Interactions between the Midgut of Culex Pipiens Quinquefasciatus and West Nile Virus from Environmental Factors to Cell...

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Interactions between the Midgut of Culex Pipiens Quinquefasciatus and West Nile Virus from Environmental Factors to Cell Binding and the Immune Response
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1 online resource (129 p.)
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english
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Anderson,Sheri Lizbeth
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Smartt, Chelsea T
Committee Members:
Day, Jonathan F
Tabachnick, Walter J
Connelly, Cynthia Roxanne
Powell, Charles A
Richards, Stephanie

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Entomology and Nematology -- Dissertations, Academic -- UF
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Entomology and Nematology thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
This dissertation explored different intrinsic and extrinsic factors that influence the West Nile virus (WNV) vector competence of two different colonies of Culex pipiens quinquefasciatus collected from Gainesville (CPQG) and Vero Beach (CPQV), FL. For the CPQG colony, results suggest that virus dose, extrinsic incubation temperature (EIT), and incubation period (IP) influenced infection rate, dissemination rate and body and leg titer. When EIT and IP were investigated in CPQV dissemination rate and the titer of bodies was influenced by EIT and IP. Body titers tended to be higher in mosquitoes with disseminated infections compared to mosquitoes with infections limited to the midgut in both colonies, however there were fewer CPQV mosquitoes with disseminated infections. Results from both of these studies suggest the two colonies manifest a midgut escape barrier and it is influenced by extrinsic factors under different conditions and show that vector competence involves midgut interactions with the virus . The next step was to examine the affect of WNV presence on blood meal digestion. Blood digestion is faster two days post-blood meal in CPQG given WNV. The influence of WNV on digestion rate prompted an investigation of gene expression changes, in particular, two immune response genes. One gene that was up-regulated, a Gram-negative bacteria protein-like gene, was altered in the presence of WNV compared to mosquitoes given a blood meal without virus and these changes were dependent on the colony. In addition, defensin was constitutively expressed in CPQG regardless of WNV indicating a role in general mosquito immunity. The binding of WNV to midgut proteins was investigated. West Nile virus binds to midgut proteins with the same apparent molecular weight in both CPQG and CPQV and a colony of Culex nigripalpus and also binds to midgut proteins that are different sizes. These results suggest that different species of mosquitoes may use different mechanisms for WNV entry into midgut cells. Results from this dissertation support that vector competence is a complicated process influenced by biological and molecular processes. Knowledge of these processes provides the first steps to investigating the differences in vector competence observed in mosquito populations.
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Includes vita.
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by Sheri Lizbeth Anderson.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
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Adviser: Smartt, Chelsea T.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

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INTERACTIONS BETWEEN THE MIDGUT OF Culex pipiens quinquefasciatus AND WEST NILE VIRUS FROM ENVIRONMEN TAL FACTORS TO CELL BINDING AND THE IMMUNE RESPONSE By SHERI LIZBETH ANDERSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1

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2011 Sheri Lizbeth Anderson 2

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To my dad, Anthony Bond Anderson (26 November 1947 11 January 2011) 3

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ACKNOWLEDGMENTS I would like to take this opportunity to thank my dissertation committee, C.T. Smartt, W.J. Tabachnick, S.L. Richards, C.R. Connelly, J.F. Day, and C.A. Powell. I have been very fortunate in having an intelle ctually stimulating, supportive, and motivating mentor in C.T. Sm artt. I am grateful to t he University of Florida for supporting me as a student through an Alumni Awar d. I am also grateful to the Florida Department of Agriculture and Cons umer Services grant (grant 00084365) and the National Institutes of Health grant (gr ant AI42164) for supporting my research. I am thankful to C. Lord, W. J. Tabachnick, C.R. Connelly J.F. Day, S.L. Richards and C.T. Smartt for allowing me to build upon the studies they began at the Florida Medical Entomology Laboratory. I am thankful that S.L. Richards, C.T. Smartt, and S.A. Yost were great teachers, collaborators, and friends. I would like to especially thank C.T. Smartt and S.A. Yost fo r the many long hours and late nights of mosquito midgut dissections. I would like to thank the members of the Smartt lab who were always supportive and helpful: T. Stenn, S. Guimaraes, and J. Erickson. I also want to thank the many people for helping me with mosquito r earing: S. Ortiz, H. Lynn, T. Stenn, T. Hope, H. Robinson, C. Westbrook, and C. Vitek. I also thank my friends (and their dogs): Evelien Van Ekert, Sulley Beh-Mahmoud, Erik Blosser, Stacey Clune, Meaghan Beaty, Amy Katherine, Bethany Bolling, Nikki Marlenee, Krystle Reagan, Janice Maloney, and family: Anthony Bond Anderson Jr., Sheryl Ann Anderson, Jamie Lee, Lua Ca t, and Gabriel Anderson for support. I welcome you to enjoy the Tales of a Schizophrenic Graduate Student. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................4 LIST OF TABLES ............................................................................................................8 LIST OF FIGURES ..........................................................................................................9 ABSTRACT ...................................................................................................................10 CHAPTER 1 INTRODUCTION AND REVIEW OF THE LI TERATURE.......................................12 Introductory Statement ............................................................................................12 Mosquitoes : Culex pipiens quinquefasciatus and Culex nigripalpus ......................13 Culex pipiens quinquefasciatus Say .................................................................14 Culex nigripalpus Theobald ..............................................................................15 West Nile Virus .......................................................................................................15 General West Nile Virus Transmission Cycle ...................................................16 Viral Characterization .......................................................................................16 Viral Invasion and Replication ..........................................................................17 West Nile Virus Genetics ..................................................................................18 Intrinsic Factors Affecting Vector Competence .......................................................18 The Mosquito Midgut ........................................................................................19 Mosquito Blood Digestion .................................................................................20 Infection and Transmission Barriers .................................................................22 Midgut infection barriers .............................................................................23 Midgut escape barriers ..............................................................................23 Mosquito Viral Receptors ........................................................................................25 Additional Factors Affe cting Vector Competence ....................................................27 Gene Expression ....................................................................................................29 Mosquito Immunity ..................................................................................................33 Immune Signaling Pathways ............................................................................33 Ribonucleic acid interference pathway .......................................................34 Toll pathway ...............................................................................................35 Immune deficiency pathway .......................................................................36 Janus kinase/signal transducor and activator of transcription pathway ......37 Immune response inhibition and gene expression changes ......................37 2 EFFECTS OF WEST NILE VIRU S DOSE AND EXTRINSIC INCUBATION TEMPERATURE ON TEMPORAL PROGRESSION OF VECTOR COMPETENCE IN Culex pipiens quinquefasciatus SAY (DIPTERA: CULICIDAE )...........................................................................................................40 Materials and Methods ............................................................................................41 5

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Mosquitoes .......................................................................................................41 Mosquito Infection ............................................................................................41 Mosquito Processing ........................................................................................42 Statistics ...........................................................................................................43 Results ....................................................................................................................43 Blood Meals and Freshly Fed Mosquitoes ........................................................43 Infection Rates .................................................................................................43 Dissemination Rates ........................................................................................44 Body and Leg Titer ...........................................................................................44 Discussion ..............................................................................................................45 3 Culex pipiens quinquefasciatus SAY (DIPTERA: CULICIDAE) MIDGUT ESCAPE BARRIER IS INFLUENCED BY EXTRINSIC INCUBATION TEMPERATURE AND I NCUBATION PERIOD......................................................51 Materials and Methods............................................................................................53 Mosquitoes....................................................................................................... 53 Mosquito In fection............................................................................................53 Mosquito Pr ocessi ng........................................................................................54 Statistical Analyse s..........................................................................................54 Result s....................................................................................................................55 Blood Meal Titer and Fres hly Fed Mosquitoes .................................................55 West Nile Virus Infection and Dissemination Rates Between Incubation Periods and Extrinsic In cubation Tem peratur es............................................55 The Effect of Incubation Period and Extrinsic Incubation Temperature on the West Nile Virus Titer in Bodi es and Legs................................................55 Discussio n..............................................................................................................56 4 BLOOD DIGESTION IN Culex pipiens quinquefasciatus SAY (DIPTERA: CULICIDAE) AFTER INFECTION WITH WEST NI LE VI RUS................................63 Materials and Methods............................................................................................65 Mosquitoes....................................................................................................... 65 Blood Meal Pr eparatio n....................................................................................65 Digestion Rate and Vect or Compet ence..........................................................66 Mosquito Pr ocessi ng........................................................................................66 Statistical Analys is............................................................................................67 Result s....................................................................................................................68 Virus Titer of Blood Meals and Freshly Fed Mo squitoes..................................68 Blood Digestion Rates in Uninfected Versus West Nile Virus-Infected Mosquitoes....................................................................................................68 Effects of Virus Dose on Early Infe ction...........................................................68 Effects of Virus Dose on Body and Leg Tite rs..................................................69 Effects of Virus Dose on Infe ction and Disseminat ion Rates............................69 Discussio n..............................................................................................................69 6

