auburn, CA) immunomagnetic be ad-sorting reagents and protocols supplied by the manufacturer. In one me thod, at both time points, we du al-labeled whole PBMC with anti-feline CD8 antibody conjugated to fluor escein isothiocyanate (FITC) (30 L/1x106 cells) (clone CAT357, Wayne and Mary Tompkins, Nort h Carolina State Univer sity, Raleigh, NC) and anti-feline CD4 antibody conjugated to biotin (30 L/1x106 cells) (clone CAT30A, Wayne and Mary Tompkins, North Carolina State University, Raleigh, NC). Purification of cells was done with anti-FITC beads and anti-biotin beads sequentially (10 L/1x107 cells; 20 L beads/1x107 cells) (Miltenyi Biotech, Auburn, CA). Labele d cells were removed by adherence to the magnetic column.

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43 In an alternate method, CD8+ T cells were se lected first by the us e of anti-feline CD8 antibody conjugated to phycoeryth rin (PE) (MCA 1347PE, Serotec Inc., Raleigh, NC) (10 L/1x106 total cells). Isolation of CD8+ T cells wa s done with anti-PE beads (20 L of anti-PE beads/1x107 total cells) (Miltenyi Biotech, Auburn, CA ) and the labeled CD8+ T cells were removed by adherence to the magnetic column. The non-adherent cells were incubated with monoclonal antibody anti-CD79a conjugated to allophycocyanin (APC) (C7252; clone HM57, DakoCytomation California Inc., Carpinteria, CA) (10 L/1x106 cells) followed by anti-APC beads (20 L/1x107 cells) (Miltenyi Biotec h, Auburn, CA). The labeled B cells were removed by adherence to the magnetic column. The non-adhe rent cells were incubated with anti-feline CD4 antibody conjugated to phycoerythrin (PE) (MCA 1346 PE, Serotec Inc., Raleigh, NC) (10 L/1x106cells) followed by anti-PE beads (20 L/1x107cells ) (Miltenyi Biotech, Auburn, CA). The purity of the selected lymphocyte populatio ns was determined by phenotype analysis using flow cytometry as described below. The purity of lymphocyte populations from blood collected at 8 weeks was about 80%. Refinement of the imm unomagnetic selection techniques used for blood samples collected at 16 w eeks resulted in purities greater than 90%. Flow Cytometry Subpopulations of PBMC, thymocytes, and lymph node cells were analyzed by dualfluorescence flow cytometry as previously descri bed (Orandle et al., 2000). Briefly, the CD4+ and CD8+ T cells were identified by incubating cells with a combination of anti-feline CD4biotin (CAT30A) and anti-feline CD8-FITC (C AT357), or anti-feline CD4-FITC (MCA 1346F) and anti-feline CD8PE (MCA 1347PE). B and T ce lls were identified by incubation of cells with a combination of mouse an ti-feline CD5FITC (MCA 2038F Serotec Inc, Raleigh, NC.) and mouse anti-feline B cell-PE (MCA 1781PE, Se rotec Inc., Raleigh, NC). After incubation with the primary antibodies, the cells were wash ed with PBS buffer containing 2% fetal calf

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44 serum, and the cells labeled with anti-feline CD4biotin were further incubated with streptavidinPE (Sigma Aldrich, St. Louis, MO .). Following the final washes with PBS/2% fetal calf serum buffer, the cells were re-suspended in isotoni c 0.25% paraformaldehyde. The samples were acquired on a FACScan cytometer (Becton Dickins on Biosciences, San Jose, CA) and analyzed using the LYSIS-II program (Becton Dickinson Bios ciences, San Jose, CA.). Lymphocytes were gated on the basis of light sca tter profiles as previously desc ribed (Orandle et al., 1997). Quantitative Real-Time PCR for FIV Provirus Genomic DNA was extracted (QIA amp DNA Mini Kit, Qiagen Inc., Valencia, CA) from thymocytes, lymph node cells, PBMC, and blood lymphocyte subpopulations for quantification of FIV provirus by real-time PCR using primers for the JSY3 gag gene. DNA concentration and purity was determined by UV spectrophotometer (A260/A280). Feline G3PDH was selected as the housekeeping gene for normalization of FIV content. The gag primers, feline G3PDH primers, and corresponding Taqman probes were design ed using Primer Express software (PE Applied Biosystems, Foster City, CA) (Norway et al., 2001)(Table 3-2). PCR analyses were conducted in a 25-l reaction volume of PCR Un iversal Master Mix (PE Applied Biosystems) containing ~100-200 ng of DNA, 900 nM of each gag and G3PDH primer, and 125 nM of the TaqMan probes. The standard curve was ge nerated by PCR on serial dilutions of a cDNA containing the JSY3 gag sequence and feline G3PDH. All samp les and the serial dilutions of the standards were assayed in duplicate. For all sa mples, the target quantity was determined from the standard curve and divided by the target quant ity of a calibrator, a 1 sample. All other quantities were expressed as an n-fold differen ce relative to the calibrator. The relative FIV provirus content was expre ssed as the ratio of FIV gag to G3PDH.

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45 Quantitative Real-Time PCR for FIV Transcription Total RNA was extracted (RNeasy Midi Kit, Qiagen Inc., Valencia, CA) from thymocytes, lymph node cells, PBMC, and blood lymphocyte subpopulations for quantification of FIV transcription by real-time PCR using primers for the JSY3 gag and rev genes. Transcription of the gag gene results in an unspliced or singly spliced product whereas transcription of the rev gene produces multiply spliced RNA products (Kiyomasu et al., 1991) (Michael et al., 1991) (Phillips et al., 1992) (Tomonaga et al., 1993b). RNA concentration and purity was determined by UV spectrophotometer (A260/A280). Reverse transcription was performed by use of the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, Foster City, CA) utilizing ~0.5 g RNA and corresponding 3 reverse gag and rev specific primers with the following cycling conditions : 75C for 5 min, 42C for 1 hour, 95C for 5 minutes, and 4C for 5 minutes. The gag primers, G3PDH primers, and corresponding Taqman probes were those described for the provi rus real-time PCR assay above (Table 3-2). The rev primers and corresponding Taqman probes were designed using Primer Express software (PE Applied Biosystems, Foster City, CA) (Table 3-2). The rev Taqman probe anneals to the sp lice junction of rev 1 and rev 2 (base pair 6513 to 8951 of JSY3), and is a minorgroove-binding probe (MGB). PCR analyses were conducted in a 25-l reaction volume of PCR Universal Master Mix (PE A pplied Biosystems, Foster City CA) containing ~100-200 ng of cDNA 900 nM of each gag rev and G3PDH primer, and 125 nM of the TaqMan probes. The standard curve was generated by PCR on se rial dilutions of a c DNA containing the JSY3 gag and rev sequences and feline G3PDH. All samples and the serial dilutions of the standards were assayed in duplicate. For all samples, targ et quantity was determined from the standard curve and divided by the target quantity of a calib rator, a 1 sample. All other quantities were

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46 expressed as an n-fold difference relative to the calibrator. The relative FIV gag and rev gene transcription products were e xpressed as the ratio of FIV gag and rev to G3PDH. Statistical Analysis A statistical software progra m (SigmaStat 3.0, SPSS Inc., Chicago, IL) was utilized for all data analyses. The data were transformed pr ior to analysis, via square root, in order to normalize any sample variability. Hematologica l profile results were analyzed by a one-way analysis of variance (One-Way ANOVA). Tran sformed variables that were not normally distributed or displayed unequal variances were compared using the ANOVA on Ranks test. A one-way repeated measure of analysis of variance (RMANOVA) was utilized to compare an individual infected set of animals across the length of infection. Transformed variables for proviral load and relative transcript comparison that had normal distribution and equal variance were compared using the Students t-test. Transformed variables that were not normally distributed or displayed unequal variances were compared using the Mann-Whitney rank sum test. Results are presented as means one standard deviation. Values with P 0.05 were considered significant. Results Blood Lymphocyte Subpopulations Blood samples were collected from the FIV-inf ected cats every 2 weeks from the time of virus inoculation at birth to 16 weeks of age. Lymphocyte subpopulations (CD4+ T cells, CD8+ T cells, and B cells) in the blood were analyzed by flow cytometry to monitor fluctuations associated with progression of FIV infection. In cats infected with JSY3 wild type (WT) there was a progressive inversion of the CD4:CD8 ratio from 2 weeks to 10 weeks post-in fection with the values being statistically different from uninfected control kittens (P 0.05) (Figure 3-6). Ther e is a similar, but less

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47 pronounced, inversion for cats infected with the ORF-A/2 deletion mutant (JSY3 ORF-A/2). At two and four weeks, the JSY3 (WT) ratio was significantly lower than JSY3 ORF-A/2 (P 0.05) (Figure 3-6). By 16 weeks post-inoculation, the CD4:CD8 ratios were similar for the 2 groups of infected cats and there wa s no statistical difference. The inversion of the CD4:CD8 ratio was associated with loss of CD4+ T cells from 2 to 8 weeks post-infection in both groups of cats (F igure 3-7). This reduction in absolute CD4+ T cells was statistically different from uninfected cats at 2 weeks, 4 weeks, 8 weeks and 12 weeks postinfection in both JSY3 (WT) and JSY3 ORF-A/2 cats (P 0.05) (Figure 3-7). The CD4+ T cell loss was greater for JSY3 (WT) infected cats fr om 6 to 10 weeks post-infection compared to cats infected with the ORF-A/2 deletion mutant virus. However, there was a statistically significant difference in JSY3 ORF-A/2 week 4 absolute CD4+ T ce lls compared to week(s) 8, 12 and 16 (P 0.05) indicating a progressive loss of CD4+ T cells. By 16 weeks post-infection, the numbers of CD4+ T cells were similar in both groups although JSY3 ORF-A/2 infected kittens exhibited statistically reduced CD4+ T ce lls compared to uninfected animals (P 0.05) (Figure 37). The number of CD8+ T cells fluctuated dur ing the 16-week course of FIV infection (Figure 3-8). At 2 weeks and 4 weeks, both JSY3 (WT) and JSY3 ORF-A/2 cats had lower CD8+ cells in whole blood than uninfected cats (P 0.05) (Figure 3-8). There was no progressive expansion in this lymphocyte subpopulation in the bl ood of either group of cats but rather an expansion at 4 weeks for JSY3 (WT) and at 10 weeks for JSY3 ORF-A/2. In contrast to the number of absolute CD8+ T cells, there was a slight expansion in the number of B cells starting around 4 weeks postinfection in the JSY3 (WT) infected group (Figure 3-9). This absolute number was maintain ed to 16 weeks post-infection in cats infected

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48 with JSY3 (WT). In contrast, a progressive decline in the number of B cells in the JSY3 ORFA/2 infected cats was monitored from 8 to 16 w eeks post-infection. At 2 weeks and 8 weeks, JSY3 ORF-A/2 infected cats had a statistically lower absolute B cell counts than uninfected cats (P 0.05) (Figure 3-9). At 4 weeks, absolute B cell count in JSY3 ORF-A/2 was statistically greater in comparison to week 16 (P 0.05) (Figure 3-9). At 16 weeks, the difference in JSY3 ORF-A/2 absolute B cell numbers approached statistical si gnificance (P=0.06). A lower absolute B cell number in the blood could suggest sequestration of the B cells within other lymphoid compartments. Thymus and Lymph Node Ly mphocyte Subpopulations Thymus and lymph node tissue samples were co llected at necropsy from the FIV-infected cats at 8 weeks and 16 weeks of age. Lym phocyte subpopulations were analyzed by flow cytometry to monitor fluctuations associated w ith progression of FIV infection. At 8 weeks, there were statistically fewer T cells in the t hymus of JSY3 (WT) infected cats, than the uninfected cats and JSY3 ORF-A/2 infected cats (Figure 3-10 (C )). This is further exemplified when cells are categorized into double nega tive T cells (DNeg), double positive T cells (DP), CD4+, and CD8+ T cells (Figure 3-10 (A-B; E-F )), in which JSY3 (WT)-infected cats had statistically less of each form of T cell at 8 weeks as compared to 16 weeks (P 0.05). JSY3 (WT) also had a decrease in the absolute number of B cells in the thymus at 8 weeks compared to JSY3 ORF-A/2 infected cats, but the slight increase relative to JSY3 ORF-A/2 cats at 16 weeks, was not significant (Figure 3-10 (D)). JSY3 ORF-A/2 maintained the same level of absolute B cells at 8 and 16 weeks which remain ed elevated above JSY3 (WT) and uninfected cats (Figure 3-10 (D)). Analysis of the lymph node samples revealed that JSY3 ORF-A/2 had a greater number of absolute CD4+ T cells, and absolute T cells co mpared to JSY3 (WT) and uninfected cats at 8

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49 weeks (Figure 3-11 (A and C)). The absolute nu mber of CD8+ T cells, and B cells at 8 weeks however were essentially equivalent between JSY3 ORF-A/2 and JSY3 (WT) infected cats, both were elevated relative to uninfected cats (Figure 3-11 (B a nd D). At 16 weeks the absolute number of CD4+ T cells and T cells is a pproximately equal between JSY3 (WT) and JSY3 ORF-A/2, both were greater than uninfected cat s (Figure 3-11 (A and C)). However, both CD8+ T cells and B cells are increased in JSY3 (W T) infected tissue, and were greater than the levels detected in JSY3 ORF-A/2 infected and uninfected ca ts at 16 weeks (Figure 3-11 B and D)). Absolute CD8+ T cells in the lymph node samples of JSY3 ORF-A/2 infected cats at 16 weeks were significantly different from uninfected CD8+ T cells at 16 weeks (Figure 3-11 (B)). Proviral Load in Lymphoid Tissues and Lymphocyte Subpopulations The proviral load was quantified in genomic DNA extracted from th ymocytes, lymph node cells, peripheral blood mononucle ar cells (PBMC), and blood lymphocyte subpopulations (CD4+ T cells, CD8+ T cells, and B cells) prepared from FI V-infected cats at 8 and 16 weeks postinfection. The relative quantity of provirus in these cell populations was determined by a realtime PCR assay specific for the gag gene of JSY3 FIV. At 8 and 16 weeks post-infection, the relative proviral load in the thymus, lymph nodes, and PBMC in cats infected with JSY3 ORF-A/2 was lower than that for cats infected with JSY3 (WT) (Figure 3-12; Figure 3-13). The differen ce in proviral load betw een the 2 groups of cats was most pronounced at 8 weeks postinfection. At this time, the proviral load in the thymus, lymph node cells and PBMC was significantly lower in cats infected with the ORF-A/2 deletion mutant (P 0.05) (Figure 3-12). On examination of gag proviral load in relation to tissue weight, JSY3 ORF-A/2 had a statistically lower ratio in the thymus at 8 weeks versus JSY3 (WT) (Figure 3-14). JSY3 (WT) ha d a statistically significant re duction in the ratio at 16 weeks