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5 THE EFFECT OF WEST NILE VIRUS INFECTION ON THE MIDGUT GENE EXPRESSION OF Culex pipiens quinquefasciatus SAY (DIPTERA: CULICIDAE )...........................................................................................................75 Materials and Methods............................................................................................76 Virus .................................................................................................................76 Mosquitoes....................................................................................................... 77 Sequence A nalyses ..........................................................................................77 Mosquito In fection............................................................................................77 Mosquito Midgut Dissection and Semi-Quantitat ive RT-P CR...........................78 Quantitative Reverse Transcription Polymerase Chai n Reacti on.....................79 Result s....................................................................................................................79 Sequence A nalysis ...........................................................................................79 Virus Titers of Blood M eals...............................................................................80 Experiment One...............................................................................................80 G1A1 temporal gene expression in midguts of Culex ppiens quinqufasciatus fed meals containing West Nil e virus or no virus...........80 G43A2 temporal gene expr ession in midguts of Culex pipiens quinqufasciatus fed meals containing WNV or no virus..........................81 Virus titers of mo squito mi dguts.................................................................81 Experiment Two...............................................................................................81 G1A1 temporal gene expression in midguts of Culex pipiens quinquefasciatus Gainesville and Culex pipiens quinquefasciatus Vero Beach fed meals containing West Nile virus or no virus.................81 G43A2 temporal gene expr ession in midguts of Culex pipiens quinquefasciatus Gainesville and Culex pipiens quinquefasciatus Vero fed meals containing West Nile virus or no virus............................82 Virus titers of mo squito mi dguts.................................................................82 Discussio n..............................................................................................................83 6 WEST NILE VIRUS-BINDING PROTEINs IN THE MIDGUT OF Culex pipiens quinquefasciatus SAY AND Culex nigripalpus THEOBALD (DIPTERA: CULICIDAE ).........................................................................................................102 Materials and Methods..........................................................................................103 Mosquitoes and Midgu t Dissecti ons...............................................................103 Protein Quantification and 1-D Electrophor esis..............................................104 Virus Overlay Binding Prot ein Assay (V OBPA)..............................................104 Results ..................................................................................................................105 Discussio n............................................................................................................105 7 GENERAL CONCLUSIONS AND FUTURE DIRE CTIONS..................................108 LIST OF REFE RENCES.............................................................................................115 BIOGRAPHICAL SKETCH ..........................................................................................129 7

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LIST OF TABLES Table page 2-1 The mean West Nile virus (WNV) titers (log10 pfu/mL) SE ................................49 2-2 Analysis of Variance results of Culex pipiens quinquefasciatus (CPQG) body 3-1 The mean West Nile virus (WNV) titers (log10 pfu/mL) SE ................................61 3-2 Analysis of Variance results of Culex pipiens quinquefasciatus (CPQV) body and leg titer (log10 pfu West Nile virus/mL) ........................................................62 4-1 The description of the Sella scale stages of bl ood digesti on ................................72 4-2 The digestion rate (mean standard error) 72 4-3 The West Nile virus titers ( pfu/mL) (mean standard error) 73 4-4 Analysis of Variance results of Culex pipiens quinquefasciatus ...........................73 8

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LIST OF FIGURES Figure page 4-1 The West Nile virus body titer (log10 pfu/mL) (mean standard error) for mosquitoes given a high or low WNV dose and held at 28 C f..........................74 5-1 Multiple protein sequence al ignments of a fragment of the Culex pipiens quinquefasciatus putative Gram-negative bacteria binding (GNBP) protein.......88 5-2 Multiple protei n sequence alignments of the complete sequence of the Culex pipiens quinquefasciatus putative defensin,G43A2 (100 amino acids) with defensin proteins from other mos quitoes ............................................................90 5-3 The RNA integrity of Culex pipiens quinquefasciatus midguts by gel electrophor esis...................................................................................................91 5-4 G1A1 gene expression analysis Culex pipiens quinquefasciatus midguts by semi-quantitativ e RT-P CR..................................................................................92 5-5 G43A2 gene expression analysis in Culex pipiens quinquefasciatus (CPQG) midguts by semi-quant itative RT -PCR................................................................93 5-6 Experiment one, Culex pipiens quinquefasciatus (CPQG) midgut West Nile virus titer at different incubat ion periods afte r infection.......................................94 5-7 Experiment two: The RNA integrity of Culex pipiens quinquefasciatus (CPQG and CPQV) midguts by semi-quantitative RT-PCR using CQ actin.......95 5-8 G1A1 gene expression analysis in mi dgut tissue from two populations of Culex pipiens quinquefasciatus (CPQG and CP QV)..........................................97 5-9 G43A2 gene expression analysis in midgut tissue from two populations of Culex pipiens quinquefasciatus (CPQG and CP QV)..........................................99 5-10 Experiment two, Culex pipiens quinquefasciatus (CPQG and CPQV) midgut West Nile virus titers at different in cubation periods afte r infection...................101 6-1 Culex pipiens quinquefasciatus (CPQG, CPQV) and Culex nigripalpus (CNG) midgut protein analysis by 1-D electrophoresis ................................................107 7-1 This figure shows the potential interaction between intr insic..............................114 9

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy INTERACTIONS BETWEEN THE MIDGUT OF Culex pipiens quinquefasciatus AND WEST NILE VIRUS FROM ENVIRONMEN TAL FACTORS TO CELL BINDING AND THE IMMUNE RESPONSE By Sheri L. Anderson August 2011 Chair: Chelsea T. Smartt Major: Entomology and Nematology This dissertation explored different intrinsi c and extrinsic factors that influence the West Nile virus (WNV) vector compet ence of two different colonies of Culex pipiens quinquefasciatus collected from Gainesville (CPQG) and Vero Beach (CPQV), FL. For the CPQG colony, results suggest that viru s dose, extrinsic incubation temperature (EIT), and incubation period (IP) influenced in fection rate, dissemination rate and body and leg titer. When EIT and IP were invest igated in CPQV dissemination rate and the titer of bodies was influenced by EIT and IP. Body titers tended to be higher in mosquitoes with disseminated infections com pared to mosquitoes wit h infections limited to the midgut in both colonies, however there were fewer CPQV mosquitoes with disseminated infections. Results from both of these studies suggest the two colonies manifest a midgut escape barrier and it is in fluenced by extrinsic factors under different conditions and show that vector competence invo lves midgut interactions with the virus The next step was to examine the affect of WNV presence on bloo d meal digestion. Blood digestion is faster two days pos t-blood meal in CPQG given WNV. 10

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The influence of WNV on digestion rate prompted an investigation of gene expression changes, in particular, two imm une response genes. One gene that was upregulated, a Gram-negative bacteria protein-like gene, was altered in the presence of WNV compared to mosquitoes given a bl ood meal without virus and these changes were dependent on the colony. In addition, def ensin was constitutively expressed in CPQG regardless of WNV indicating a ro le in general mosquito immunity. The binding of WNV to midgut proteins was investigated. West Nile virus binds to midgut proteins with the same apparent mo lecular weight in both CPQG and CPQV a nd a colony of Culex nigripalpus and also binds to midgut protei ns that are different sizes. These results suggest that different spec ies of mosquitoes may use different mechanisms for WNV entry into midgut cells. Results from this dissertation support t hat vector competence is a complicated process influenced by biological and mole cular processes. Knowledge of these processes provides the first steps to investi gating the differences in vector competence observed in mosquito populations. 11