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50 compared to 8 weeks (P 0.05). This result could not be demonstrated for the lymph node however (P=0.065) (Figure 3-15). By 16 weeks postinfection, the proviral load in the thymus, lymph nodes, and PBMC were similar between the 2 groups of infected kittens (Figure 3-13). For cats infected with JSY3 (WT), the proviral load was significantly lo wer in all the lymphoid tissues at 16 weeks post-infection compared to 8 weeks (Table 3-3). Fo r cats infected with JSY3 ORF-A/2, there were no significant differences in proviral load in the lymphoid tissues between 8 and 16 weeks post-infection (Table 3-3). Similar to the total lymphocyte population in the blood, the provira l load in the blood lymphocyte subpopulations (CD4+ T cells, CD8+ T cells, B cells) was lower in cats infected with the ORF-A/2 deletion mutant compared to cats inf ected with the wild type parent virus (Figure 3-16). At 16 weeks post-infecti on, the proviral loads in the CD4+ and CD8+ T cells were similar for the 2 groups of infected cat s, but the B cells in the JSY3 ORF-A/2 infected cats still had a significantly (P=0.025) lower proviral load (Figure 3-17). For each group of infected cats, there were no significant differences in proviral load of the lymphocyte subpopulations between 8 and 16 weeks post-infection (Table 3-4). Transcription of gag and rev in Lymphoid Tissues FIV gag and rev transcripts were quantified in R NA extracted from thymocytes, lymph node cells, and PBMC collected from infected ca ts at 8 and 16 weeks post-infection. The relative quantity of viral transcripts in these cell populations was determined by a real-time PCR assay specific for the gag and rev genes of JSY3 FIV. Transcription of the gag gene produces full-length transcri pts that are translated into proteins necessary for assembly of fully infectious virions. Comp ared to cats infected with JSY3 (WT) virus, the relative amount of gag transcripts in the thymus, lymph nodes, and PBMC were

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51 reduced in cats infected with the ORF-A/2 deletion mutant at both 8 and 16 weeks post-infection (Figure 3-18). The relative amount of gag transcripts were significantly lower in the thymus (P=0.048), lymph node (P=0.005) and PBMC (P< 0.001) at 8 weeks (Figure 3-18 (A)), and in the thymus (P=0.026) and lymph node (P=0.029) at 16 w eeks (Figure 3-18 (B)). For cats infected with JSY3 (WT), the relative quantity of gag transcripts was significantl y greater in the thymus compared to lymph node (P=0.008) and PBMC (P=0.039) at 8 weeks post-infection, and the PBMC contained significantly more gag transcripts than the lymph node (P=0.015) (Table 3-5). Similarly, for cats infected with JSY3 ORF-A/2, the relati ve quantity of gag transcripts was also significantly greater in the thymus compared to lymph node (P=0.014) and PBMC (P=0.007) at 8 weeks post-infection (Table 3-5). Th ere were no significant differences in gag transcript quantity between the thymus, lymph node, and PBMC at 16 weeks for either group of infected cats (Table 3-5). Transcription of the rev gene occurs early after FIV inte gration into the host cell genome, and the resultant rev protein regulates the releas e of other viral transcript s from the nucleus to the cytoplasm for translation into proteins for assembly of inf ectious virions (Kiyomasu et al., 1991) (Michael et al., 1991) (Phillips et al., 1992) (Tomonaga et al., 1995). Compared to cats infected with JSY3 (WT) virus, the relative amount of rev transcripts in the thymus, lymph nodes, and PBMC were lower in cats infected with the ORF-A/2 deletion mutant at both 8 and 16 weeks post-infection (F igure 3-18 (C) and (D)). The relative amount of rev transcripts were significantly lower in th e lymph node (P=0.010) and PBMC (P< 0.001) at 8 weeks (Figure 3-18 (C)). For cats infected with JSY3 (WT), the relative quantity of rev transcripts was greater in the thymus than lymph node and PBMC at both 8 and 16 weeks postinfection, but these differences we re not statistically si gnificant (Table 3-6) For cats infected

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52 with JSY3 ORF-A/2, the relative quantity of rev transcripts was significantly (P 0.05) greater in the thymus than lymph node and PBMC at 8 weeks post-infection (Tab le 3-6). In addition, the relative quantity of rev transcripts was significantly (P< 0 .05) greater in the lymph node and PBMC at 16 weeks compared to 8 weeks post-infection (Table 3-6). Transcription of gag and rev in Blood Lymphocyte Subpopulations The relative quantity of gag and rev transcripts was determined in CD4+ T cells, CD8+ T cells, and B cells in the blood of infected cats at 8 and 16 weeks post-infection. Similar to the lymphoid tissues the relative quantities of gag and rev transcripts were lower in all 3 lymphocyte subpopulati ons of cats infected with JSY3 ORF-A/2 at 8 weeks postinfection compared to JSY3 (WT) infected cats (Figure 3-19). At 16 weeks post-infection, the relative quantities of gag transcripts in CD4+ and CD8+ T cells were similar for the 2 groups of cats, whereas the quantity in B cel ls was still significantly (P= 0.03) lower in the cats infected with the ORF-A/2 deletion mutant (Fi gure 3-19 (B)). In contrast to the gag transcripts, the relative quantity of rev transcripts remained lower in a ll 3 lymphocyte subpopul ations in the JSY3 ORF-A/2 infected cats at 16 weeks (Figure 3-19 (D)). The rev transcripts were significantly lower in the CD4+ T cells (P= 0.014) and B cells (P=0.007) (Figure 3-19 (D)). For cats in both groups, there were no signifi cant differences in th e relative quantity of gag or rev transcripts in the CD4+ T cells, the CD8+ T cells, and the B cells at 8 weeks postinfection, 16 weeks post-infecti on, or in comparison of quantities at 8 weeks versus 16 weeks post-infection (Tables 3-7 and Table 38). The only exception was that the rev transcripts were significantly (P=0.01) greater in th e B cells than CD4+ T cells for JSY3 (WT) infected cats at 16 weeks post-infection (Table 3-8).

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53 Discussion Close examination of the thymus subpopulations by flow cytometry re vealed a significant reduction in the absolute number of T cells in JS Y3 (WT) infected cats at week 8 compared to the JSY3 ORF-A/2 infected or uninfected cats. JS Y3 (WT) infected cats at week 8 had a significant decrease in the numbe r of CD4+ T cells, CD8+T cells, CD4+CD8+ (DP) T cells and CD4-CD8-(DNeg) T cells in the thymus compared to JSY3 (WT) infected cats at 16 weeks (P 0.05). JSY3 ORF-A/2 infected samples had equivalent absolute CD8+ T cells to JSY3 (WT) infected at 16 weeks. This information is di fferent than previously reported (Norway et al., 2001), where a reduced expansion of CD 8+ thymocytes was seen in JSY3 ORF-A/2 infected cats at 14 weeks post-infection. Perhaps the sampli ng at 16 weeks, compared to 14 weeks, with greater number of JSY3 ORF-A/2 infected cats in these ex periments, accounted for this observation. Thymus samples of JSY3 ORF-A/2 infected have consistently more DP and DNeg T cells compared to JSY3 (WT) or uninfected cats at 16 weeks. This observation of more DP T cells in the thymus of JSY3 ORF-A/2 infected cats, compared to JSY3 (WT) infected, is consistent with the previous report (Norway et al., 2001). In terms of thymic and ly mph node proviral load, JSY3 ORF-A/2 infected cats had statistically lower proviral load at 8 weeks compared to JSY3 (WT) infected, but by 16 weeks, there is no difference suggesting that the level of proviral load had pa ralleled or that a percentage of JSY3 (WT) infected cells have decreased by ce ll death. This observation is different than the previous report of a significan tly reduced proviral load in JSY3 ORF-A/2 infected thymus of 14 week old cats (Norway et al., 2001). Perhaps th e sampling at 16 weeks, compared to 14 weeks, with greater number of JSY3 ORF-A/2 infected cats in these experiments, accounted for this observation.

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54 Examination of the level of gag and rev gene expression in the thymus and lymph node demonstrated that JSY3 ORF-A/2 infected cats consistently expressed lower gene expression and this difference was statistically significant for gag RNA at 8 and 16 weeks. Despite the fact that JSY3 ORF-A/2 infected thymus and lymph nodes had more T cells (in the thymus, DNeg, DP) there was no equivalence to JSY3 (WT) infected in the production of gag or rev transcripts at 16 weeks. This suggests JSY3 ORF-A/2 produced fewer RNA products despite clinical (PBMC) parameters of chronic FIV infection (d ecreased absolute CD4+ T cell count; inverted CD4:CD8 ratio). Analysis of the PBMC flow cytometry data demonstrates the progressive decline in the CD4+:CD8+ ratio, with a loss of CD4+ T cells in both JSY3 (WT) and JSY3 ORF-A/2 infected cats. This decline in CD4+ T cells is more severe in JSY3 (WT) in fected cats earlier (week 2) in the course of infection compared to JSY3 ORF-A/2 (week 8). This observation is consistent with the previous report of a delay in the decline of the CD4:CD8 ratio in JSY3 ORF-A/2 infected cats (Norway et al., 2001). By 16 weeks of infection, the relative CD4:CD8 ratio of JSY3 (WT) and JSY3 ORF-A/2 infected cats are equivalent with approximately equal number of CD4+ T cells and CD8+T cells. JSY3 (WT) in fected cats experienced a mild expansion of B cells in the PBMCs at 4 weeks that JSY3 ORF-A/2 infected cats do not. In contrast, JSY3 ORF-A/2 cats exhibit a mild increase in ab solute B cell numbers at 4 weeks with a gradual decline in those B ce ll numbers during the following weeks, nearly approaching statistical significance at 16 weeks (P=0.06). The reason for the lower abso lute B cell numbers in the circulating PBMCs of JSY3 ORF-A/2 infected cats is unknown, but may reflect a generalized sequestration and compartmentalizati on of B cells in the thymus and lymph node, as suggested by flow cytometry data. In JSY3 ORF-A/2 infected thymus, B cell levels were

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55 monitored that consistently remained higher than JSY3 (WT) infected th ymus at 8 and 16 weeks although not statistically signifi cant. In the lymph node, JSY3 ORF-A/2 infected B cell numbers are also higher than JSY3 (WT) and do not change between week 8 and 16, although not statistically significant. However, by 16 weeks of JSY3 (WT) infection, increased B cell numbers are observed in both the thymus and th e lymph node possibly indi cating an influx of B cells into the tissues. The mechanism by which ORF-A/2 enhances viral replication in PBMCs is unknown. FIV-p34 is a natural ORF-A/2 mutant that has a stop codon in the middle of the ORF-A/2 gene (Talbot et. al., 1989). When the stop codon is converted to a tryptophan codon, FIV-p34 is capable of replicating in feline PBMCs impli cating ORF-A/2 as an important factor for replicating in primary cells (Water s et. al. 1999). Analysis of JSY3 ORF-A/2 sorted PBMCs demonstrates a reduction in the proviral load with a significant difference in B cell proviral load between JSY3 (WT) at 16 weeks. Reduced gag and rev gene expression was seen in all of the JSY3 ORF-A/2 subpopulations, with CD8+ T cells demonstrating statis tically significant differences from JSY3 (WT) at 8 weeks and JSY3 ORF-A/2 B cells expressing fewer gag and rev transcripts at 16 weeks. It may be that OR F-A/2 has multiple functions which promote virus replication, as seen in HIV accessory genes. Gemeniano et. al. (2003) reported ORF-A/2 is required for virus particle formation/virus in fectivity, and similar to Vpr, induces a G2 cell cycle arrest (Gemeniano et. al., 2003; Ge meniano et. al., 2004). The low level of viral transcripts in JSY3 ORF-A/2 infected animals may reflect the reduc tion in proviral load and may be related to the problem of particle formation and or virus infectivity (Gemeniano et al., 2003). Yang et. al. (1996) previously reported that during acute phase of infectio n, proviral load was only found in the CD4+ T cell subset. Data presented here de monstrates provirus in all three subsets during

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56 acute phase of infection. It is possible that a difference in sampling time point 2 weeks, compared to 8 weeks, may account for this differe nce. Yang et. al. (1996) also suggested that the progressive decline in CD4+ T cells whil e CD8+ T cells and B cells numbers remain consistent during the chronic phase indicate that FIV infection is late nt and non-cytolytic in CD8+ and B cells. However, data presented he re supports active gene expression in CD8+ T and B cells at 16 weeks. The use of neonatal kitte ns instead of adult cats, could account for this difference in results. In summary, T cell and B cell numbers were in creased in the thymus at 8 and 16 weeks of JSY3 ORF-A/2 infected cats compared to JSY3 (WT) infected cats. Despite these increases in cell numbers, by 16 weeks, JSY3 ORF-A/2 infected cats have near equivalent levels of proviral load but, reduced gag and rev mRNA, compared to JSY3 (WT) infected cats. JSY3 ORF-A/2 infected cats absolute values in lymph node subpopulations did not change markedly between week 8 and week 16 and although the numbers of cells were consistent, this did not result in an increase in the rate of gag and rev gene expression in the lymph node at 8 or 16 weeks. Analysis of the PBMC revealed that B cell numbers in the blood of JSY3 ORF-A/2 infected cats were lower than JSY3 (WT) infected cats, and that these B cells, when sorted and analyzed for proviral load and gene expre ssion, revealed lower proviral lo ad and viral gene expression throughout the JSY3 ORF-A/2 infection. Specifically, B cells exhibited a statistically significant reduction in proviral lo ad and viral gene expression during chronic infection. The reason for this is unknown although the mean absolute number of B cells at 16 weeks for cats infected with JSY3 ORF-A/2 (768 cells/L) is lower th an JSY3 (WT) (2,954 cells/L) and could be attributable. However, upon examinati on of the absolute mean values of CD8+ and CD4+ T cells at 8 weeks, there is equivale nt number of cells between JSY3 (WT) and

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57 JSY3 ORF-A/2 yet lower proviral load and gene expression in JSY3 ORF-A/2 infected cats. Whether ORF-A/2 contributes to th e shift from CD4+ cells during acute infection, to the B cells in the chronic phase of infec tion, remains to be determined.

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58 Figure 3-1. Construction of the CMVdriven JSY3 (WT) molecular clone.

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59 Figure 3-2. Construction of the CMV-driven JSY3 ORF-A/2 molecular clone.

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60 Figure 3-3. Schematic of viral events depic ting what transpired utilizing the CMV driven plasmids. Upon transfection of the 293 T ce lls, the CMV promoter would be utilized to drive initial expression of the virus a nd that promoter is subsequently removed during viral reverse transcription and asse mbly. The intact FIV LTR then drives expression in the feline CD4E cell line.

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61 NOVAK AORFA#1/RMN4R LENGT H: 462 CHECK: 2527 .. 1 CTTTGTGTTG CTATATCA AA ATCTAATAAC TCTTCAGCTT CTTCTGGTCC 51 TATCCATTGT CTATTGGCTG CAAATCCTTC TGCCAT AATT ATTGTTGCAA 101 ATGAAATATT ATTATAAGTA TTTCTAAGCA GTAG TTATTG ATAATGTAGA 151 TTGCAACTGC CAATAGTAGA A TTTGCAACA GAACCAACAT AAACAGTATT 201 TTGTTTGGGG TTTCTTAAAT CT ATGTCTCC AAACTAAT CC TTGTAGTAAT 251 CTAATAACTT TGTCCCTTTC TA ATTGATGT GCTAATACAA ATATTCTGAT 301 AGCTTTTTCC TTTTCTAGTT TCTTA-GACCT TATTAAAT--T GTTAGTATGTC 351 TTCCATTCAT AGGCTCCCTG AC CATAATAG ACTCCAGCTG GGCTTGGATT 401 GAATGACCTC CAAATCAGCA GGCGTTCTGT AAGGAGAAAC AAAGCGTTGA 451 TTACAGCATC CT Figure 3-4. ORF-A/2 gene DNA sequence data from the PCR product of JSY3 ORF-A/2 infected CD4E cells. Side-by-side comparis on of this sequence with JSY3 wild type ORF-A/2 gene revealed four base pair deleti ons (indicated by dash marks (-)) from site-directed-mutagenesis as described previously.

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62 NOVAK WT#2/RMN4R LENGTH: 465 CHECK: 5005 .. 1 TTTGTGTTGC TATATCAAAA TCTAATAACT CTTCAGCTTC TTCTGGTCCT 51 ATCCATTGTC TATTGGCTGC AAATCCTTCT GCCATAATTA TTGTTGCAAA 101 TGAAATATTA TTATAAGTAT TTCTAAGCAG TAGTTA TTGA TAATGTAGAT 151 TGCAACTGCC AATAGTAGAA TTTGCAACAG AACCAAC ATA AACAGTATTT 201 TGTTTGGGGT TTCTTAAATC TA TGTCTCCA AACTAATCCT TGTAGTAATC 251 TAATAACTTT GTCCCTTTCT AATTGATGTG CTAATAC AAA TATTCTGATA 301 GCTTTTTCCT TTTCTAGTTT CTTA GTGACC TTATTAAATA ATGTTAGTAT 351 GTCTTCCATT CATAGGCTCC CTGACCATAA TAGACT CCAG CTGGGCTTGG 401 ATTGAATGAC CTCCAAATCA GCAGGCGTTC TGTAAGGAGA AACAAAGCGT 451 TGATTACAGC ATCCT Figure 3-5. ORF-A/2 gene DNA sequence data from the PCR product of JSY3 (WT) infected CD4E cells. Side-by-side compar ison of this sequence with JSY3 ORF-A/2 PCR product reveals an intact ORF-A/2 gene.

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63 Table 3-1. The sequences of the primers us ed for conventional PCR analyses for FIV. Primer Sequence (5' to 3') Use FIV-1 TGA CCG TGT CTA CTG CTG CT Conventional PCR for gag FIV-5 CAC ACT GGT CCT GAT CCT TTT FIV-2 CCA CAA TAT GTA GCA CTT GAC CConventional PCR for gag FIV-4 GGG TAC TTT CTG GCT TAA GGT G (nested PCR) RMN 3 AGT GGC GAG GAT GCT GTA AT Conventional PCR for ORF-A/2 RMN 4 CCT GGA TTT AGT GGC CCT TC

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64 Table 3-2. The sequences of the primers and pr obes used for quantitative real time PCR analyses for FIV. Primer or Probe Sequence (5' to 3') Use F-GAG AGC CCT CCA CAG GCA TCT C R-GAG TGG ACA CCA TTT TTG GGT CAA Probe-GAG 6-FAM-ATT CAA ACA GCA AAT GGA GCA CCA CAA TAT G-TAMRA F-G3PDH CCA TCA ATG ACC CCT TCA TTG Real-time qRTPCR for G3PDH R-G3PDH TGA CTG TGC CGT GGA ATT TG Probe-G3PDH 6FAM-CCT CAA CTA CAT GGT CTA CAT GTT CCA GTA TGA TTC C-TAMRA F-REV CGG AAT GAA CTT CAA GAG GTA AAA CTA Real-time qRTPCR for rev R-REV TCA TAT TCT TGA AGG CTT TCT TCC TT Probe-REV 6FAM-CAG GTA A AA AGA AAA AAA GAC AAA-MGBNFQ

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65 Figure 3-6. Absolute CD4:CD8 T ce ll ratios from birth to 16 week s. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference between uninfected and JSY3 (WT) infected kittens (P 0.05). (**) denotes a statistically significant differen ce between JSY3 (WT) and JSY3 ORF-A/2 infected kittens (P 0.05). Figure 3-7. Absolute CD4+ T cells in PBMC from birth to 16 weeks. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference between uninfected, JSY3 (WT), and JSY3 ORF-A/2 infected kittens (P 0.05). (**) denotes a statistically significant di fference between JSY3 (WT) and uninfected kittens (P 0.05). (***) denotes a statistica lly significant difference between JSY3 ORF-A/2 and uninfected kittens (P 0.05). () denotes a statistically significant difference between JSY3 ORF-A/2 week 4 compared to week(s) 8, 12, 16 (P 0.05).

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66 Figure 3-8. Absolute CD8+ T cells in PBMC from birth to 16 weeks. The data are expressed as mean one standard deviation. (*) denot es a statistically significant difference between uninfected and JSY3 ORF-A/2 infected kittens (P 0.05). Figure 3-9. Absolute B cells in PBMC from birt h to 16 weeks. The data are expressed as mean one standard deviation. (*) denotes a st atistically significant difference between uninfected and JSY3 ORF-A/2 infected kittens (P 0.05). (**) denotes a statistically significant difference between JSY3 ORF-A/2 week 4 compared to week 16 (P 0.05).

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67 Figure 3-10. Absolute thymocyte subpopulations at 8 and 16 weeks as determined by flow cytometry. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference at 8 weeks between uninfected, JSY3 (WT), and JSY3 ORF-A/2 infected kittens (P 0.05). The solid line with (*) denotes a statistically significant increase in JSY3 (WT) from 8 weeks.

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68 Figure 3-11. Absolute lymphocyte subpopulations at 8 and 16 weeks as determined by flow cytometry. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference at 16 weeks between uninfected and JSY3 ORFA/2 infected kittens (P 0.05).

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69 Figure 3-12. Relative pr oviral load of the FIV gag gene in thymus (TH), lymph node (LN) and blood (peripheral blood mononuclear cells (PBM Cs)) of 8-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens were infected with 104 TCID50 of JSY3 (WT) (n=3 kittens) or JSY3 ORF-A/2 (n=4) kittens. The relative proviral load of FIV JSY3 gag gene in thymocytes, lymph node cells and peripheral blood mononuclear cells was determined by real-time PCR assay. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference between JSY3 (WT) and JSY3 ORF-A/2 infected kittens (P 0.05). Figure 3-13. Relative proviral load in thymus (TH), lymph node (LN) and peripheral blood mononuclear cells (PBMCs) 16-week old ki ttens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens were infected with 104 TCID50 of JSY3 (WT) (n=3 kittens) or JSY3 ORF-A/2 (n=6) kittens. The relative proviral load of FIV JSY3 gag gene in thymocytes, lymph node cel ls and peripheral blood mononuclear cells was determined by real-time PCR assay. The data are expressed as mean one standard deviation.

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70 Figure 3-14. Relative proviral load per gram t hymus weight at 8 and 16 weeks. The relative proviral load of FIV JSY3 gag gene in thymocytes was determined by real-time PCR assay. The data are expressed as mean one standard deviation. (*) denotes a statistically significant differen ce between JSY3 (WT) and JSY3 ORF-A/2 infected kittens (P 0.05). (**) denotes a statistically significant difference between week 8 JSY3 (WT) and week 16 JSY3 (WT). Figure 3-15. Relative proviral load per gram ly mph node weight at 8 and 16 weeks. The relative proviral load of FIV JSY3 gag gene in lymph node cells was determined by real-time PCR assay. One way analysis of varian ce (ANOVA) did not re veal statistical significance (P=0.065). Students t-test did not reveal a ny statistical significance between JSY3 (WT) week 8 and JSY3 ORF-A/2 week 8 (P=0.057).

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71 Table 3-3. The FIV proviral load in the thymus, lymph nodes, a nd peripheral blood lymphocytes of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2). JSY3 (WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks Thymus 1.64 (0.77)c,e 0.33 (0.27)d 0.14 (0.10) 0.30 (0.35) Lymph Node 1.21 (0.20)f 0.71 (0.77) 0.08 (0.06) 0.50 (0.82) PBMC 2.10 (0.55)g 0.79 (0.86) 0.05 (0.03) 0.33 (0.41) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Genomic DNA was extracted from the thym us, lymph nodes, and peripheral blood lymphocytes (PBMC)of kittens at 8 w eeks (n=3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) post-infection. Provirus gag gene in the genomic DNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of provirus gag gene. d The relative amount of provirus gag gene was significantly reduced in the thymus at 16 weeks compared to 8 weeks post-infection (P 0.05). e The relative amount of provirus gag gene at 8 weeks was significantly increased in JSY3 (WT) infected thymus than JSY3 ORF-A/2 thymus (P 0.05). f The relative amount of provirus gag gene at 8 weeks was significantly increased in JSY3 (WT) infected lymph nodes than JSY3 ORF-A/2 lymph nodes (P 0.05). g The relative amount of provirus gag gene at 8 weeks was significantly increased in JSY3 (WT) infected PBMC than JSY3 ORF-A/2 PBMC (P 0.05).

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72 Figure 3-16. Relative pr oviral load of the FIV gag gene in CD4+, CD8+, and B cells of the blood of 8-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens were infected with 104 TCID50 of JSY3 (WT) (n=3 kittens) or JSY3 ORF-A/2 (n=4) kittens. The relative proviral load of FIV JSY3 gag gene in CD4+, CD8+ and B cells was determined by real-time PCR assay. The data are expressed as mean one standard deviation. Figure 3-17. Relative pr oviral load of the FIV gag gene in CD4+, CD8+, and B cells of the blood of 16-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens were infected with 104 TCID50 of JSY3 (WT) (n=3 kittens) or JSY3 ORF-A/2 (n=6) kittens. The relati ve proviral load of FIV JSY3 gag gene in CD4+, CD8+ and B cells was determined by real-time PCR assay. The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference between JSY3 (WT) and JSY3 ORF-A/2 infected kittens (P 0.05).

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73 Table 3-4. The FIV proviral load in CD4+ T cells, CD8+ T cells and B cells in the peripheral blood of cats neonatally infected with JS Y3 (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2). JSY3(WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks CD4+ T cells 2.30 (2.84)c 0.33 (0.03) 0.05 (0.06)0.16 (0.19) CD8+ T cells 1.51 (1.21) 0.62 (0.47) 0.02 (0.02)0.23 (0.22) B cells 2.08 (0.21) 1.71 (0.80) 0.06 (0.01)0.20 (0.13) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Genomic DNA was extracted from CD4+ T cells, CD8+ T cells, and B cells purified by immunomagnetic selection from the peripheral blood of kittens at 8 weeks (n=3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) postinfection. Provirus gag gene in the genomic DNA was qua ntified by a real time PCR assay. c Mean (standard deviation) for the relative amount of provirus gag gene.

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74 Figure 3-18. Relative gene expression of FIV gag and rev in the thymus (TH), lymph node (LN) and blood (PBMC) of 8-week and 16-week ol d kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens we re infected at birth with 104 TCID50 of JSY3 (WT) (n=6 kittens) or JSY3 ORF-A/2 (n=10) kittens. The thymus, lymph nodes, and blood were harvested from kittens at 8 weeks (JSY3 (WT) =3 kittens, JSY3 ORFA/2=4 kittens) and 16 weeks (JSY3 (WT) =3 kittens, JSY3 ORF-A/2=6 kittens) post-infection. The relative e xpression patterns of FIV JSY3 gag and rev gene in thymocytes, lymph node cells, and peri pheral blood mononuclear cells were determined by real-time PCR assay. (A) Relative expression pattern of JSY3 gag at 8 weeks. (B) Relative expression of JSY3 gag at 16 weeks. (C) Relative expression of JSY3 rev at 8 weeks. (D). Relative expression of JSY3 rev at 16 weeks. The data are expressed as mean one standard deviati on. (*) The relative expression in kittens infected with JSY3 ORF-A/2 was significantly (P 0.05) less than in kittens infected with JSY3 (WT).

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75 Table 3-5. Transcription of the FIV gag gene in the thymus, lymph nodes, and peripheral blood lymphocytes of cats neonatally infected w ith JSY3 wild type (WT) FIV and the ORFA/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3(WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks Thymus 1.55 (0.68)c,d 1.31 (1.04) 0.49 (0.49)f 0.28 (0.21) Lymph Node 0.20 (0.04) 0.35 (0.22) 0.05 (0.04) 0.10 (0.08) PBMC 0.53 (0.15)e 0.28 (0.13) 0.03 (0.04) 0.13 (0.19) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from the thymus, lymph nodes, and peripheral blood lymphocytes (PBMC) of kittens at 8 weeks (n =3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) post-infection. JSY3 gag RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of gag RNA. d The relative amount of gag RNA was significantly gr eater in the thymus than in the lymph node and PBMC at 8 weeks post-infection (P 0.05). e The relative amount of gag RNA was significantly gr eater in the PBMC th an in the lymph node at 8 weeks post-infection (P 0.05). f The relative amount of gag RNA was significantly gr eater in the thymus than in the lymph node and PBMC at 8 weeks post-infection (P 0.05).

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76 Table 3-6. Transcription of the FIV rev gene in the thymus, lym ph nodes, and peripheral blood lymphocytes of cats neonatally infected w ith JSY3 wild type (WT) FIV and the ORFA/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3(WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks Thymus 2.45 (3.32)c 1.56 (1.29) 0.32 (0.33)d 0.29 (0.20) Lymph Node 0.14 (0.06) 0.44 (0.39) 0.02 (0.02) 0.06 (0.04)ePBMC 0.14 (0.03) 0.35 (0.43) 0.01 (0.01) 0.08 (0.08)f a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from the thymus, lymph nodes, and peripheral blood lymphocytes (PBMC) of kittens at 8 weeks (n =3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) post-infection. JSY3 rev RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of rev RNA. d The relative amount of rev RNA was significantly greater in the thymus than in the lymph node and PBMC at 8 weeks post-infection (P 0.05). e The relative amount of rev RNA was significantly greater in the lymph node at 16 weeks compared to 8 weeks post-infection (P 0.05). f The relative amount of rev RNA was significantly greater in the PBMC at 16 weeks compared to 8 weeks post-infection ( P 0.05).