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CHAPTER 1 INTRODUCTION AND REVIEW OF THE LITERATURE Introductory Statement Vector competence is the ability of the mosquito to become infected with and transmit a virus (Hardy et al 1983, Tabachnick 1994). Becaus e the mosquito midgut is the first point of contact for an arbovirus when a mosquito takes an infectious blood meal (Hardy et al. 1983), the interaction between the midgut and the virus is an integral step in the transmission potential that impacts vector competenc e. Biological processes in the mosquito (i.e. intrinsic factors) c ontribute to vector competence and include several barriers that the virus must cross before it can be transmitted (Black et al. 2002). Processes outside of the mosquito (i.e extrinsic factors) such as virus dose (Kramer et al. 1981, Richards et al. 2007a, Anderson et al. 2010), extrinsic incubation temperature (EIT) (Kramer et al. 1983, Ric hards et al. 2009, 2010, Anderson et al. 2010), and incubation period (IP) (Dohm et al. 2002, Kilpatri ck et al. 2008, Anderson et al. 2010) also impact vector competence. To further complicate matters, it is known that different species and populations (Lorenz et al. 1984, Tabachnick et al. 1985, Richards et al. 2009, 2010) of mosquitoes show diffe rential vector competence, however, the genes and molecular processes that contribute to the variation in vector competence at the level of the mosquito midgut are largely unknown. Many studies have focused on the impact of a single intrinsic or extrinsic factor on a mosquitos vector competence (Kramer et al. 1981, 1983, Richards et al. 2007a), however it has been demonstrated by studies that examine multiple factors that these factors can interact in complex and unpredictable ways (Richards et al. 2009, 2010, 12

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Anderson et al. 2010) thus assigning the trait of vector competence to a single process is complicated. The research presented in this dissertation explores the interaction of the midgut of Culex pipiens quinquefasciatus Say with West Nile virus (WNV, family Flaviviridae genus Flavivirus) and explores the impact on vector competence. The hypothesis tested here is that in addition to intrinsi c and extrinsic factors, there are molecular processes in the mosquito that influence the vector competence of Cx. p. quinquefacsciatus for WNV. The following literatu re review provides background information about the mosquito species and the virus used in these studies, the mosquito midgut, including its role as a barrier to viral infection, explains intrinsic and extrinsic factors, and discusse s what is known to date about virus binding receptors, gene expression and mosquito immune respons e to viral infection (Chapter 1). Chapters 2 and 3 both examine the affect of EIT and IP on the infection and dissemination rates, and body and leg ti ters of two different colonies of Cx. p. quinquefasciatus Chapter 4 examines the impact of the presence of WNV on the rate of blood digestion in the midgut of Cx. p. quinquefasciatus Chapter 5 examines the impact of WNV on the gene expression of two immune response genes in the midgut of Cx. p. quinquefasciatus. Chapter 6 examines t he midgut proteins of Cx. p. quinquefasciatus and Cx. nigripalpus Theobald that bind to WN V. Chapter 7 is a summary of the research and di scusses future directions. Mosquitoes : Culex pipiens quinquefasciatus and Culex nigripalpus Culex spp. mosquitoes are important vect ors of arboviruses throughout the world (Hayes 1989). Saint Louis encep halitis virus (SLEV, family Flaviviridae genus Flavivirus ) and West Nile virus (WNV, family Flaviviridae genus Flavivirus ) cause 13

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symptoms ranging from flu-lik e to encephalitis and death in humans and death in horses (Hayes 1989). Throughout their distribution, both Culex pipiens quinquefasciatus and Culex nigripalpus are considered prim ary vectors of SLEV (Chamberlain et al. 1964, Dow et al. 1964, Sudia and Chamberlain 1964, Moore et al. 1993, Lillib ridge et al. 2004) and WNV in the United States (Blackmore et al. 2003, Rutledge et al. 2003, Vitek et al. 2008). Culex pipiens quinquefasciatus Say Culex pipiens quinquefasciatus is distributed throughout the southern United States below 36 N (Barr 1957). It is found in Alabam a, Arizona, California, Colorado, Florida, Georgia, Illinois, Indiana, Iowa, Kansas, Kent ucky, Louisiana, Maryland, Mississippi, Missouri, Nebraska Nevada, North Carolina, Ohio, Oklahoma, South Carolina, Tennessee, Texas, Utah, Virginia, and West Virginia (Dar sie and Ward 2005). Culex p. quinquefasciatus is an anthropophilic mosquito found in water with high quantities of organic matter and artifici al containers (Barr 1967). Female Cx. p. quinquefasciatus is an opportunistic feeder and will feed on a variety of bird and mammal species, including humans, which contri butes to its significance as a vector (Apperson et al. 2004, Zinser et al. 2004, Molaei et al. 2005). Culex pipiens quinquefasciatus female adults are medium-sized brown mosquitoes with darker brown tarsi, thoraces, and proboscises (Darsie and Ward 2005). The head is light brown with antennae about the same length as the proboscis (Darsie and Ward 2005). The abdomen has basal pale, narrow, rounded bands that resemble half-moons (Darsie and Ward 2005). 14

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Culex nigripalpus Theobald Culex nigripalpus is distributed throughout the southeastern United States occurring in Arkansas, Florida, Georgia, Kentucky, Louisiana, North Carolina, South Carolina, and Texas (Darsie and Ward 2005) with abundant populations occurring in central and southern Florida (Nay ar 1982). Mosquitoes of this species will oviposit in a variety of habitats including acid cypre ss swamps, salt marshes (Provost 1969) and containers such as untreated pools and tires, although they prefer to oviposit in intermittently-flooded, shallow freshwater habi tats (Nayar 1982, Day and Curtis 1994). Culex nigripalpus shows a seasonal shift in host preference, with preferential feeding on birds in the spring and early summer and swit ching to feeding on a higher proportion of mammals in the late summer and fall (Edman and Taylor 1968). Such a switch in host preference makes this species an ideal amplification vector as well as placing it into contact with humans and horses for epidemic and epizootic transmission of SLEV and WNV (Day 2001, 2005) and a bridge vector for eastern equine encephalitis virus (Nayar 1982). Culex nigripalpus females are medium-sized brow n mosquitoes with dark brown legs that have reflective bronze to me tallic blue-green scales (Nayar 1982). The proboscis is dark (Nayar 1982). The abdomen has brown to black scales with reflective bronze to metallic blue-green scales. Occa sionally, there are narrow white basal bands present on some abdominal segments (Nayar 1982). West Nile Virus West Nile virus is endemic to Africa, Asia, Australia, and Europe (Brinton 2002, Kramer et al. 2008, Brault 2009) West Nile virus has re-emerged in recent years causing substantial epidemics all over the world, includi ng epidemics in France, Israel, 15

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Italy, Morocco, Romania, Russia, and the United States since 1996 (Peterson and Roehrig 2001). West Nile vi rus spread from the United States to the Caribbean, Canada, Central and South Amer ica, and Mexico (Lanciotti et al. 1999, Hayes et al. 2005, Komar and Clark 2006, Morales et al. 2006) and can now be found on every continent except Antarctica (Kramer et al. 2008). General West Nile Virus Transmission Cycle In North America, WNV has a broad range of vectors and hosts. It has been isolated from over 62 species of mosquitoes (CDC 2007) in 11 genera (Brault 2009) and 284 species of birds (Kramer et al. 2008), alligators (Rodrigues and Maruniak 2006, Unlu et al. 2010), and squirrels (Tiawsiris up et al. 2010, Padgett et al. 2007) which allows WNV a broad geographical distribution. In the enz ootic cycle, WNV is passed between passerine birds and ornithophilic mosquitoes in the genus Culex. Different Culex spp. are the primary vectors of WNV in different regions of the United States. In the northeastern and north central states, Culex pipiens pipiens L. is the primary vector of WNV (Savage et al. 1999). Culex tarsalis Coquillett is the primary WNV vector in the western states (Bolling et al. 2007, Kramer et al. 2008). Culex pipiens quinquefasciatus is the primary vector of WNV in the southern states (Lillibridge et al. 2002) and Cx. nigripalpus is an important vector in Florida (Blackmore et al. 2003, Rutledge et al. 2003, Vitek et al. 2008). Viral Characterization West Nile virus was first isolated from a woman in Uganda in 1937 (Smithburn et al. 1940). West Nile virus is classified in the family Flaviviridae genus Flavivirus and belongs to the Japanese enceph alitis virus serogroup (Calisher et al. 1989). This 16

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serogroup includes SLEV, Murray Valley encephal itis virus, Japanese encephalitis virus (JEV), dengue virus (DENV), and yellow fever virus (Mackenzie et al. 2002). Viral Invasion and Replication Flaviviruses have icosahedral symmetr y with a 40-50 nanometer (nm) diameter and are surrounded by a host-derived envelope covered with glycoprotein polymers. The genome consists of 11 kilobase pairs (kb) of positive-sense RNA (Linderbach and Rice 2001, Mukhopadhyay et al. 2005). The open reading frame codes for one polyprotein, which is further cleaved into three structural proteins: core protein, membrane protein, and envelope protein; and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Linderbach and Rice 2001, Mukhopadhyay et al. 2005). Arboviruses usually replicate within mosquito tissues prior to being transmitted to a host in what is known as biological transmission (Higgs and Beaty 2005). In rare instances an arbovirus may be mechanically transmitted to a host as a result of being present on the mouthparts without actually replicating within the mosquito (Higgs and Beaty 2005). Biological transmission occurs after a mosquito ingests an infectious blood meal. Virions attach to mosquito cellular receptor s on midgut epithelial cells via glycoproteins on the virus envelope, i.e. virus attachment protei ns (VAPs) (Linderbach and Rice 2001, Salas-Benito et al. 2007). The virus penetrates the cell by receptormediated endocytosis (Chu et al. 2006) or pinocytosis, although, lysosomes can also uncoat the virus once it has entered the cell (Brinton 2002). The positive-sense RNA is released and translation of non-structural proteins is required for transcription and replication to occur. After the virus enters th e mosquito midgut cells, viral titers fall for a short period of time known as the eclipse phase (McLean 1955, White and Fenner 17

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1994). Subsequently the titer increases due to viral replication and, depending on the mosquito-virus system, the mosquito may be in fected for its entire lif e at a stable titer (McLean 1955, Hardy et al. 1983, Mellor 2000). Conversely, some mosquitoes may not be infected their entire lives (Girard et al. 2005) at a stable titer (Smartt et al. 2009, Anderson et al. 2010). Once replication begi ns, the virions assemble and bud from the cell membrane and the virus is infective to other cells at this point (Hardy et al. 1983, Brinton 2002). West Nile Virus Genetics The WNV introduced into New York in 1999 (Lanciotti et al. 1999) was most closely related to a lineage I strain that origi nated in Israel (Ebel et al. 2001). The strain was termed NY99 (Lanciotti et al. 1999). In 2001, a single nucleotide mutation resulted in a more virulent form of WNV, termed WN02 (Ebel et al. 2004, Davis et al. 2005). This mutation (NS3-249) has arisen independent ly at least three times (Brault 2009). est Nile virus 02 (WN02) is transmitted earlier by Culex pipiens pipiens L. and Culex tarsalis Coquillet (Moudy et al. 2007, Kilpatrick et al. 2008) and is more virulent in mice (Jerzak et al. 2005) and birds (Brault et al. 2004) compared to NY99. New York 99 has been completely replaced by WN02 in the Unit ed States (Ebel et al. 2004, Moudy et al. 2007). Intrinsic Factors Affect ing Vector Competence Vector competence includes an arthropods su sceptibility and ability to transmit a pathogen (Hardy et al. 1983). Intrinsic vector competence includes the factors within the arthropod that permit an arthropod vector to become infected with and transmit a virus (Kramer et al. 1981, Hardy et al. 1983, De Foiart et al. 1987). In trinsic factors that affect vector competence include barriers to infection and dissemination that are 18

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influenced by both genetic and environmental fa ctors (Hardy et al. 1983, Tabachnick 1994, Bosio et al. 1998, 2000, Beerntsen et al. 2000), and endogenous mosquito substances such as digestive enzymes (Hardy et al. 1983). The Mosquito Midgut The mosquito midgut consists of a narro w anterior region, a flask-like posterior region and the midgut epit helial cells surrounded by basal la mina (Clements 1992). It is considered the first formidable barrier to vector competence in a mosquito. This is in part due to the fact that blood digestion occurs in the mosquito midgut (Hardy et al. 1983, Clements 1992). When a virus is inges ted with a blood meal, the virus infects and multiplies within posterior midgut epithelial cells (Whitfield et al. 1973). The distribution and number of cells that become infected can vary from species to species and due to the type of virus (Whitfield et al. 1973, Hardy et al. 1983). Once virus has entered the posterior midgut with the blood meal, virions attach to cellular receptors, penetrate the cells, and are presumably uncoated by membrane fusion (Hardy et al. 1983). Initiation of viral infection in the midgut requires a high enough viral titer to infect midgut cells (Bates and Roca-Garcia 1946, Chamberlain and Sudia 1961, Jupp 1974, Kramer et al. 1981, Hardy et al. 1983) and ingestion of a low virus titer may result in lower infection, disseminati on, and transmission in some mosquitoes (Kramer et al. 1981). The initial virus titer needed to infe ct midgut cells can vary for the same virus in different mos quitoes (Chamberlain et al. 1959), different viruses in the same mosquito species (Hardy et al. 1983), and between individual mosquitoes (Kramer et al. 1981). Once the virus has entered the midgut epithelial cells, the titer of virus in the mosquito drops and remains low for a short period of time and then peaks within a few days (McLean 1955, Hardy et al. 1983). Once virus has begun 19

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replicating, it must reach a high enough level to disseminat e out of the midgut (Kramer et al. 1981, Hardy et al, 1983). Virions mu st pass through the basal lamina of the epithelial cells unidirectionally from the luminal to the abluminal region (Hardy et al. 1983). Viruses acquire an envelope during ma turation occurs by crossing through the basal lamina and varies by virus and mosqui to species during dissemination (Hardy et al. 1983). Once the virus has escaped into the hemocoel, secondary amplification in different cells and tissues can occur (Whitfiel d et al. 1973, Hardy et al. 1983, Girard et al. 2004) before the salivary glands bec ome infected with the virus. West Nile virus escape the posterior midgut epithelial cells in Cx. p. quinquefasciatus after replicating to a high enough titer (Kramer et al. 1981) and proceed to infect the fat body in t he abdomen, thorax, and head, anterior midgut epithelial cells, and muscle tissue (Girard et al. 2004). The fat body is considered the most important tissue for secondary amplificat ion to occur (Girard et al. 2004). Salivary glands become infected at approximately the same time as anterior midgut epithelial cells and WNV virions spread from cell to cell in the posterior direct ion from the cardia epithelium to the anterior midgut epithelial cells (Girard et al. 2004). Similar to midgut epithelial cells, few salivary gland cells in t he lateral lobes become infected (Whitfield et al. 1973, Girard et al. 2004) and the virions mo ve from the distal portion of the gland to the apical portion (Hardy et al. 1983). Mosquito Blood Digestion Digestion of vertebrate blood in female mosquitoes involves proteolytic enzymes that cleave proteins used in vitellogenesis and egg development (Clements 1992). The midgut is the primary site of blood digestion in mosquitoes. Blood digestion is a two20