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77 Figure 3-19. Relative gene expression of the FIV gag and rev gene in CD4+, CD8+ and B lymphocytes in the blood of 8-week old a nd 16-week old kittens infected with JSY3 (WT) and JSY3 ORF-A/2 virus. The kittens we re infected at birth with 104 TCID50 of JSY3 (WT) (n= 6 kittens) or JSY3 ORF-A/2 (n=10) kittens. Blood was collected from kittens at 8 weeks (J SY3 (WT) = 3 kittens, JSY3 ORF-A/2= 4 kittens) and 16 weeks (JSY3 (WT) = 3 kittens, JSY3 ORF-A/2= 6 kittens) post-infection. The CD4+, CD8+ and B lymphocytes were purif ied from the peripheral blood mononuclear cells (PBMC) by immunomagnetic selection. The relative expression of the FIV gag and rev gene in each lymphocyte populat ion was determined by real-time PCR assay. (A) Relative expression of JSY3 gag at 8 weeks. (B) Relative expression of JSY3 gag at 16 weeks. (C) Relative expression of JSY3 rev at 8 weeks. (D) Relativ e expression of JSY3 rev at 16 weeks. The data are expressed as mean one standard deviation. The rela tive expression in kittens infected with JSY3 ORF-A/2 was significantly (P 0.05) less than in kittens infected with JSY3 (WT).

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78 Table 3-7. Transcription of the FIV gag gene in CD4+ T cells, CD8+ T cells, and B cells in the peripheral blood of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3 (WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks CD4+ T cells 0.176 (0.079)c 0.125 (0.057) 0.004 (0.002) 0.042 (0.051) CD8+ T cells 0.254 (0.167) 0.049 (0.005) 0.002 (0.002) 0.107 (0.214) B cells 0.234 (0.077) 0.198 (0.041) 0.001 (0.002) 0.031 (0.026) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from CD4+ T cells, CD8+ T cells, and B cells purified by immunomagnetic selection from the peripheral blood of kittens at 8 weeks (n=3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) postinfection. JSY3 gag RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of gag RNA

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79 Table 3-8. Transcription of the FIV rev gene in CD4+ T cells, CD8+ T cells, and B cells in the peripheral blood of cats neonatally infected with JSY3 wild type (WT) FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3 (WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks CD4+ T cells 0.027 (0.032)c 0.012 (0.004) 0.002 (0.004) 0.002 (0.003) CD8+ T cells 0.033 (0.034) 0.037 (0.029) 4.44 E-4 (0.001) 0.016 (0.030) B cells 0.011 (0.002) 0.057 (0.015)d 5.06 E-5 (7.16 E-5) 0.002 (0.003) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from CD4+ T cells, CD8+ T cells, and B cells purified by immunomagnetic selection from the peripheral blood of kittens at 8 weeks (n=3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) postinfection. JSY3 rev RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of rev RNA. d The relative amount of rev RNA was significantly greater in the B cells than in the CD4+ T cells at 16 weeks post-infection (P 0.05).

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80 CHAPTER 4 VISUALIZATION OF VIRUS-INFECTED CELLS AND ACTIVELY REPLICATING THYMOCYTES AND LYMPHOCYTES OF JSY3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACUTE AND CHRONIC STAGES OF INFECTION Introduction Portions of the thymus and the lymph node were examined by immunohistochemistry to determine the percentage of cells expressing FIV p24 Gag protein during acute and chronic FIV infection. P24 is a major core protein and is pres ent after successful vira l infection. The thymus, which is responsible for T cell pr oduction, is the principle target during FIV infection (Beebe et al., 1994). The lymph nodes also serve as sec ondary sites for virus st orage and trafficking (Beebe et al., 1994). Previous studies (Norway et al., 2001) have shown that the chronic (14 weeks) JSY3 (WT) FIV infection of the thymus resulted in equivalent follicular hyperplasia between JSY3 (WT) and JSY3 ORF-A/2 but JSY3 ORF-A/2 cats displayed lower level of viral p24Gag-positive cells compared to JSY3 (W T) and that FIV p24 Gag positive cells were distributed evenly throughout the thymus but excluded from the lymphoid follicles. By examining the percentage of cells expressing vira l p24 we were able to vi sualize the distribution of virus production and quantit ate differences between JSY3 wild type (WT) and JSY3 ORFA/2 infected kittens at acut e and chronic FIV infection. The thymus and lymph node sections were al so stained with Ki67 antibody to determine the mean number of cells and distribution of cells that were under going active cell cycle replication (those cells not in Go arrest) during acute a nd chronic infection. The Ki67 protein is essential for the cell cycle and it s removal halts cell proliferation (Schluter et al., 1993). It can be utilized as an index of pr oliferation by immunohistochemist ry on the thymus and lymph node sections. Typically the antibody is utilized to determine a corr elation between low or high Ki67 index and low or high grade histopathology of neoplasms. The Ki67 antibody was utilized to

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81 visualize the distribution a nd degree of cell replication between JSY3 (WT) and JSY3 ORF-A/2 tissues as well as to correlate the degree of vi ral p24 protein expression to cell replication. The Ki67 antibody has not been prev iously used in FIV studies. Materials and Methods Immunohistochemistry Assay for FIV p24 Protein and Ki67 Protein Five micron paraffin-embedded sections of thymus and lymph node were stained with hematoxylin and eosin for morphological analys is. For immunohistochemistry, 5-m frozen sections were fixed in ice cold ethanol for 5 mi nutes and rinsed in PBS buffer. Sections were incubated at room temperature for 30 minutes with 1% normal horse serum blocking solution and blotted, followed by a 30 minute incubation with 10g/mL of mono clonal antibody to FIV p24 Gag protein (clone PAK3-2C1; Custom Monocl onals International, We st Sacramento, CA.) or 0.8 g/mL of monoclonal antibody to Ki -67 antigen (clone MIB-1 (M 7240); Dako Cytomation Inc., Carpinteria, CA). For a nega tive control, thymus a nd lymph node sections were incubated with 1% normal horse serum instead of the FIV p24 antibody or Ki67 antibody. All sections were developed using the Vectastain Univer sal Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturers instructions and stained with diaminobenzidine chromagen enhanced with nickel Sections were then rinsed in water and counterstained with Harriss hematoxylin. The p2 4 sections were examined microscopically, at a 10x objective, for measurements of follicular, medullary (for th ymus sections) and total area using the Image J NIH software program (http://rsb.info.nih.gov/ij/download.html ). The results were reported as number of FIV p24-positive cells identified per unit of designated area. The Ki67 sections were examined microscopica lly on 100x power and a total of 200 cells were counted in a minimum of three (maximum six) random, non-overlapping fields using the Image

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82 J NIH software program (http://rsb.info.ni h.gov/ij/download.html ). The results were reported as the number of Ki67-positive cells identified per 200 total cells counted per average field. Statistical Analysis A statistical software progra m (SigmaStat 3.0, SPSS Inc., Chicago, IL) was utilized for all data analyses. The data were transformed pr ior to analysis, via square root, in order to normalize any sample variability. Transformed variables that ha d normal distribution and equal variance were compared using the one-way analysis of variance (One-Way ANOVA). Transformed variables that were not normally di stributed or displayed unequal variances were compared using the ANOVA on Ranks test. Values with P 0.05 were considered significant. Results H&E sections showed similar changes with in infected thymuses of JSY3 and JSY3 ORFA/2 infected cats. Observable depletion of t hymocytes was variable, but overall the cortices were well populated and the corticomedullary junc tions were clearly visible. Lymphoid follicles were a prominent feature within thymuses of bot h groups of cats and were formed either within the medullary areas or abutting th e cortical surface. H&E sections of lymph nodes for all cats contained abundant prominent secondary lymphoid follicles, but with an overall mild to moderate reduction in cortical cellularity. Initial immunohistochemical slides exhibi ted mild homogenous brown extracellular background staining within the germinal centers of secondary lymphoid follicles, particularly within lymph node sections. However, attempts to incorporate a step to quench endogenous peroxidase activity resulted in abrogation of an tibody staining and were discontinued. The low level of background staining did not impair th e evaluation of positive cellular staining. The distribution of p24-positively staining cells within the JSY3 (WT) and JSY3 ORFA/2 infected thymic sections was largely limite d to lymphoid follicles with smaller numbers of

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83 cells scattered throughout the medulla. Positive p24 cells were rare within the thymic cortex. Uninfected tissues did not stain positively wi th the p24 FIV antibody. Positive p24 staining cells were distributed evenly throughout the follicles (F igure 4-1.) (Table 4-1). Within the lymph node, positive cells were a prominent feature within the mantles of the se condary follicles, but with large numbers of cells also present and ev enly distributed throughout the remainder of the section (Figure 4-2.) (Table 4-2). As p24-positive cells were over represented with in follicles, histologic measurements of area were made to standardize for their relative c ontribution to the total tissue area. Cells were counted and categorized as follicular or nonfollicul ar, and results were tabulated as number of positive cells per unit area (Table 4-1; Table 4-2) No statistical difference between the number of p24 positive cells within the lymph nodes or thymuses of JSY3 and JSY3 ORF-A/2 infected cats was measured. The distribution of Ki67 positively staining cell s within the thymic sections was largely limited to cortices with smaller numbers of cel ls scattered throughout the medulla. Ki67 positive cells were rare within the thymic follicles (Figur e 4-3; Figure 4-4). Unin fected tissues did stain positively with the Ki67 antibody and this distribution was the same as JSY3 and JSY3 ORFA/2 infected cats (Table 4-3). Within the ly mph node, positive cells were a prominent feature within the mantles of the secondary follicles, bu t with a large numbers of cells also present evenly distributed throughout the rema inder of the section (Figure 4-5). Ki67 positive cells were over represented within the cortices of the thymus and evenly distributed in the lymph nodes. As a result of this distribution, the Ki67 sections were examined microscopically on 100x power and a total of 200 cells were counted in a minimum of three (maximum six), random, non-overlapping fields. Cells were counted and categorized as cortical

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84 or medullary for the thymus, uncategorized for th e lymph node, and the results were tabulated as number of positive cells per 200 total cells per fi eld (Table 4-3; Table 4-4). Quantitation of positive cells showed no statistical differences be tween the number of Ki67 positive cells within lymph nodes and thymuses of JSY3 and JSY3 ORF-A/2 infected cats. Discussion The results from these experiments demonstrat e FIV-infected thymocytes distribute to the lymphoid follicles, slightly to th e medulla and rarely to the cort ex. These results differ from prior immunohistochemistry experi ments with JSY3 (WT) and JSY3 ORF-A/2 infected kittens which saw a consistent thymic distribution of p24 positive cells and did not see lymphoid follicular distribution during late nt infection (Norway et al., 2 001). Other work using the NCSU1 FIV strain with cats of differing routes of infec tion or timing of inoculat ion, also noted lack of FIV expression from thymic follicles utilizing in situ hybridization technique s (Orandle et al., 1997). However, additional studies from our la boratory have confirmed FIV expression from thymic lymphoid follicles by in situ hybridization techniques (la boratory observation; data not shown). Likewise, other in situ hybridization and immunohistoc hemistry data have shown a similar distribution pattern (fo llicular and medullary) in HIV/AIDS patients (Prevot et al., 1992) (Burke et al., 1995) and SIV inf ected monkeys (Li et al., 1995). In our recent experiments we utilized frozen tissue, and not paraffin sections, as done previously. In addition, we rem oved the step to quench endogenous peroxidase activity because it attenuated antibody staining. In order to valid ate our results, thymic tissues used in the Norway et. al. study (Norway et al., 2001) we re stained with the FIV p24 antibody using the most current protocol. The staining patterns of th ose tissues were in agre ement with the current results (Figure 4-6). This diffe rence in protocol could account fo r the differences in the results of the two studies. Perhaps w ith frozen tissue sections a nd ethanol fixation, the p24 Gag

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85 antibody could not penetrate to la bel the infected cells. Altern atively, cells could harbor the virus without active vi ral replication and not label with p24 antibody. With regards to the mean number of positive cells per unit area, we saw no statistical differences between JSY3 (WT) and JSY3 ORF-A/2 thymus or lymph node at either time point. At week 8, JSY3 (WT) staining exhibited mo re p24-positive cells per area compared to JSY3 ORF-A/2 but by 16 weeks of age both groups demonstrated a similar number of thymocytes positively labeling per unit ar ea. In the lymph node however, JSY3 ORF-A/2 displayed more p24 positive cells compared to JSY3 (WT) at week 8 and week 16. Since lymph nodes are a major site of antigen trafficking (Janeway Jr. et al., 2001), this supports the observation that there are additional cell types (CD8+, B cell, macrophages, and plasmacytoid dendritic cells (PDCs) as seen in HIV/SIV) pres ent that are responsible for viral replication and dissemination. This observation has been confir med by others (English et al., 1993) (Zhang et al., 2005) (Brown et al., 2007). Pe rhaps with the mutation in the ORF-A/2 gene, and subsequent decreased viral replication and ge ne expression, there is a localiza tion of these antigen presenting cells, such as plasmacytoid dendritic cells (PDCs) (Cella et al., 1999) (Yoneyama et al., 2004), in the inflamed lymph node. This can also be s upported by our DLEC imm unohistochemistry data which demonstrated a greater mean number of DLEC-positive cells (cell surface marker for plasmacytoid dendritic cells) at either 8 or 16 weeks, in JSY3 ORF-A/2 lymph node, compared to JSY3 (WT) lymph node (Chapter 6). Immunohistochemical analysis of the thym us with the Ki67 antibody demonstrated primarily cortical labeling, indisc riminant medullary staining and exclusion from the follicles. This data signifies active cell cy cling within the cortices of th e thymus. JSY3 (WT) infected initially exhibited intense labeling of the cort ices, however by 16 weeks, this mean value was

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86 less than uninfected age-matched c ontrols. This could be partially reflect indirect thymocyte cell death secondary to cell signaling events (such as activation of apoptosis due to caspase signaling as seen in HIVnot necessarily due to direct FIV infection of the cortical cells) (Meissner et al., 2006) or perhaps thymocyte entr ance into a quie scent state (Go) where Ki67 protein expression is undetectable. The mean number of positive thymocytes of JSY3 ORF-A/2 did not vary between week 8 and week 16 and remain elevated compar ed to uninfected controls from respective time points. This elevated level of replicating thym ocytes cannot be the resu lt of increased thymic mass relative to uninfected, or JSY3 (WT), as th ymic mass values did no t change significantly over time. This data is inte rpreted to reflect that the ORF-A/2 mutation did not appear to alter cell replication in the cortical thymocytes. Ki67 labeling of the lymph node sections dem onstrated a generalized positive staining both within the secondary follicle s, and the cortical areas of the lymph nodes. In JSY3 ORF-A/2 animals, an overall reduced level of Ki67 ce ll proliferation was obser ved at both time points while there was an increase number of Ki67 positive cells for both uninfected and JSY3 (WT). There are an overall reduced number of Ki67 positive cells for JSY3 ORF-A/2 in contrast an increased number of p24 positive cells at week 16 This trend may suggest that a population of JSY3 ORF-A/2 infected cells were not actively re plicating cells compared to JSY3 (WT).

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87 Table 4-1. Mean number of p24+ cells per thymic area. Animal Group Mean number positive cells/unit area 8 week JSY3 (WT) (n=3) 2.03E-05 8 week JSY3 ORF-A/2 (n=4) 1.02E-05 16 week JSY3 (WT) (n=3) 1.57E-05 16 week JSY3 ORF-A/2 (n=6) 1.48E-05 Table 4-2. Mean number of p24+ cells per lymph node area. Animal Group Mean number positive cells/unit area 8 week JSY3 (WT) (n=3) 2.81E-05 8 week JSY3 ORF-A/2 (n=4) 3.73E-05 16 week JSY3 (WT) (n=3) 3.18E-05 16 week JSY3 ORF-A/2 (n=5) 5.84E-05

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88 Table 4-3. Mean number of Ki67+ cel ls per 200 counted thymic cells. Animal Group Mean number positive cortical cells/200 Mean number positive medullary cells/200 8 week Uninfected (n=2) 43.0820.50 8 week JSY3 (WT) (n=3) 88.3343.00 8 week JSY3 ORF-A/2 (n=3) 71.9425.00 16 week Uninfected (n=4) 53.2922.14 16 week JSY3 (WT) (n=3) 49.6735.17 16 week JSY3 ORF-A/2 (n=6) 74.1132.04 Table 4-4. Mean number of Ki67+ cel ls per 200 counted lymph node cells. Animal Group Mean number positive cells/200 8 week Uninfected (n=1) 34.50 8 week JSY3 (WT) (n=3) 46.55 8 week JSY3 ORF-A/2 (n=4) 37.90 16 week Uninfected (n=1) 62.00 16 week JSY3 (WT) (n=3) 58.00 16 week JSY3 ORF-A/2 (n=4) 37.19

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89 Figure 4-1. 10X. Histologic secti on of the JSY3 (WT) infected Week 8 thymus demonstrating p24 antibody-stained cells (black ) within the follicles (F) and the medullary (M) areas of the lobule. Note the lack of p24+ cells within the cortex (C).

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90 Figure 4-2. 10X. Histologi c section of the JSY3 ORF-A/2 infected Week 8 lymph node demonstrating p24 antibody-stained cells (bl ack) localized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C).

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91 Figure 4-3. 20X. Histol ogic section of a JSY3 ORF-A/2 infected Week 16 thymus demonstrating Ki67 antibody-stained cells (bla ck). Note the cells localize to the cortex (C) and sparsely to the medulla (M). Figure 4-4. 100x. Histol ogic section of JSY3 ORF-A/2 infected Week 16 thymus demonstrating Ki67 antibody-stained cells (black) (arrows) within the cortex.

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92 Figure 4-5. 100X. Histologic section of the JSY3 (WT) infected Week 8 lymph node demonstrating Ki67 antibody-stai ned cells (black) (arrows).

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93 Figure 4-6. 10X. Histologic sec tion of the thymus demonstr ating p24 antibody-stained cells (black) within the follicles (F) and the medu llary (M) areas of the lobule. Note the lack of p24 positive cells within the cortex (C). This samp le is representative of our protocol utilizing JSY3 ORF-A/2 infected tissue (Week 14) from (Norway et al., 2001).

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94 CHAPTER 5 MEASUREMENT OF CYTOKINES IL-4, IL-7, IL-15, INTERFERON-ALPHA AND INTERFERON-GAMMA IN JSY3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACUTE AND CHRONIC FIV INFECTION Introduction Cytokines are important in maintaining ha rmony within the immune system. Cytokines are proteins that are made and secreted by cells and have the ability to affect the behavior of other cells via autocrine, paracrin e or endocrine mechanisms (Janeway Jr. et al., 2001). They are usually made in response to an activating stimulus and genera te their response by binding to receptors (Janeway Jr. et al., 2001). Infections wi th lentiviruses disrupt the cytokine profile of the target cells. Fo r example in HIV infection, anti-viral (Th1) cytokines such as IFNand IL-2 are decreased with time, while others such as IL-4, IL-10, also know as humoral cytokines (Th2), are increased (Kedzierska et al., 2001). Change s in cytokine profiles contribute to viral pathogenesis. For example in HIV, increases in IL-4 production cause CD4+ T cells to differentiate into a Th2 phenotype, as opposed to a Th1, and these cells may produce additional IL-4, contributing to a decreased cell-mediated immunity which is crucial for virus elimination (Santana et al., 2003). Numerous studies have been performed in orde r to elucidate the role of cytokines during lentiviral infection. In FIV, Liang et. al. (Lia ng et al., 2000) were able to show that interferongamma (IFN) and IL-10 expression is up-regulated in the thymus and lymph nodes of FIVinfected cats. Ohashi et. al (Ohashi et al., 1992) demonstrat ed that the pro-inflammatory cytokine IL-6, which is responsible for polycl onal B cell activation and proliferation, remains elevated in FIV infection compared to uninfected cats. Cytokines of particular interest to this study are IL-4, IL-7, IL-15, IFN, and IFNand their level of gene expression in the thymus and the lymph node. Acutely and chronically

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95 infected -JSY3 (WT) and JSY3 ORF-A/2 kittens were utilized to determine if there was a difference in cytokine gene e xpression during acute and chroni c FIV infection. Although we observed a decreased level of viral gag and rev gene expression in the thymus and lymph nodes samples with the ORF-A/2 deletion of the molecular clone JSY3 (Chapter 3), we observed equivalent pathologic changes in the thymus (lymphoid follicular hyperpla sia) of JSY3 (WT) and JSY3 ORF-A/2 infected samples (Cha pter 4). Since no statisti cal differences between JSY3 (WT) and JSY3 ORF-A/2 infected cats were observe d in the amount of FIV p24 Gag antibody labeling of thymus or lymph node tissue, nor di stribution of p24, we hypothe sized that cytokine composition of the thymus and lymph node may in fact be equivalent between JSY3 (WT) and JSY3 ORF-A/2 infected kittens. Materials and Methods Cytokine Primers and Probes Cytokine primers and probes were designed as described previously in, Cytokine Profiles and Viral Replication within the Thymuses of Neonatally Feline Immunodeficiency VirusInfected-Cats (Kolenda-Roberts, 2006). Their sequences and specificity were confirmed and validated. Quantitative Real Time PCR for Cytokine Transcription Total RNA was extracted from thymic and lymph node samples from acutely (week 8) and chronically-infected (week 16) JSY3 (WT) and JSY3 ORF-A/2 cats (RNeasy Midi Kit, Qiagen Inc., Valencia, CA). Additional JSY3 (WT) infected thymus RNA at weeks 8 and 16 were kindly provided through collaborative work w ith Calvin Johnson and Holly Kolenda-Roberts (Kolenda-Roberts, 2006). Total RNA was also extracted from uninfected age-matched thymus samples; lymph node samples however, were not available. RNA concentration and purity was determined by UV spectrophotometer (A260/A280). RNA samples were treated with DNase I

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96 (Sigma, St. Louis, MO, USA). One microgram samp les of extracted RNA were used to generate complementary DNAs (cDNAs) in a 20 l synthesis reaction usi ng random hexamer primers (First Strand cDNA Synthesis Kit, Roche, Indi anapolis, IN, USA). Feline G3PDH was selected as the housekeeping gene for normalization of cytokine mRNA content. The feline G3PDH primers, cytokine primers, and corresponding Taqman probes were designed using Primer Express software (PE Applied Biosystems, Foster C ity, CA) (Table 4-1 and Table 5-1). Realtime RT-PCR analyses were conducted using the ABI 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA), utilizing a 25l reaction volume of PCR Universal Master Mix (PE Applied Biosystems, Fo ster City, CA) containing ~100-200 ng of cDNA 900 nM of G3PDH primer, and 125 nM of the TaqMan probes. The standard curve was generated by PCR on serial dilutions of a thymic cDNA samp le from a selected 6 week-old, FIV-infected animal. All samples and the serial dilutions of th e standards were assayed in triplicate. For all samples, target quantity was determined from the standard curve and divided by the target quantity of a calibrator, a 1 sample. A ll other quantities were expressed as an n -fold difference relative to the calibrator, and the same calibrator thymic sample was used for all cytokine experiments. The relative feline cytokine ge ne transcription products we re expressed as the ratio of cytokine mRNA to G3PDH mRNA content. Statistical Analysis A statistical software progra m (SigmaStat 3.0, SPSS Inc., Chicago, IL) was utilized for all data analyses. The data were transformed pr ior to analysis, via square root, in order to normalize any sample variability. Transformed variables that ha d normal distribution and equal variance were compared using the one-way analysis of variance (One-way ANOVA). Transformed variables that were not normally di stributed or displayed unequal variances were compared using the one-way analysis of variance on ranks (ANOVA on Ranks). A one-way

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97 repeated measures ANOVA (RM-ANOVA) was perf ormed to analyze cytokine differences, between each group of cats, over time. Results are presented as means one standard deviation. Values with P 0.05 were considered significant. Results IL-4 IL-4 gene expression was compared over time and against age-matc hed controls (thymus only). Lymph node and thymic IL-4 gene expr ession was measured at 8 weeks and 16 weeks post-JSY3 (WT) and JSY3 ORF-A/2 infection. Arithmetic means with the corresponding standard deviations are repr esented in Table 5-2 and 5-3. During acute infection in the thymus, IL-4 tr anscript levels remain indistinguishable between JSY3 (WT), JSY3 ORF-A/2 and uninfected cats (Figure 5-1; Table 5-3). However, at 16 weeks post-infection, JSY3 ORF-A/2 infected cats exhibi t decreased IL-4 expression, statistically lower than agematched uninfected controls (P 0.05). There is an approximate 21fold difference between the JSY3 ORF-A/2 infected and uninfected cats; a 4-fold difference between the JSY3 (WT) and uninfected cats at 16 weeks. Based on th is data it appears that both JSY3 (WT) and ORF-A/2-deleted virus exer t a suppressive effect on IL-4 production in the thymus especially in JSY3 ORF-A/2 infected cats. Uninfect ed cats at 16 weeks demonstrate increased IL-4 production which suggests an age-related occurrence within the developing thymus. Relative levels of IL-4 expre ssion in the lymph node were no t statistically different at either 8 or 16 weeks when comparing JSY3 (WT) to JSY3 ORF-A/2 (Figure 5-2; Table 5-2). In both JSY3 (WT), and JSY3 ORF-A/2, there is a 3-fold and a 2-fold difference between week 8 thymus versus week 8 lymph node (elevated le vels in the lymph node compared to thymus)

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98 (Table 5-3). However, in JSY3 ORF-A/2 infected cats, we obser ved a 6-fold increase in week 16 lymph node IL-4 gene expression compared to week 16 thymic IL-4 levels (Table 5-3). IL-7 IL-7 gene expression was compared over time and against age-matc hed controls (thymus only). Lymph node and thymic IL-7 gene expr ession was measured at 8 weeks and 16 weeks post-JSY3 (WT) and JSY3 ORF-A/2 infection. Arithmetic means with the corresponding standard deviations are repr esented in Table 5-2 and 5-3. Changes in thymic IL-7 gene expression over time were not statistically significant in either wild type or ORF-A/2 infected cats (Figur e 5-3). As with IL-4, there was an increase in IL-7 transcripts in the uninfected population at 16 weeks. This increase may suggest an agerelated change associated with thymus maturation. In the lymph node the relative le vel of IL-7 RNA in JSY3 (WT) was statistically increased at 16 weeks, compared to 8 week post-infection (P 0.05)(Figure 5-4). This was reflected as a 3fold increase in the level of IL-7 gene expres sion in JSY3 (WT) at 16 weeks compared to a 2fold increase in JSY3 ORF-A/2 at 16 weeks. IL-15 IL-15 gene expression was compared over time and against age-matched controls (thymus only). Lymph node and thymic IL-15 gene expr ession was measured at 8 weeks and 16 weeks post-JSY3 (WT) and JSY3 ORF-A/2 infection. Arithmetic means with the corresponding standard deviations are repr esented in Table 5-2 and 5-3. IL-15 gene expression at 8 weeks in the JSY3 (WT) thymus was comparable to uninfected controls although JSY3 ORF-A/2 thymic expression is reduced nearly 3-fold (Figure 5-5). JSY3 ORF-A/2 thymic IL-15 transcripts remain re latively unchanged between time points and are significantly reduced at 16 weeks, in compar ison to JSY3 (WT) at 16 weeks; this difference

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99 is statistically significant (P 0.05). The elevation of JSY3 (W T) IL-15 gene expression and statistically significant expansion of CD8+ (SP) T cells at 16 weeks (Chapter 3) that is not seen in JSY3 ORF-A/2 thymocyte populations supports previous work that demonstrated a relationship between high IL-15 levels and an en hanced number, function and survival of CD8+ (SP) T cells (Lodolce et al., 2002). IL-15 levels are characteristic ally low in the sera of HIVinfected/AIDS patients and suggest a loss of regu lation of the immune system (Ahmad et al., 2003). No differences in lymph node IL-15 levels were found to be statistically significant between WT and ORF-A/2 cats at either time point (Figur e 5-6). Since IL-15 is important in lymphoid homeostasis and involved with transm itting signals to various innate immune cell types it is likewise unchanged in either group (Lodolce et al., 2002). Interferon Gamma (IFN) Interferon-gamma expression was compared ove r time and against age-matched controls (thymus only). Lymph node and thymic IFNgene expression was measured at 8 weeks and 16 weeks post-JSY3 (WT) and JSY3 ORF-A/2 infection. Arithmetic means with the corresponding standard deviations are grap hically represented in Table 5-2 and 5-3. No statistically significant differences in thymic IFNgene expression could be determined between JSY3 (WT), JSY3 ORF-A/2, and uninfected animals at either 8 or 16 weeks (Figure 5-7). At 16 weeks, a 4-fold diffe rence was observed between JSY3 (WT) infected thymus and uninfected cats; a 2-fold difference was observed between JSY3 ORF-A/2 and uninfected. There was only a 2-fold difference between JSY3 (WT) and ORF-A/2 infected cats at 16 weeks. The uninfected IFN16 week time point is similar to the trends observed for IL-4 and IL-7. IFNexpression level is abrogated by cats inf ected with FIV, and infected animals express IFNat a level 4-times, or 2-times less than uninfected controls. Since the predominant