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step process, whereby proteins are cleaved by endopeptidases into large peptides. These large peptides are furt her cleaved by exopeptidases. The endopeptidases are trypsin and chymotry psin. Trypsins are serine proteases (Clements 1992) and there ar e at least seven isoforms in the midgut of Aedes aegypti (L.) (Brackney et al. 2010). The S1.A subfamily of serine proteases includes trypsin, chymotrypsin, collagenase, and elastase (Blo w 1997). Trypsin is the principal enzyme involved in blood digestion (Clements 1992) and is secreted in a biphasic manner, early (1-3 hour (h) and late (8-36 h post-blood meal (hpbm)) in Ae. aegypti (Felix et al. 1991). In Cx. p. quinquefasciatus late trypsin starts increasing in the posterior midgut 6 hpbm, peaks at 36 h, and declines through 72 hpbm (Okuda et al. 2002) upon completion of the gonotrophic cycle when eggs ar e mature and can be ovipos ited (Okuda et al. 2002, Garcia-Rejon et al. 2008). Chymotrypsin is pr esent at low levels during blood digestion in adult mosquitoes (Borovsky 1986, Clements 1992, Okuda et al. 2002). In Cx. p. quinquefasciatus chymotrypsin is secreted about 6 hpbm, peak at about 36 hpbm, declines to low levels by 48 hpbm, and ce ases production by 72 hpbm (Okuda et al. 2002). Other enzymes involved in blood protein digestion include aminopeptidases and elastases. In Cx. p. quinquefasciatus aminopeptidase activity begins soon after the blood meal is imbibed (Okuda et al. 2002). Activity of aminopeptidase mimics that of chymotrypsin, peaking at 36 hpbm and declines slowly until it is gone 72 hpbm (Okuda et al. 2002). Elastase levels begin build ing 6 h pbm, peak 36 hpbm, and disappear by 72 hpbm (Okuda et al. 2002). Esterases and glycosidases also contribute to blood 21

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digestion since there are lipids and sugars pres ent in blood as well as protein (Clements 1992). After imbibing a blood meal, the peritrophic matrix (PM) forms around the meal to protect the midgut epithelium either from mechanical da mage or pathogens (Clements 1992). The PM is fully formed by 18 hpbm in Cx. p. quinquefasciatus (Okuda et al. 2002). It is known that proteolytic digestive enzymes, such as trypsin, in the midgut interact with arboviruses including La Crosse virus (LACV, family Bunyaviridae genus Bunyavirus ) (Ludwig et al. 1989) and dengue virus (DENV, family Flaviviridae genus Flavivirus ) (Molina-Cruz et al. 2005). In Ae. aegypti trypsin increases DENV replication in the midgut and dissemination out of the midgut into the hemocoel (Molina-Cruz et al. 2005). Virions also influence the formation of the PM. The PM begins forming earlier (1 hpbm) and is thicker at 6 and 12 hpbm in Ae. aegypti fed a DENV-infected blood meal compared to mosquitoes given an uninfected blood meal (Suwanmanee et al. 2009). In this case, DENV presence results in modifica tions of the PM structure, suggesting that virus presence in the blood meal influences physiological processes involved in PM formation whichsuggests that the presence of virus in the blood meal of mosquitoes changes the physiology of the mosquito. Infection and Transmission Barriers Basic barriers to arbovirus infection and transmission in mosquitoes include: 1) midgut infection barriers (MIB), 2) midgut escape barriers (MEB), 3) salivary gland infection barriers (SIB), and 4) salivary gland escape barriers (SEB) (Hardy et al. 1983). The extent of these barrier s depends on intrinsic and extrinsic factors and differs for 22

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different virus-vector systems. The following examples illustrate observations for the midgut barriers in different mosquitoes infected with different arboviruses. Midgut infection barriers A midgut infection barrier (MIB) results when a pathogen fails to replicate within the midgut epithelial cells and this is the mo st frequently reported ba rrier to arboviral infection in mosquitoes (Kramer et al. 1981, Hardy et al. 1983). This barrier accounts for the refractoriness of some species of mosquitoes for an arbovirus (Kramer et al. 1981). Hypotheses to explain the MIB include: 1) inactivation of the virus by digestive enzymes, 2) absence of viral receptors in th e midgut epithelial cells, or 3) abortive replication of the virus in midgut epithelial cells. A MIB can be bypassed by inoculating mosquitoes with virus directly into the hemocoel i.e. intrathoracic inoculation, so that the virus can replicate and disseminate to ot her tissues including the salivary glands (Houk et al. 1986). Mosquitoes that ar e susceptible to infection and capable of transmitting the virus when inoc ulated but not when orally fed an infectious blood meal, demonstrate a MIB. For example, Cx. tarsalis is a competent vector of western equine encephalitis virus (WEEV, genus Togaviridae family Alphavirus ), whereas Cx. p. pipiens is refractory to the same virus (Houk et al. 1986). When Cx. p. pipiens is inoculated intrathoracically with WEEV, mo squitoes become infected at approximately the same rate as Cx. tarsalis demonstrating that the MIB in Cx. p. pipiens is caused by the inability of WEEV to absorb, penetrate, re plicate and/or escape the midgut epithelial cells. Midgut escape barriers Arboviruses may be trapped by the midgut epit helial cells, fail to replicate to high enough levels in midgut cells, or abortive replic ation occurs resulting in a midgut escape 23

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barrier (MEB) which prevents the arbovirus fr om exiting the midgut and disseminating to other tissues and organs (Kramer et al. 1981, Hardy et al. 1983, Black et al. 2002). Three hypotheses may explain the presence of a MEB. 1) The initial virus titer may be below some threshold needed to infect, multip ly and escape the midgut epithelial cells, 2) although the virus can multiply in the mi dgut epithelial cells, it fails to escape the midgut (Kramer et al. 1981), 3) abortive replication caused by defective interfering particles or other host factor s occurs (Hardy et al. 1983). Evidence supporting the presence of a MEB is the observation of low number of virions disseminated to the legs and lo w transmission rate despite a high midgut infection rate (Kramer et al. 1981, Hardy et al. 1983). Virus in legs of mosquitoes with infected bodies indicates that the virus has disseminated (Kramer et al. 1981, Turell 1984); e.g. has escaped the midgut. In additi on, intrathoracic inoculation of a virus resulting in transmission can also support t he presence of an MEB to a particular virus just as it does the presenc e of a MIB (Paulson et al. 1989). Aedes triseriatus (Say) given a blood containing LACV have a high rate of midgut infection (Paulson et al. 1989) but show lo w dissemination to the salivary glands and therefore an MEB. Intr athoracic inoculation of Ae. triseriatus with LACV bypasses the MEB as shown by the increase in transmission rate. Culex tarsalis infected with low titers of WEEV di splay midgut barriers to infection (Kramer et al. 1981, Mahmood et al. 2006), although the MEB is more important because WEEV fails to escape the midgut and disseminate when midgut epithelial cells do become infected. These findings indicated that in some mosquito species-arbovirus relationships a dose-dependent MEB may play an important role in vector competence. 24

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Mosquito Viral Receptors To isolate potential viral receptors for mosquito-borne viruses, cell culture, affinity chromatography, and various virus overlay methods have been used. However, few putative viral receptors have been identified in mosquitoes. Several factors contribute to the difficulty in isolating viral receptors from mosquitoes including: 1) different kinds of molecules can act as receptors (Mendoza et al. 2002), 2) multiple receptors may be used by a single virus for entry into a cell, 3) different viruses can use the same receptor to enter a cell, 4) different tissues can have different viral receptors for entry into cells (Ren et al. 2007), and 5) different serotypes can use different receptors (de Lourdes Muoz et al. 1998, Reyes-del Valle et al 2004). Candidate cellular receptors have been identified for DENV (de Lourdes Muoz et al. 1998, Mendoza et al. 2002, Reyesdel Valle et al. 2004, Mercado-Curiel et al 2006, 2008, Liu et al. 2008), WNV (Chu et al. 2004, 2005, Ren et al. 2007), and Japanese encephalitis virus (JEV, family Flaviviridae genus Flavivirus ; Chu et al. 2005, Ren et al. 2007, Das et al. 2009). Many of these receptors have been identified us ing mosquito cell culture. Candidate receptor proteins for the binding and entry of WNV into Aedes albopictus (Skuse) C6/36 cells include four protei ns that are 140 kDa, 95 kDa, 70 kDa, and 55 kDa in size (Chu et al. 2005). The 140 kDa and 55 kDa proteins had a lower affinity for binding WNV than the 95 kDa and 70 kDa proteins indicating that these proteins may only associate with the receptor complex. Polyclonal antibodies against the 95 kDa and 70 kDa proteins inhibited ent ry of WNV into the C6/36 cells and because they also interact with WNV, JEV, and DENV-2 in C6/36 cells they may form part of a general mosquito-flavivirus binding complex (Chu et al. 2005). 25