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100 cell type that expresses IFNis the CD8+ (SP) thymocyte, a nd given the expansion of the CD8+ (SP) T cells in the JSY3 (WT) in fected thymus at 16 weeks (Chapt er 3) we would expect to see an elevation in IFNrelative to uninfected or ORF-A/2 kittens. The reduction in IFNexpression in both groups likel y indicates viral suppression. No statistically significant differences in IFNlevels were observed in the lymph nodes at 8 and 16 weeks (Figure 5-8). As seen in the thymus at 16 weeks, JSY3 ORF-A/2 infected cats exhibited slightly higher levels of IFNexpression with a 3-fold difference between JSY3 (WT) and JSY3 ORF-A/2 infected cats. Interferon Alpha (IFN) Interferon-alpha expression was compared ove r time and against age-matched controls (thymus only). Lymph node and thymic IFNgene expression was measured at 8 weeks and 16 weeks post JSY3 (WT) and JSY3 ORF-A/2 infection. Arithmetic means with the corresponding standard deviations are graphically represented in Table 5-2 and 5-3. IFNtranscript levels in th e thymus at 8 weeks were reduced in JSY3 (WT) and JSY3 ORF-A/2 infected cats in comparison to 8 w eek controls. A 69-fold reduction in JSY3 (WT) IFNgene expression and a 16-fold reduction in JSY3 ORF-A/2 IFNgene expression were observed at 8 weeks, compared to uninfected controls at 8 weeks. Only a 1-fold difference was observed between JSY3 (WT) and JSY3 O RF-A/2 infected thymus at 8 weeks (Figure 5-9). At 16 weeks, the IFNgene expression of uninfected contro ls increased approximately 7-fold, in comparison to uninfected cats at 8 weeks. There was an 86-fold increase in IFNgene expression in uninfected cats at 16 weeks, in co mparison to JSY3 (WT) at 16 weeks, and an 82fold increase in comparison to JSY3 ORF-A/2 infected cats at 16 weeks (Figure 5-9). A statistically significant difference was seen betwee n JSY3 (WT) infected cats and uninfected cats at 16 weeks (P 0.05). No statistically significant differe nces could be measured at 16 weeks

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101 between JSY3 ORF-A/2 infected and uninfected cats, or ORF-A/2 infected cats and JSY3 (WT) infected cats a lthough a 1-fold difference between WTand ORF-A/2-infected at 16 weeks is observed. Both JSY3 (WT) and ORF-A/2 cats exhibited decreased IFNgene expression in the thymus during acute and chroni c infection, with JSY3 (WT) virus exhibiting a much larger reduction. This data is similar to what is seen in HI V patients with viral depletion of type I interferons (IFN, ) (Martinelli et al., 2007) ove r the course of infection. Lymph node IFNgene expression in JSY3 (WT) at 8 weeks was lower than JSY3 ORFA/2 infected cats at 8 weeks (Table 5-3). At 16 weeks post-infection, ho wever, both wild typeand ORF-A/2 infected lymph node IFNgene expression levels were approximately equal (Figure 5-10). Discussion IL-4 is important for maintain ing the thymocyte population. It does not participate in the immune response within the thymus, but is a mediator between the thymocyte and thymic epithelial cells (TEC ) (Yarilin et al., 2004). The down-regul ation of IL-4 gene expression in JSY3 ORF-A/2 cats at 16 weeks, compared to unin fected cats at 16 weeks post-infection, is statistically significant (Figure 5-1). This s uggests that despite JSY3 ORF-A/2 being a replication mutant with low viral gag and rev gene expression in the thymus at 16 weeks (Chapter 3), the virus exerts a suppressive e ffect on thymocyte maturation and development during chronic FIV infection. IL-4 has been shown to promote memory CD8+ survival (Khaled et al., 2002). It is produced by activated CD4+ T cells, mast cel ls, basophils and NK cells which up-regulates MHC II expression on B cells, macrophages and prom otes T and B cell activation (Paul, 1991). In view of this information it is interesting to note that lymph node IL-4 gene expression is elevated, especially in ORF-A/2 mutants, above JSY3 (WT) in fected cats at 16 weeks. In the

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102 absence of uninfected controls it is difficult to assess the signifi cance of this observation. In HIV-1 patients, IL-4 production is increased alongside IgE, re sembling an allergic patient (Becker, 2007). This response is thought to be due to HI V-1 virions shedding gp120, which contains a superantigen (supra llergen) domain that enables vira l glycoproteins to bind the Vh3 domain of IgE molecules bound to dendritic ce lls, mast cells, basophils, and plasmacytoid dendritic cells. This interaction increases th e expression of cytokine s IL-4, 5, 10, and 13 from these hematopoietic cells. Perhaps a similar event may be occurring with FIV gp120, and this may, in part, explain our lymph node findings. Within the thymus, IL-7 has been implicated in T-cell homeostasis; data has shown that IL-7 modulates low-affinity peptide-induced prolif eration, which is critical in the regulation of T-cell homeostasis (Yarilin et al., 2004). Pr imary sources of IL-7 are non-marrow-derived stromal and epithelial cells of the thymus and bone marrow (Yari lin et al., 2004). Both JSY3 (WT) and JSY3 ORF-A/2 infected cats had decreased IL-7 gene expression at 8 and 16 weeks suggesting a viral-induced suppressi ve effect on IL-7 gene expre ssion. This suppressive effect could alter the thymus microenvironment and perh aps T-cell regulation, as IL-7 has been shown to assist in the differentiation and growth of the CD4-CD8(double negative) T cells. Specifically at 16 weeks, we observe an incr eased production of IL-7 transcripts in the uninfected kittens, as we saw in IL-4 at this time point, sugge sting IL-7 plays a role in the maturation of the thymus. In the lymph node at 16 weeks, there was a stat istically significant in crease in IL-7 gene expression in JSY3 (WT) infected cats compared to 8 weeks. This data su pports the role of IL-7 in T-cell regeneration in a respons e to a peripheral T cell depleti on. IL-7 is also required for survival of naive CD4+ and CD8+ cells and memory CD8+ cells (Schluns et al., 2003).

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103 The cytokine IL-15 is critical in lymphoid homeostasis, in addition it appears to be important in transmitting signals to various innate immune cell types (Natural killer cells (NK), NK T cells and / intraepithelial lymphocytes (IELS) (L odolce et al., 2002) (Ma et al., 2000). In vivo IL-15 is responsible for ly mphoid proliferation, protection from cell death, cell homing, and it is absolutely required for the developmen t of memory T-cell popula tions. It also has a supportive role for CD8+ T cellspreferentially enhancing the number of CD8+ T cells, their function and survival. Data from Lodolce et al. (2002) has shown that IL-15 receptor dependent signals are required for CD8+ T cell proliferation in response to bystander stimuli, and these signals are delivered by cells other than T cells. This occurs via conserved microbial motifs, such as poly I: C (viral mimic) which bind to toll-like receptors (TLRs) on the cell surface, and this binding leads to th e release of type I interferons (IFN/ ) (Lodolce et. al., 2002). These interferons then st imulate the release of IL-15 from stromal cells and other accessory cell types (such as monocytes). The aforementioned sequence of events results in increased IL-15 gene expression. At 16 weeks, the JSY3 (WT) thymus has a statistically significant expansion of CD4-CD8+ (SP) thymocytes compared to 8 weeks (Chapter 3), that JSY3 ORF-A/2 infected cats do not experience, and JSY3 (WT) infected cats experiences an increase in IL-15 gene expre ssion in the thymus at 16 weeks. (IL-15 gene expression in JSY3 ORF-A/2 infected cats is reduced at both 8 and 16 weeks). The only disparity in this correlate resides in the level of IFNgene expression. IFNgene expression, however; is low in JSY3 (WT) thymus samples at 16 weeks (Figure 5-9). This is indicati ve of viral inhibition since interferon production can be in hibited at various stages in vi ral replication as seen with adenovirus, SV40 virus, influenza, vaccinia and ot her retroviruses, like HIV (Stark et al., 1998).

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104 IFNgene expression may also be reduced by the infection and the depletion of IFNproducing cells such as seen in HI V-1 patients (Meyers et al., 2007). Interferon-gamma is produced by NK cells, thymoc ytes, as well as activated T cells. It stimulates mononuclear phagocytes as well as cytotoxic Tlym phocytes in response to viral infection (Le et al., 2000). In the thymus, it induces thymic epithelial cell (TEC) activation which is critical for T cell development and matu ration (Yarilin et al., 2004). In our data, we observed a slight increase in IFNgene expression in the thymus of JSY3 ORF-A/2 infected cats at 8 weeks compared to uninfected cats. In the uninfected animals at 16 weeks, however, there was an increase in IFNgene expression compared to JSY3 (WT) or JSY3 ORF-A/2 (Figure 5-7). This suggests that there is viral suppression of IFNgene expression in both JSY3 (WT) and JSY3 ORF-A/2 kittens at 16 weeks post-infe ction, and that the rise in IFNin uninfected kittens at 16 week s suggests a role of IFNin thymic maturation. No notable differences in IFNgene expression were seen in the lymph nodes. Four of the cytokine gene expression an alyses show higher ge ne expression values, although not statistically significant, in the thymus of uninfected kittens at 16 weeks (IL-4, IL-7, IFN, IFN). The cytokine IL-15 is the exception with a reduction in th e uninfected samples relative to JSY3 (WT) infected (Figure 5-5). These elevations in cytokine gene expression in uninfected animals at 16 weeks highlight the impo rtance of the cytokine microenvironment in the development and maturation of the thymus and represent a generalized increase in thymopoietic activity in this age group. Since we did not have uninfected lymph node samples available at the time of this experiment it is more difficult to draw any conc lusions from our results. The only notable result was an increase in IL-7 gene expression in JSY3 (WT) samples that was statistically significant

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105 at 16 weeks compared to 8 weeks (Figure 5-4). Th is elevation supports a role of IL-7 in T-cell regeneration and restoration after periphe ral depletion during ch ronic FIV infection. Table 5-1. Primers and probes utilized for the cytokine real time quantitative PCR. Primer or Probe Sequence (5 to 3) IL-4 forward primer TTC ACG GAA CAG GTC CTG TTT IL-4 reverse primer TG C TCC ACC AAA TTC CTC AAA IL-4 probe 6FAM-CCA TGC TGC T GA GGT TCC TGT CGATAMRA IL-7 forward primer GCC CT G TGA AAC TCT TGA TAA GTG IL-7 reverse primer TCG TGC TGC TCA CAA GTT GAA IL-7 probe 6FAM-AGA TTG AAT TCC TCA CTG TTA TTC ACT TTG ACA AAG TG TAMRA IL-15 forward primer AAC TGA AGC TTG CAT TCC TGT CT IL-15 reverse primer T CC TGC CAG TTT GCC TCT GT IL-15 probe 6FAM-CAT TTT GAG CTG TAT CAG TGC AGG TCT TCC TAA-TAMRA IFN-alpha forward primer TCC GGT GGA GAC CAG TCC C IFN-alpha reverse primer TTC TGG TTC GTC ACG TGC A IFN-alpha probe 6FAM-CAA GGC CCA AGC CCT CTC GGT GTAMRA IFN-gamma forward primer ATG ATG ACC AGC GCA TTC AA IFN-gamma reverse primer TTT ACT GGA GCT GGT ATT TAA CAA CTT ATC IFN-gamma probe 6FAM-AGC ATG GAC ACC ATC AAG GAA GAC ATG C-TAMRA Note: Reprinted with permission from Kolenda-Roberts, H. 2006. Cytokine profiles and viral replication within the thymuses of neonatally feline immunodeficiency virus-infected cats. PhD dissertation (Page 34, Table 4-1). University of Florida, Gainesville, Florida.

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106 Figure 5-1. IL-4 gene expression in the thymus. The thymus was harvested from kittens at 8 weeks (Uninfected (n=5); JSY3 (WT) (n=8); JSY3 ORF-A/2 (n=4)) and 16 weeks (Uninfected (n=3); JSY3 (WT) (n=10), JSY3 ORF-A/2 (n=5)). The data are expressed as mean one standard deviation. (*) denotes a statistically significant difference between JSY3 ORF-A/2 and uninfected kittens at 16 weeks post-infection (P 0.05). Figure 5-2. IL-4 gene expression in the lym ph node. The lymph nodes were harvested from kittens at 8 weeks (JSY3 (WT) (n=3); JSY3 ORF-A/2 (n=4)) and 16 weeks (JSY3 (WT) (n=2), JSY3 ORF-A/2 (n=5)). The data are expressed as mean one standard deviation. No statistically sign ificant differences were observed.

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107 Figure 5-3. IL-7 gene expression in the thymus. The thymus was harvested from kittens at 8 weeks (Uninfected (n=5); JSY3 (WT) (n=8); JSY3 ORF-A/2 (n=4)) and 16 weeks (Uninfected (n=3); JSY3 (WT) (n=11), JSY3 ORF-A/2 (n=6)). The data are expressed as mean one standard deviation. No stat istically significant differences were observed. Figure 5-4. IL-7 gene expression in the lym ph node. The lymph nodes were harvested from kittens at 8 weeks (JSY3 (WT) (n=3); JSY3 ORF-A/2 (n=4)) and 16 weeks (JSY3 (WT) (n=3), JSY3 ORF-A/2 (n=6)). The data are expressed as mean one standard deviation. (*) denotes a stat istically significant differen ce between JSY3 (WT) at 8 weeks compared to 16 weeks post-infection (P 0.05).

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108 Figure 5-5. IL-15 gene expression in the thymus. The thymus was harvested from kittens at 8 weeks (Uninfected (n=5); JSY3 (WT) (n=8); JSY3 ORF-A/2 (n=4)) and 16 weeks (Uninfected (n=3); JSY3 (WT) (n=11), JSY3 ORF-A/2 (n=6). The data are expressed as mean one standard deviation. (*) denot es a statistically significant difference between JSY3 WT at 16 weeks compared to JSY3 ORF-A/2 kittens at 16 weeks post-infection (P 0.05). Figure 5-6. IL-15 gene expre ssion in the lymph node. The ly mph nodes were harvested from kittens at 8 weeks (JSY3 (WT) (n=3); JSY3 ORF-A/2 (n=4)) and 16 weeks (JSY3 (WT) (n=3), JSY3 ORF-A/2 (n=6)). The data are expressed as mean one standard deviation. No statistically sign ificant differences were observed.

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109 Figure 5-7. IFNgene expression in the thymus. The t hymus was harvested from kittens at 8 weeks (Uninfected (n=5); JSY3 (WT) (n=8); JSY3 ORF-A/2 (n=4)) and 16 weeks (Uninfected (n=3); JSY3 (WT) (n=11), JSY3 ORF-A/2 (n=6). The data are expressed as mean one standard deviation. No stat istically significant differences were observed. Figure 5-8. IFNgene expression in the lymph node. The lymph nodes were harvested from kittens at 8 weeks (JSY3 (WT) (n=3); JSY3 ORF-A/2 (n=4)) and 16 weeks (JSY3 (WT) (n=3), JSY3 ORF-A/2 (n=6)). The data are expressed as mean one standard deviation. No statistically sign ificant differences were observed.

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110 Figure 5-9. IFNgene expression in the thymus. The t hymus was harvested from kittens at 8 weeks (Uninfected (n=6); JSY3 (WT) (n=7); JSY3 ORF-A/2 (n=4)) and 16 weeks (Uninfected (n=3); JSY3 (WT) (n=11), JSY3 ORF-A/2 (n=6). The data are expressed as mean one standard deviation. (*) denot es a statistically significant difference between uninfected kittens at 16 weeks compared to JSY3 WT infected kittens at 16 week s post-infection (P 0.05). Figure 5-10. IFNgene expression in the lymph node. The lymph nodes were harvested from kittens at 8 weeks (JSY3 (WT) (n=3); JSY3 ORF-A/2 (n=4)) and 16 weeks (JSY3 (WT) (n=3), JSY3 ORF-A/2 (n=6)).The data are expressed as mean one standard deviation. No statistically sign ificant differences were observed.

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111 Table 5-2 Transcription of cytoki ne genes (IL-4, IL-7, IL-15, Interferon-gamma, Interferonalpha) in the thymus of cats neonatally inf ected with JSY3 wild type FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3 (WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks IL-4 0.53 (0.39)c 1.57 (1.28) 0.59 (0.73)0.32 (0.31)d IL-7 0.81 (0.55 ) 2.08 (2.84) 1.73 (1.83)2.40 (2.22) IL-15 1.23 (0.89) 1.96 (1.20)e 0.38 (0.34)0.28 (0.14) IFN3.58 (2.90) 20.59 (41.36)f 14.94 (14.63)21.54 (19.03) IFN2.29 (1.98 ) 3.68 (3.40) 3.56 (4.41)5.48 (5.55) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from the thymus of kittens at 8 weeks and 16 weeks post-infection. Relative cytokine RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of cytokine RNA. d The relative amount of IL-4 RNA was significantly greater in uni nfected thymus at 16 weeks versus JSY3 ORF-A/2 at 16 weeks post infection (P 0.05). e The relative amount of IL-15 R NA was significantly greater in JSY3 (WT) thymus at 16 weeks post infection versus JSY3 ORF-A/2 at 16 weeks post infection (P 0.05). f. The relative amount of IFNRNA was significantly greater in the thymus of uninfected kittens at week 16 compared to JSY3 (WT) at week 16 (P 0.05).

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112 Table 5-3 Transcription of cytoki ne genes (IL-4, IL-7, IL-15, Interferon-gamma, Interferonalpha) in the lymph nodes of cats neonatally infected with JSY3 wild type FIV and the ORF-A/2 deletion mutant of JSY3 (JSY3 ORF-A/2 ). JSY3 (WT)a JSY3 ORF-A/2 8 weeksb 16 weeks 8 weeks 16 weeks IL-4 1.35 (1.32)c 0.82 (0.33) 1.08 (0.17) 1.90 (1.64) IL-7 2.21 (0.99) 5.99 (1.49)d 2.86 (1.47) 5.00 (1.65) IL-15 0.48 (0.39) 0.84 (0.99) 0.81 (0.73) 0.57 (0.31) IFN2.92 (3.16) 27.55 (26.77) 5.93 (7.21) 26.64 (25.13) IFN1.74 (1.28) 1.55 (3.61) 1.03 (1.36) 5.01 (6.74) a Kittens were infected at birth with JSY3 w ild type (WT) FIV (n=6) or the ORF-A/2 deletion mutant of JSY3 FIV (JSY3 ORF-A/2) (n=10). b Total RNA was extracted from the lymph nodes of kittens at 8 weeks (n=3 for JSY3 (WT), n=4 for JSY3 ORF-A/2) and 16 weeks (n=3 for JSY3 (WT), n=6 for JSY3 ORF-A/2) postinfection. Relative cytokine RNA was quantified by a real time PCR assay. c Mean (standard deviation) for the relative amount of cytokine RNA. d The relative amount of IL-7 RNA was significantly greater in JSY3 (WT) at 16 weeks compared to JSY3 (WT) at 8 weeks post infection (P 0.05).

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113 CHAPTER 6 VISUALIZATION OF PLASMACYTOID DE NDRITIC CELLS (PDC) WITHIN THE THYMUS AND LYMPH NODES OF FIV JS Y3 WILD TYPE (WT) AND JSY3 ORF-A/2 INFECTED KITTENS DURING ACU TE AND CHRONIC INFECTION Introduction Previous studies on HIV-1 have discovered in terferon-producing cells (IPCs)/plasmacytoid dendritic cells (PDCs) that are important medi ators of innate immunity (Zhang et al., 2005). These cells act to suppress vira l replication through the secret ion of interferon (IFN)-alpha (Zhang et al., 2005) (Gurney et al., 2004). In vitro experiments have found that these cells can suppress HIV and that this cell population is seve rely reduced in the peripheral blood of HIVinfected individuals (Barron et al., 2003) (Donaghy et al., 2001) In addition, it was found that PDCs are lost from lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysio logy of AIDS (Brown et al., 2007). The thymus has been shown to harbor a subset of resident PDC, however, their func tion within the normal thymus remains unclear (Fohrer et al., 2004). A recombinant human dendritic cell l ectin (rhDLEC) has been developed and commercially developed antibodies are availabl e facilitating the study of the interferon producing cell (IPC)/plasmacytoid dendritic cell type (PDC). However, previous reports indicated that anti-human DLEC antibodies di d not cross-react with PDC from the peripheral blood of the rhesus macaque (Chung et al., 2005). The experiments described in th is chapter are a con tinuation of data in itially provided by Holly Kolenda-Roberts (Kolenda-Roberts, 2006), on the polyclonal anti -human DLEC-derived antibody. These results provide prel iminary data to characterize this cell type in JSY3 (WT) and JSY3 ORF-A/2 infected cats during acu te (8 weeks) and chronic ( 16 weeks) FIV infection. The results demonstrate that the rhDLEC polyclonal antibody does cross-react with a subset of feline

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114 thymic and lymph node cells. These DLEC-positive cells seem to be IFNproducing cells that are localized in the lymphoid fo llicles of the thymus and the s econdary mantles of the lymph nodes. PDCs and IFNcells are depleted from the thymus over the course of JSY3 (WT) and JSY3 ORF-A/2 FIV infection. JSY3 ORF-A/2 infected cats demonstrate higher IFNpositive cells and DLEC-positive cells in the lymph node at week 8 compared to JSY3 (WT) infected cats at 8 weeks. By 16 weeks, JSY3 ORF-A/2 infected cats have an equivalent number of IFNpositive cells to JSY3 (WT) infected cats and a higher number of DLEC-positive cells compared to JSY3 (WT) infected cats, in th e lymph node, indicating a loss of IFNexpression in JSY3 ORF-A/2 infected cats. Materials and Methods Immunohistochemistry Assay for DLEC and IFNFive micron paraffin-embedded sections of JSY3 (WT) and JSY3 ORF-A/2 thymus and lymph node were stained with hematoxylin and eosin for morphological analysis. Uninfected thymus samples were from age-matched controls Uninfected lymph node sections were not available for comparison. For immunohistochemist ry, 5-m frozen sections were fixed in ice cold ethanol for 5 minutes and rinsed in PBS buffer. Sections were incubated at room temperature for 30 minutes with 1% normal horse serum blocking solution and blotted, followed by a 30 minute incubation with 10 g/mL of either anti-rhDLEC polyclonal antibody (R&D Systems, Minneapolis, MN, USA), or a polyclonal anti-human IFNantibody (PBL Biomedical Laboratories, Piscataway, NJ, USA). For a nega tive control, thymus and lymph node sections were incubated with 1% normal horse serum in stead of the anti-rhDLEC polyclonal antibody or the anti-human IFNantibody. All sections were devel oped using the Vectastain Universal Elite ABC Kit (Vector Laboratories Inc., Burl ingame, CA) according to the manufacturers instructions and stained with di aminobenzidine chromagen and enhanced with nickel. Sections

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115 were then rinsed in water and counterstained with Harriss hemat oxylin. The slide sections were examined microscopically, at a 10x objective, fo r measurements of follicular, medullary (for thymus sections) and total area using the Image J NIH software program (http://rsb.info.nih.gov /ij/download.html ). The results were reported as number of positivelystained cells identified per unit of designated area. Statistical Analysis A statistical software progra m (SigmaStat 3.0, SPSS Inc., Chicago, IL) was utilized for all data analyses. The data were transformed pr ior to analysis, via square root, in order to normalize any sample variability. Transformed variables that ha d normal distribution and equal variance were compared using the one-way analysis of variance (One-Way ANOVA). Transformed variables that were not normally di stributed or displayed unequal variances were compared using the ANOVA on Ranks test. Values with P 0.05 were considered significant. Results Initial immunohistochemical slides exhibi ted mild homogenous brown extracellular background staining within the germinal centers of secondary lymphoid follicles, particularly within lymph node sections. However, attempts to incorporate a step to quench endogenous peroxidase activity resulted in abrogation of an tibody staining and were discontinued. The low level of background staining did not impair th e evaluation of positive cellular staining. Experiments utilizing the anti-human-IFNantibody resulted in mild positive staining of cells localized along the corticomedullary junc tion, and the endothelial cells, in uninfected thymus samples (Figure 6-1). JSY3 (WT) and JSY3 ORF-A/2 infected samples also had these cells present, but the predominance of positive cells were present within the lymphoid follicles which were secondary to viral infection (Figure 6-2; Figure 6-3). There were no visible

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116 differences in the distribution of positive ce lls in the thymus between JSY3 (WT) and JSY3 ORF-A/2 infected cats at 8 or 16 weeks. Within the lymph node, IFN-positive cells were a prominent feature within the mantles of the secondary follicles, but with large numbers of cells also present and evenly distributed throughout the remainder of the section (Figure 6-4). Immunohistochemistry experiments utilizing the anti-rhDLEC antibody revealed positive staining in the JSY3 (WT) and JSY3 ORF-A/2 infected thymus with DLEC-positive cells localized within the lymphoid follicles (Figures 6-5 and 6-6). Lymph node samples displayed intense DLEC staining in the man tles of the secondary follicles with scattering of positive cells throughout the cortex (Figure 6-7). Statistical analysis of the thymus data showed that there were no signi ficant differences at either time point, in the number of IFNpositively staining cells pe r unit area between JSY3 (WT) and JSY3 ORF-A/2 infected cats. At week 8, JSY3 ORF-A/2 infected thymus samples contained a greater mean number of IFNpositive cells per total t hymus area measured, than JSY3 (WT) infected or uninfected cats (Table 6-1). However, by 16 weeks, JSY3 ORF-A/2 and JSY3 (WT) infected cats have less IFNpositive cells per thymus area than uninfected cats (Table 6-1). This same trend is evident in th e lymph node samples (Table 6-2). No comparison can be made, however, between the mean number of IFNpositive cells in uninfected lymph node, JSY3 (WT) infected, and JSY3 ORF-A/2 infected, as uninfected lymph node samples were not available at the time of the experiment. For thymus tissues stained with anti-rhDLEC, 8-week-old JSY3 (WT) infected samples contained statistically more DLEC-positively staining cells than 16-week-old JSY3 (WT) infected animals (P 0.05) (Table 6-3). No other statisti cal differences were found. Unlike the

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117 mean number of IFN-positive cells in the thymus at 8 weeks (Table 6-1), JSY3 ORF-A/2 infected cats had lower DLEC-positive cells in the thymus at 8 weeks, than JSY3 (WT) or uninfected cats at 8 weeks (Table 6-3). The opposite trend is seen in the lymph node samples wherein JSY3 ORF-A/2 infected cats at 8 weeks had a greater mean number of DLEC-positive cells per area, compared to JSY3 (WT) infect ed (Table 6-4). In addition, at 16 weeks, JSY3 ORF-A/2 infected lymph node samples also contain a greater number of DLEC-positive cells per area versus JSY3 (WT) (Table 6-4). No comparison can be made however, between the mean number of DLEC-positive cells in uni nfected lymph node, JSY3 (WT), and JSY3 ORFA/2, as uninfected lymph node samples were no t available at the time of the experiment. Discussion These immunochemistry experiments support pr evious data (Kolenda-Roberts, 2006) that the rhDLEC polyclonal antibody does cross-react with a subset of feline thymic and lymph node cells. These cells were present in both JSY3 (WT) and JSY3 ORF-A/2 infected cats during acute and chronic FIV infec tion. Both rhDLEC and IFNgave a similar distribution pattern, analogous to the FIV p24 Gag staini ng (Chapter 4). This distri bution data suggests that FIVinfected-p24-positive cells may in fact be plasmacy toid dendritic cells that express IFN-alpha. In support of this is original data from Holly Kolenda-Roberts (Kole nda-Roberts, 2006;KolendaRoberts, 2006) double-labeled immunohistochemistry performed on JSY3 (WT) infected thymus samples which stained positively for both DLEC and IFN. Her results suggest that DLECpositive cells may in fact be interferon producing cells (IPCs). These findings support previous HIV-1 reports on PDCs (Zhang et al., 2005). In terms of IFNimmunohistoche mistry, JSY3 ORF-A/2 infected thymus and lymph node at week 8 consistently had a greater number of positive cells than JSY3 (WT) infected or uninfected samples, but this difference was not st atistically significant. At 16 weeks however,

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118 the mean number of IFN-positive cells in JSY3 ORF-A/2 and JSY3 (WT) infected cats were equivalent in the thymus and lymph node samples. This overall reduction in the number of cells in the thymus expressing IFNin JSY3 ORF-A/2 and JSY3 (WT), compared to uninfected samples at week 16, suggest a down-regulation of IFNproduction. In Chapter 5, IFNgene expression data demonstrat ed the suppression of IFNgene expression in the thymus of JSY3 (WT) and JSY3 ORF-A/2 infected cats at 16 weeks. Th is data supports the reduction seen in the number of IFNpositive cells in JSY3 (WT) and JSY3 ORF-A/2 infected thymus at 16 weeks. In the lymph node samples at 16 weeks, we obs erve approximately equivalent numbers of IFNpositive cells in JSY3 (WT) and JSY3 ORF A/2 infected cats, although the lack of uninfected lymph node sections makes it difficult to assess how significant this equivalence is. Similarly, in Chapter 5, we reported e quivalent relative levels of IFNgene expression from the lymph node of JSY3 (WT) and JSY3 ORF-A/2 infected cats at 16 weeks. DLEC immunohistochemistry revealed a deple tion of the PDC cell types from the thymus and lymph node over the course of FIV infec tion. Specifically, JSY3 (WT) infected DLECpositive cells in the thymus decreased significantly from week 8 to week 16 (P 0.05). JSY3 ORF-A/2 infected cats had lower DLEC-positive cells in the thymus at week 8 compared to uninfected or JSY3 (WT) infected cats. Howe ver, by 16 weeks of infection, JSY3 (WT) and JSY3 ORF-A/2 infected cats had equivalent numb ers of DLEC-positive cells in the thymus, lower numbers than uninfected cat s. In the lymph node, no statistic al differences were seen but JSY3 ORF-A/2 maintained higher numbers of DLEC-positive cells compared to JSY3 (WT). In summary, rhDLEC polyclonal antibody does cr oss-react with a subset of feline thymic and lymph node cells. These DLEC-positive cells seem to be IFNproducing cells that are

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119 localized in the lymphoid follicles of the thymus and the secondary mantles of the lymph nodes. JSY3 ORF-A/2 infected cats appear to have higher numbers of IFNproducing cells in the thymus and lymph node at 8 weeks compared to 16 weeks. By 16 weeks, however, both JSY3 (WT) and JSY3 ORF-A/2 infected cats have equivalent numbers of IFNpositive cells in both the thymus and lymph node. JSY3 ORF-A/2 infected cats have lower DLEC-positive cells in the thymus at 8 weeks versus JSY3 (WT) infected cats, but e quivalent numbers to JSY3 (WT) infected cats, at 16 weeks. It must be propos ed that some other cell type is producing IFNin the JSY3 ORF-A/2 infected thymus at 8 weeks, to co rrelate the low DLEC numbers at 8 weeks. Clearly PDCs and IFNcells are depleted from the thymus over the course of JSY3 (WT) and JSY3 ORF-A/2 FIV infection. The number s of DLEC in lymph node of JSY3 ORF-A/2 infected cats remains elevated into 16 weeks compared to JSY3 (WT). This suggests a more dramatic PDC depletion in JSY3 (WT) infected lymph nodes much like what is seen in the peripheral lymph nodes of advanced SIV infection (Brown et al., 2007).