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In addition to the 95 kDa and 70 kDa proteins that allow DENV-2 entry into C6/36 cells (Chu et al. 2005), other putative rec eptors of DENV include 45 kDa and 40 kDa proteins that bind DENV-4 in C6/36 cells (Reyes-del Valle et al. 2004), the brush boarder membrane of the midgut of Ae. albopictus (Liu et al. 2008), and mosquito extracts from larval, p upal, midgut, salivary gland, and ovarian tissues of Ae. aegypti (Mendoza et al. 2002). Other pr oteins that have been identif ied as putative receptors for DENV include a 80 kDa and an 67 kDa protein found to bind DENV-2 in C6/36 cells (de Lourdes Muoz et al. 1998). All four se rotypes of DENV also bind to 80 kDa and 67 kDa proteins in C6/36 cells and mo squito midgut extracts from Ae. aegypti (MercadoCuriel et al. 2006). All four serotypes also bind to 77 kDa, 58 kDa, 54 kDa, and 37 kDa proteins in salivary gland extracts from Ae. aegypti (Cao-Lormeau 2009). Dengue virus1 and DENV-4 bind to 67 kDa, 56 kDa, 54 kDa, 50 kDa, and 48 kDa proteins in salivary extracts from Aedes polynesiensis Marks (Cao-Lormeau 2009). Flavivirus binding protein studies indicate that: 1) different tissues may have different proteins that bind to viruses (de Lourdes Muoz et al. 1998, Cao-Lormeau 2009), 2) different viruses may bind to differ ent proteins (de Lourdes Muoz et al. 1998, Chu et al. 2005), and 3) different virus sero types may bind to different proteins (de Lourdes Muoz et al. 1998, Reyes-del Valle et al. 2004). The converse also exists and the same sized proteins in different tiss ues may bind to the same virus (de Lourdes Muoz et al. 1998, Mercado-Curiel et al. 2006, 2008). Although observations support the possibility of general flavivirus receptor s (Chu et al. 2005, Ren et al. 2007), there have been no investigations to confirm this hypothesis. 26

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Midgut proteins isolated from three colonies of Ae. aegypti that differ in DENV-2 vector competence (Bosio 1998, 2000, Bennett et al. 2005) were identified using virus overlay binding protein analysis (VOPBA) and two-dimensional electrophoresis (Mercado-Curiel et al. 2008). The colonies ar e known to be susceptible to (no barriers), refractory to (MIB), or rest ricted to the midgut (MEB) for DENV-2 infection. Virus overlay binding protein analysi s revealed that midguts of Ae. aegypti (susceptible and refractory strains) had a prot ein of apparent molecular weight of 67 kDa that bound to DENV-2. The colony having DENV restricted only to the midgut had a binding protein of 64 kDa. Using polyclonal antibodies against these proteins, immunoblots showed that the protein (67/64 kDa) was the same in each colony. Specific antibodies generated to the 67/64 kDa protein were conjugated to a fluorescent antibody and examined using confocal microscopy. This showed that t he proteins bound to the basal lamina of the midgut. These are the first results to s uggest the presence of a protein molecular marker for vector compet ence (Mercado-Curiel et al 2008) since this protein is the same putative receptor already characteriz ed for DENV (Mercado-Curiel et al. 2006). Additional Factors Affect ing Vector Competence Factors such as extrinsic incubation tem perature (EIT), virus dose, and mosquito age have also been shown to influence vector competence in complicated ways (Hardy et al. 1983, Reisen et al. 2006, Richards et al. 2007a, 2009, 2010, Anderson et al. 2010). Extrinsic incubation temperature does not affect all mosquito-virus associations in the same manner (Dohm et al. 2002). In general, Cx. p. quinquefasciatus infected with WNV (Richards et al. 2007b, 2010, Ande rson et al. 2010) and SLEV (Hurlbut 1973, Richards et al. 2009) showed higher rates of infection and dissemination when held at higher EITs than their cohor ts held at lower EITs. Culex tarsalis was also found to 27

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transmit WNV more efficiently when held at hi gher EITs (Reisen et al. 2006, Kilpatrick et al. 2008). Culex pipiens pipiens infected with WNV also showed higher rates of infection and dissemination when held at hi gher EIT and dissemination occurred earlier than in the group held at the highest EI T compared to groups held at lower temperatures (Dohm et al. 2002, Kilpatrick et al. 2008). Culex pipiens pipiens were also able to transmit Rift Valley fever virus (RVFV, family Flaviviridae genus Flavivirus ) earlier when held at higher EITs compared to mosquitoes that were held at lower EITs (Turell et al. 1985). However, higher EIT does not always increase infection and dissemination. The converse has also been reported. Culex tarsalis were less competent vectors of WEEV when held at higher EITs co mpared to mosquitoes held at lower EITs (Kramer et al. 1983) and the impact of EIT changes over time (Kilpatrick et al. 2008, Anderson et al. 2010). Viral dose also influences vector com petence in complex ways (Richards et al. 2007a, 2009, 2010). Several studies illustrate this complexity For example, when viral doses were less than 3.7 logs 10 plaque-forming units (pfu) WNV/mL, Cx. p. quinquefasciatus infection and dissemination rates were much lower than mosquitoes that were given doses of 5.8 logs 10 pfu WNV/mL demonstrating that virus dose can have an effect. However, when the same colony of Cx. p. quinquefasciatus were fed blood meals containing freshly propagated WNV (Anderson et al. 2010), mosquitoes fed either a higher (7.0 logs 10 pfu WNV/mL) or lower (5.9 logs 10 pfu WNV/mL) dose did not exhibit differences in the infection rates. However the dissemination rates were lower in mosquitoes given the lower virus dose (A nderson et al. 2010). Feeding previously frozen virus results in lower infection and dissemination rates compared to feeding 28

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freshly propagated virus showing that even t he condition of the virus preparation can influence vector competence (Richards et al. 2007b). The age of the adult mosquito at the time of virus infection also influences vector competence. The influence of mosquito age illustrates the complexity of environmental effects. Different environmental factors c an influence the effects of other genetic and environmental factors on vector competence in a complex m anner. For example, the effect of mosquito age was dependent on the origin of the colony tested (Richards et al. 2009, 2010), type of virus, EIT, and viru s dose for two different colonies of Cx. p. quinquefasciatus infected with SLEV or WNV. There are differences in how the MIB and MEB respond to temperature and age since at different ages and different EITs, the dissemination rates for Cx. p. quinquefasciatus were different from the infection rates. These observations demonstrate the complexity of the effects of the environment on vector competence for WNV. Richards et al (2009) observed similar complexities with SLEV. Gene Expression Identifying the specific mosquito genes that influence vector competence has been a long sought objective in vector biolog y. An understanding of the specific genetic mechanisms and the contributing non-genetic env ironmental factors influencing vector competence is essential for providing gr eater predictability about which species and vector populations pose health threat s and under which circumstances (Tabachnick 1994). However, reaching this objective is very difficult and currently, although it is clear that both genetic and non-genetic processes are involved, the specific processes remain elusive in all vector-pathogen systems. For example, the susceptibility of a mosquito to a particular virus may have many different genes contributing to infection 29

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(Tabachnick, 1994, Beerntsen et al. 2000). Since an individuals phenotype for susceptibility to infection or transmission can be characteri zed quantitatively, i.e., the amount of virus in the indivi dual varies quantitatively, th e phenotype is likely influenced by several genes that can be considered quantit ative trait loci (QTL) (Bosio et al. 1998, 2000, Tabachnick 1994). The QTL correla ting to DENV-2 competence have been mapped in two colonies of Ae. aegypti (Bosio et al. 1998, 2000, Bennett et al. 2005). Two QTL influence a MIB and one QTL influences a MEB for DENV-2. There may be as many as three QTL involved in dissemination of DENV-2 in Ae. aegypti (Bennett et al. 2005). Genes that contribute to viru s-vector competence remain unknown and research is ongoing to elucidate these genes. One way to approach identifying genes infl uencing vector competence is to evaluate gene expression differences between infected and uninfected individuals (Morlais et al. 2003, Sanders et al. 2005, Smar tt et al. 2009, Bartholomay et al. 2010). An important starting tool for gene expression studies is having the organisms complete genome sequence. The comp lete genome sequences of Anopheles gambiae Giles (Holt et al. 2002), Ae. aegypti (Nene et al. 2007), and Cx. p. quinquefasciatus (Arensburger et al. 2010) are available and gene sequences can be found in web-based databases. Genome sequences enable comparative phylogeno mic analyses between three genera of mosquitoes and Drosophila melanogaster Meigen (Arensburger et al. 2010, Bartholomay et al. 2010). Tools such as microarray analysis (Sanders et al. 2005), differential display analysis (Morlais et al. 2003, Smartt et al. 2009), high throughput gene expression, and reverse geneti cs (Xi et al. 2009) can elucidate gene changes in response to pathogen infection. 30