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120 Table 6-1. Mean number of IFNpositive cells as measured per total thymus area. Animal Group Mean number positive cells/unit area 8 week Uninfected (n=2) 1.55E-06 8 week JSY3 (WT) (n=3) 1.94E-05 8 week JSY3 ORF-A/2 (n=4) 3.83E-05 16 week Uninfected (n=2) 2.40E-06 16 week JSY3 (WT) (n=3) 1.05E-05 16 week JSY3 ORF-A/2 (n=6) 1.21E-05 Table 6-2. Mean number of IFNpositive cells as measured per total lymph node area. Animal Group Mean number positive cells/unit area 8 week JSY3 (WT) (n=3) 2.45E-05 8 week JSY3 ORF-A/2 (n=4) 3.74E-05 16 week JSY3 (WT) (n=3) 1.10E-05 16 week JSY3 ORF-A/2 (n=6) 1.96E-05

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121 Table 6-3. Mean number of DLEC-positive ce lls as measured per total thymus area. Animal Group Mean number positive cells/unit area 8 week Uninfected (n=2) 2.27E-06 8 week JSY3 (WT) (n=3) 2.19E-05a 8 week JSY3 ORF-A/2 (n=4) 1.10E-05 16 week Uninfected (n=4) 2.30E-06 16 week JSY3 (WT) (n=3) 8.95E-06 16 week JSY3 ORF-A/2 (n=6) 8.35E-06 a. The mean number of DLEC-positive cells in JS Y3 (WT) thymus at week 8 is significantly greater than week 16 JSY3 (WT) thymus (P 0.05). Table 6-4. Mean number of DLEC-positive cell s as measured per total lymph node area. Animal Group Mean number positive cells/unit area 8 week JSY3 (WT) (n=3) 4.37E-05 8 week JSY3 ORF-A/2 (n=4) 7.18E-05 16 week JSY3 (WT) (n=3) 3.62E-05 16 week JSY3 ORF-A/2 (n=4) 5.91E-05

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122 Figure 6-1. 10X. Histologic secti on of an uninfected thymus at 16 weeks stained with anti-IFNantibody. The arrows demonstrate how the endothelial cells stain lightly. Figure 6-2. 10X. Histologic secti on of a JSY3 (WT)-infected thym us at 8 weeks demonstrating IFNantibody-stained cells (black) within th e follicles (F) and the medullary (M) areas of the lobule. Note the lack of IFNpositive cells within the cortex (C).

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123 Figure 6-3. 10x. Histolog ic section of a JSY3 ORF-A/2-infected thymus at 16 weeks demonstrating IFNantibody-stained cells (black) within the follicles (F) and the medullary (M) areas of the lobule. Note the lack of IFNpositive cells within the cortex (C). Figure 6-4. 10X. Histol ogic section of a JSY3 ORF-A/2-infected lymph node at 8 weeks demonstrating IFNantibody-stained cells (black) lo calized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C). The homogenous brown staining in the center of the follicles was typical of the lymph node samples and was not considered positive when counting for IFN-positive cells.

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124 Figure 6-5. 10x. Histolog ic section of a JSY3 ORF-A/2-infected thymus at 16 weeks demonstrating DLEC-antibody-stained cells (black) within the lymphoid follicles (F) of the lobule. Note the lack of DLEC -positive cells within the cortex (C). Figure 6-6. 10x. Histologic secti on of a JSY3 (WT)-infected thym us at 8 weeks demonstrating DLEC-antibody-stained cells (black) within the lymphoid follicles (F) of the lobule. Note the lack of DLEC-positive cells within the cortex (C).

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125 Figure 6-7. 10X. Histol ogic section of a JSY3 ORF-A/2-infected lymph node at 8 weeks demonstrating DLEC antibody-stained cells (b lack) localized to the mantle of the secondary follicles (SF) (arrow) and within the cortex (C). The homogenous brown staining was typical in the center of the follicles of the lymph node samples and was not considered positive when counting for DLEC-positive cells.

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126 CHAPTER 7 CONCLUSIONS In conclusion, the goal of this research project was to more fully characterize the role of the FIV ORF-A/2 gene expression in vivo so as to understand the mechanism of FIV pathogenesis in different lymphoid populations. In order to accomplish this, we analyzed proviral load, viral gene expression, and the local ization of virus-infected and actively replicating cells from acute to chronic phase of FIV infection. We selected th e neonatally-infected cat as the infection model, utilizing the highly pathogenic molecular clone JSY3, in the presence or absence of a functional ORF-A/2 gene. These experiments demonstrate that the ORF-A/2 gene deletion results in diminished gene expression in the thymus, lymph no de and PBMC (Chapter 3). JSY3 ORF-A/2 infected cats had greater T cell and B cell numbers in the thym us at 8 and 16 weeks compared to JSY3 (WT) infected cats. Despite these hi gher cell numbers, by 16 weeks, JSY3 ORF-A/2 infected cats have near equivalent levels of proviral load, but, reduced gag and rev transcription products, compared to JSY3 (WT) infected cats. JSY3 ORF-A/2 infected cats absolute values in lymph node subpopulations did not change markedly be tween week 8 and week 16 and although the absolute number of cells remained equivalent, th is did not result in an increase in the rate of gag and rev gene expression in the lymph node at 16 weeks. Analysis of the PBMCs revealed that B cell numbers in the blood of JSY3 ORF-A/2 infected cats were significantly lower relative to the JSY3 (WT) infected cats, and that these B cel ls, when sorted and analyzed for proviral load and gene expression, revealed lo wer levels of proviral load an d viral gene expression throughout the JSY3 ORF-A/2 infection period. Specifically, B ce lls exhibited a statistically significant reduction in proviral load and vi ral gene expression during chroni c infection. In the sorted PBMCs, the relative level of B cell proviral lo ad and viral gene expression were severely

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127 hindered by the ORF-A/2 mutation. The reason for this is unknown although the mean absolute number of B cells at 16 week s for cats infected with JSY3 ORF-A/2 (768 cells/L) is lower than JSY3 (WT) (2,954 cells/L) and could be a ttributable. However, upon examination of the absolute mean values of CD8+ and CD4+ T cells at 8 weeks, there is equivalent number of cells between JSY3 (WT) and JSY3 ORF-A/2 yet lower proviral load and gene expression in JSY3 ORF-A/2 infected cats. Whether ORF-A/2 c ontributes to the shift from CD4+ cells during acute infection, to the B cells in the chronic phase of infection, remains to be determined. Our immunohistochemistry data described in Chapter 4, dem onstrated that FIV-infected thymocytes distribute to the lymphoid follicles, sli ghtly to the medulla and rarely to the cortex. Likewise, other in situ hybridization and immunohistochemi stry data have shown a similar distribution pattern (follicular a nd medullary) in HIV/AIDS patient s (Prevot et al., 1992) (Burke et al., 1995) and SIV-infected monkeys (Li et al., 1995). The staining patterns of tissues previously reported (Norway et al ., 2001) were in agreement with our current results. At week 8, JSY3 (WT) staining exhibited more p24-pos itive cells per area compared to JSY3 ORF-A/2infected, but by 16 weeks of age both groups dem onstrated a similar number of thymocytes positively labeling per unit area. In the lymph node however, JSY3 ORF-A/2 displayed more p24-positive cells compared to JSY3 (WT) at we ek 8 and week 16. Additional cell types (CD8+, B cell, macrophages, and plasmacytoid dendritic cells (PDCs) (as seen in HIV/SIV) may be present that are responsible for viral replicat ion and dissemination. Th is observation has been confirmed by others (English et al., 1993) (Zhang et al., 2005) (Brown et al., 2007). Perhaps with the mutation in the ORF-A/2 gene, and subsequent decrease d viral replication and gene expression, there is a localization of these an tigen presenting cells, such as plasmacytoid dendritic cells (PDC) (Cella et al., 1999) (Yoneyama et al., 2004) in the inflamed lymph node.

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128 This can also be supported by our DLEC immun ohistochemistry data which demonstrated a greater mean number of DLEC-positive cells (ce ll surface marker for plasmacytoid dendritic cells (PDCs)) at either 8 or 16 weeks, in JSY3 ORF-A/2 infected lymph node, compared to JSY3 (WT) infected lymph node (Chapter 6). The ORF-A/2 mutation did not appear to alter cell replication (Ki67) in the cortical thymocytes (Chapter 4). There are an overa ll reduced number of Ki67-positive cells in the lymph node for JSY3 ORF-A/2 infected cats in contrast to an increased numb er of p24-positive cells at week 16. This data suggests that a population of JSY3 ORF-A/2-infected cells were not actively replicating cells compared to JSY3 (WT) This data does not suggest, however, that these JSY3 ORF-A/2 infected cells have entered a G2-arrest, as in vitro work has previously reported (Gemeniano et al., 2004). Four of the cytokine gene expression an alyses show higher ge ne expression values, although not statistically significant, in the thymus of uninfected kittens at 16 weeks (IL-4, IL-7, IFN, IFN). The cytokine IL-15 is the exception with a reduction in th e uninfected samples relative to JSY3 (WT) infected. These elevations in cytokine gene expression, in uninfected animals at 16 weeks, highlight the importanc e of the cytokine microenvironment in the development and maturation of the thymus and re present a generalized in crease in thymopoietic activity in this age group. IL-4 gene expression in the thymus of JSY3 ORF-A/2 infected cats was lower than JSY3 (WT) infected and was significan tly less than uninfected cats at 16 weeks. This suggests that despite JSY3 ORF-A/2 being a replicati on mutant, with low viral gag and rev gene expression in the thymus at 16 weeks (Chapter 3), the vi rus may exert a suppressi ve effect on thymocyte maturation and development during chronic FIV in fection. IL-4 has also been shown to be

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129 produced by activated CD4+ T cells, mast cel ls, basophils and NK cells which up-regulates MHC II expression on B cells, macrophages and prom otes T and B cell activation (Paul, 1991). Despite higher T and B cell numbers in the thymus of JSY3 ORF-A/2-infected cats at 16 weeks, the level of IL-4 gene expression is low, suggesti ng that the percentage of activated T cells and B cells must be low. The low IL-4 gene expressi on appears correlated to the low level of IL-15 gene expression in the thymus of JSY3 ORF-A/2 infected cats at 16 we eks. IL-15 is responsible for the induction of B cell prolif eration and differentiation (Armitage et al., 1995); in HIV-1 IL15 may promote polyclonal B-cell activation and hypergammaglobulinaemia (Kacani et al., 1999). In Chapter 6, we found the rhDLEC polyclona l antibody to cross-react with a subset of feline thymic and lymph node cells. These DLEC-positive cells seem to be IFNproducing cells that are localized in the lymphoid follicles of the thymus and the secondary mantles of the lymph nodes. JSY3 ORF-A/2 infected cats appear to have higher numbers of IFNproducing cells in the thymus and lymph node at 8 weeks co mpared to JSY3 (WT). By 16 weeks, however, both JSY3 (WT) and JSY3 ORF-A/2 infected cats have equivalent numbers of IFNpositive cells. JSY3 ORF-A/2 infected cats have lower DLECpositive cells in the thymus at 8 weeks versus JSY3 (WT), but equivalent numbers at 16 weeks. It must be proposed that some other cell type is producing IFNin the JSY3 ORF-A/2 infected thymus at 8 weeks to correlate the low DLEC numbers at 8 weeks. Clearly PDCs and IFNcells are depleted from the thymus over the course of JSY3 (WT) and JSY3 ORF-A/2 FIV infection. The numbers of DLEC in lymph node of JSY3 ORF-A/2 infected cats remains elevated into 16 weeks compared to JSY3 (WT) (Chapter 6). This suggests a more dramatic PDC

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130 depletion in JSY3 (WT) infected lymph nodes much like what is seen in the peripheral lymph nodes of advanced SIV infection (Brown et al., 2007). The combined results of these experiments sugge st that future studies are needed in order to define the role of the ORF-A/2 gene product and how it relates to FIV pathogenesis of the B cell. It appears there is a significant reduction in the number of circulating B cells, the level of B cell proviral load, and viral gene expression in the PBMCs of JSY3 ORF-A/2 infected cats. The reason for the lower absolute B cell numbers in the circulating PBMCs of JSY3 ORF-A/2 infected cats is unknown, but may reflect a gene ralized sequestration a nd compartmentalization of B cells in the thymus and lymph node, as s uggested by flow cytometry data. Measurement of IL-4 and IL-15 cytokine gene e xpression in the thymus of JSY3 ORF-A/2 infected cats revealed lower levels compared to JSY3 (WT) and uninfec ted cats. Both of these cytokines are known to have roles in B cell activation, proliferation, and differentiation.

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144 BIOGRAPHICAL SKETCH Janelle Marisa Novak was born on November 6, 1976 in Evanston, IL. She attended Michigan State University, gradua ted with Honors, and received a Bachelor of Sciences degree in Physiology in the year 1998. She then atte nded the University of Florida, College of Veterinary Medicine, in 2001, to pursue her Ph.D. in the Department of Infectious Diseases and Pathology under the supervision of Ayalew Mergia In 2004, Janelle began enrollment in the Doctor of Veterinary Medicine (D.V.M.) program at the Univer sity of Florida, College of Veterinary Medicine. After co mpletion of both degrees, Janelle will pursue a residency in anatomic pathology.