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Transcript expression in the midguts of susceptible and refractory Ae. aegypti provided a non-infectious blood m eal and a blood meal containing Plasmodium gallinaceum were compared and twenty-two messages were observed to be expressed differently (Morlais et al 2003). Eight sequences were mapped between two QTL that have been shown to be related to susceptibility to Plasmodium infection in Ae. aegypti A comparision between refractory and susc eptible mosquitoes showed that seven genes were differentially expressed. Differ entially expressed genes can result from over-expression, differential regulation, sex-specificity, and blood digestion (Morlais et al. 2003). In addition to parasite infection, diffe rential gene expression has been examined in mosquitoes infected with virus. The transcription level of midgut genes was changed when Ae. aegypti was fed a blood meal containing Sindbis virus (SINV, family Togaviridae genus Alphavirus) compared to mosquitoes provided an uninfected blood meal (Sanders et al. 2005). Changes included al tered levels of transcripts that produce chitin-binding proteins, vesicle transporters, and innate immunity molecules, such as Toll-like receptors. The midguts of Cx. p. quinquefasciatus exposed to a 6.8 logs 10 pfu WNV/mL blood meal showed 26 cDNAs whose expressi on differed from mosquitoes given an uninfected meal. Twenty-one of the comple mentary DNAs (cDNAs) were up-regulated and five were down-regulated after virus ingesti on (Smartt et al. 2009). Four of the cDNAs have been sequenced and three have s equence similarities to cDNAs found in mosquito immune response pathways: G12A2 (94% identity with a Cx. p. quinquefaciatus leucine-rich repeat containing protein and 32% identity to Toll-like 31

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receptors from Ae. aegypti Smartt et al. 2009), G43A 2 (100% identity with a Cx. p. quinquefaciatus defensin A-like protein; unpublished), G1A1 (98% identity with a Cx. p. quinquefaciatus Gram-negative bacteria-binding prot ein; unpublished), and G35A (98% identity with a Cx. p. quinquefasciatus chorion peroxidase; unpublished). Temporal gene expression studies show that G12A2 message changes in Cx. p. quinquefasciatus midguts that have been exposed to WNV compared to mosquitoes given uninfected blood meals. The increases in message ex pression corresponded to incubation periods in which WNV midgut titer was lowest (Smartt et al. 2009). When the genomes of An. gambiae Ae. aegypti, and Cx. p. quinquefasciatus were compared, 500 immunity genes in 39 families were identified in Cx. p. quinquefasciatus which included C-type lectins, fibrinogen-rela ted proteins, and serine protease inhibitors (Bartholomay et al. 2010). Genome-wide microarray anal ysis revealed an increase in gene transcription in WNV-infected Cx. p. quinquefasciatus (Bartholomay et al. 2010). In the midgut, 22 transcripts were changed 7 dpi and 309 transcripts were changed in whole bodies (Bartholomay et al. 2010). At 14 dpi, 539 infection response genes were changed in the midgut and 490 were changed in t he carcass (Bartholomay et al. 2010). Components of the Toll, immune deficiency (Imd), and Janus kinase signal transducers (JAK-STAT) pathway were among the genes ac tivated (Bartholomay et al. 2010). These gene expression studies suggest that mosquito innate immunity has a role in virus infection. Genes involved in immune pathways have been shown to be activated in Ae. aegypti infected with Plasmodium (Morlais et al. 2003), SINV (Sanders et al. 2005), and DENV (Xi et al. 2010). Onyong nyong virus (ONNV, family Togaviridae genus Alphavirus ) activated immunity genes in An. gambiae (Sim et al. 32

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2007) and WNV activated immune response genes in Cx. p. quinquefasciatus (Smartt et al. 2009, Bartholomay et al. 2010). These results also suggest that mosquitoes use multiple immune pathways against different types of viruses in addition to ribonucleic acid interference (RNAi) (Keene et al. 2004, Khoo et al. 2010). Mosquito Immunity Immune Signaling Pathways Immune signaling pathways direct immune responses to a range of pathogens in insects (Cirimotich et al. 2010). Three pat hways (i.e. Toll, Imm une Deficiency [Imd] and Janase Kinase-Signal Transducer and Trans ducer of Transcription [JAK/STAT]) originally described in Drosophila melanogaster have been identified in An. gambiae (Christophides et al. 2002). In addition, the RNAi pathway has also been characterized in mosquitoes (Keene et al. 2004, Sanchez-Vargas et al. 2004, Khoo et al. 2010). Innate immunity may play a role in limiting virus infection (Xi et al. 2009) and vector competence in mosquitoes (Keene et al. 2004, Smartt et al. 2009, Xi et al. 2010). The insect immune system is comprised of defense mechanisms that are triggered by pattern recognition receptor (PRR) molecule s which bind to specif ic features of the pathogens, known as pathogen-associated molecu lar patterns (PAMPs ) (Warr et al. 2008). Pattern Recognition Receptors c an bind directly to pathogens and cause phagocytosis, an encapsulation response, or t hey can trigger a serine protease cascade that will end in the production of antimicrobial peptides (A MPs). Two serine protease pathways have been characterized in Drosophlia : Toll and Imd. In general, mosquitoes have a more complicated immune response than Drosophila (Cirimotich et al. 2010). Gram-positive bacteria and fungi activate the Toll pathway and Gram-negative bacteria activate the Imd pathway in Drosophila (Brandt et al. 2008). The Toll and Imd pathways 33

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are not specifically activated by Gram-pos itive bacteria, Gram-negative bacteria, and fungi in mosquitoes (Cirim otich et al. 2010). Several immune pathways are induced when mosquitoes are exposed to arbovir uses including RNAi (Keene et al. 2004, Sanchez-Vargas et al. 2004, Khoo et al. 2010) JAK/STAT, Toll, and Imd (Fragkoudis et al. 2009, Souza-Neto et al. 2009). Ribonucleic acid interference pathway The ribonucleic acid interference (RNAi) pathway is a natural immune pathway that has been shown to be involved in arbovirus infection in mosquitoes. Interfering RNA (RNAi) can degrade the double-stranded RNA (dsRNA) replication-intermediate of RNA viruses (i.e. alphavirues and flavivirus es) and keep the cell from becoming infected (Travinity et al. 2004, Sanchez-Vargas et al. 2004). Long pieces of dsRNA are introduced into an organism in the form of viral genomes (Travanty et al. 2004). The dsRNA is recognized by the Rnase III enzym e Dicer. Dicer cleaves the dsRNA into small pieces called small interfering RNAs (siRNA). Double-stranded siRNA molecules are unwound by helicase and integrated in to the RNA-induced silencing complex (RISC). The complex guides the Argonaut e endonuclease to the target messenger RNA (mRNA) for cleavage. The benefit to the cell is destru ction of dsRNAs that are often associated with viral replication. The first example of arbovirus inhibiti on by the RNAi pathway was observed in An. gambiae infected with ONNV (Keene et al. 2004). When mosquitoes were co-injected intrathoracically with ONNV-e green fluore scent protein (eGFP), a recombinant ONNV with a GFP gene inserted for easy visualizat ion, along with dsRNA from the ONNV genome, viral replication was inhibited. Mosquitoes co -injected with ONNV-eGFP and 34

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dsRNA derived from An. gambiae Argonaute2 (AnAGO2) to inactivate part of the RISC complex were more permissive to infection and dissemination. To implicate components of the RNAi pathway in the anti-viral defense mechanism, Dicer2 ( Drc2 ) and Argonaute2 ( Ago2 ) were silenced in mosquitoes (Campbell et al. 2008). Aedes aegypti mosquitoes with silenced genes and control mosquitoes with active genes were cha llenged with SINV. Mosquitoes that had silenced RNAi components had high levels of vi rus infection. Control mosquitoes had levels of siRNA consistent with activation of the RNAi pathway (Campbell et al. 2008). When the RNAi pathway was selectively impaired in the Ae. aegypti midgut during blood feeding on a SINV infected meal, th e MIB and MEB were impaired (Khoo et al. 2010). Mosquitoes had higher intensity of in fection, higher infection rates, and higher dissemination rates compared to control mosquitoes (Khoo et al. 2010). Evidence suggests that the RNAi pathway is suppress ed by alphaviruses during infection (Khoo et al. 2010). Toll pathway Both the Toll and Imd pathway lead to the production of immuno-inducible effector molecules also known as antimicrobial peptides (AMPs). More than 20 effector molecules in seven classes have been identified in Drosophila (Lemaitre and Hoffmann 2007). Effector molecules in Drosophila include: drosomycins and metchnikowin (active against fungi), defensin (active against Grampositive bacteria), attacins, cercropins, drosocin, and diptericins (active agains t Gram-negative bacteria) (Hoffmann 2003). The Drosophila Toll pathway is activated by the binding of a secretion recognition molecule (i.e. peptidoglycan recognition protein-SA (PGRP-SA or SD), Gram-negative bacteria binding protein 1 (GNBP1), or Gr am-negative bacteria binding protein-3 35

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(GNBP3) to the Toll receptor (Lemaitre and Hoffmann 2007). This causes the activation of Spetzle processing enzyme (SPE) which cleaves Spetzle. Mature Spetzle binds to Toll allowing a dimmer to form at the plasma membrane which recruits Death Domain-containing proteins (i.e. MYD88, Tube, and Pelle). Through an unknown mechanism, Cactus is phosphorylated and degraded by a proteosome. This releases Dorsal and/or Dif which are translocated to the nuclease. Dorsal and Dif act as transcription factors to trigger the production of drosomycin and other AMPs. The Toll pathway in mosquitoes does behave differently (Cirimotich et al. 2010). The mosquito Toll pathway can be activated by fungi, Grampositive bacteria, viruses (Xi et al. 2009), and Plasmodium (Frolet et al. 2006). And the pathway uses REL1, which is an ortholog of Drosophila Dorsal (Cirimotich et al. 2010). When the gene expression prof iles of DENV-infected Ae. aegypti bodies and midguts were examined 10 days post-infection (dpi), many functional gene classes were differentially expressed, incl uding components from the Toll pathway (X i et al. 2009). There was a stronger and br oader gene response in bodies 10 dpi compared to midguts, indicating that the virus has reac hed peak levels in the body tissue and is declining in the midgut. Rel1, the Ae. aegypti ortholog of Dif ( Drosophila ), is upregulated in infected mosquitoes compared to uninfected mosquitoes. Immune deficiency pathway In Drosophila the Immune Deficiency (Imd ) pathway is activated by the binding of diaminopimelic-type peptidoglycans (DAPtype PGN) to PGRP-LC or LE (Hoffmann 2003, Lemaitre and Hoffmann 2007, Costal et al 2009). PGRP-LC or LE interact with RIP (receptor-interacting protein) homotypic interaction motif-like motifs (RHIM). Through an unknown signal adaptor protein, Imd is activated and the pathway is 36

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bifurcated to converge again and activate Relish. On one fork, Drosophila transforming growth-factor activated kinase (Dtak1) activates Kenny (IKK ) or Ird5 (IKK). Kenny or Ird5 phosphorylates Relish. On the other fork, Drosophila Fas-associated death domain protein (Dfadd) interacts with the caspase (D redd). Dredd cleaves Relish, releasing Rel from its negative regulator, Casper. Rel translocates into the nucleus and acts as a transcription factor to trigger the production of effector mo lecules which are AMPs such as defensins. These are the peptides that act on invaders such as bacteria, fungi, and Plasmodium in mosquitoes. Janus kinase/signal transducor and act ivator of transcription pathway The Janus Kinase/Signal Transducer and Ac tivator of Transcription (JAK/STAT) pathway (Souza-Neto et al. 2009) in Drosophila is initiated by the extracellular binding of the unpaired ligand (Upd) to the rec eptor Domeless (Dome) which causes a conformational change and the self phospohyla tion of the JAKs (Hop). This activates Hop which, in turn, phosphorylates Dome. The result is docking sites which induce the phophorylation and dimerization of STAT. STAT is translocated into the nucleus of the cell where it induces transcription of spec ific target genes. There are two negative regulators of STAT: 1) protein inhibitor of activated STAT (PIAS) and 2) suppressor of cytokine signaling (SOCS). Orthologs to the JAK/STAT pathway have been isolated from Ae. aegypti Drosophila orthologs of the negative regulators PIAS and SOCS (SUMO) have also been isolated from Ae. aegypti (Souza-Neto et al. 2009). Immune response inhibition and gene expression changes Research suggests that the antivir al immune response is complicated in mosquitoes and that several pathways are in volved. When the receptor (Dome) and the JAKs (Hop) of the JAK/STAT pat hway are silenced using dsRNA, Ae. aegypti become 37

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more susceptible to DENV infection whereas mosquitoes in which the negative regulator (PIAS) is knockeddown using dsRNA are more resistant to DENV infection (Souza-Neto et al. 2009). The same can be seen when components of the Toll pathway are silenced. When Cactus, the negative regul ator of Dif, is silenced with dsRNA in Ae. aegypti DENV infection is suppressed in the midgut, supporting the Toll pathway involvement in DENV antiviral im mune responses (Xi et al. 2009). Aedes aegypti immune-competent cells, Aag2, were used to detect small changes in gene expression to DENV infection (Sim and Dimopolous 2010). Viral infection activated components of the Toll pathway. Dengue virus infected cells that were challenged with Gram-positive and Gram-negative bacteria were less capabl e of producing defensin and cecropin and bacterial cells were able to grow to a higher optical density compared to uninfected cells (Sim and Dimopolous 2010). Dengue titers were not changed in cells challenged with Gram-negative bacteria compared to cells that were infected with DENV, but unchallenged with bacteria, supporting the hypot hesis that DENV may suppress the Toll pathway rather than evade it. Aag2 cells challenged with Gram-negative bacteria resulted in higher DENV titers compared to ce lls that were unchalle nged. This supports evidence that the Imd pathway (activated by Gram-positive bacteria) plays a role in regulating the replication of DENV (Sim and Domopolous 2010). This is further supported by evidence that silencing Casper in Ae. aegypt i increased midgut viral titer (Xi et al. 2009). Early in DENV infection in Ae. aegypti several effector molecules including, defensin-A and C are down-regulated 24 h and 3 days post-infection (dpi) (Ramirez and Domopolous 2010). When Cactus in silenced by dsRNA, the DENV titer in midguts is lower 3 dpi compared to mosq uitoes injected with double-stranded green 38

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fluorescent protein (dsGFP). When MyD88 is silenced with dsRNA, midgut DENV titer is higher compared to dsGFP-injected mo squitoes (Ramirez and Domopolous 2010). This is further evidence t hat the Toll pathway plays an im portant role in mosquito antiviral immunity. In Cx. p. quinquefasciatus infected with WNV, significant changes are evident in 22 transcripts in the midgut 7dpi (Bartholomay et al. 2010) At 14 dpi, several genes related to Toll, Imd, and JAK/STAT pathway s are activated in WNV-infected mosquitoes (Bartholomay et al. 2010). Surprisingl y, no components of the RNAi pathway were activated 7 or 14 dpi (Bartholomay et al. 2010) This supports the findings of others that more than one pathway may respond to arbovirus infection in mosquitoes (Souza-Neto et al. 2009, Xi et al. 2009, Bartholomay et al. 2010, Ramirez and Domopolous 2010, Sim and Domopolous 2010). This review summarizes the peer-reviewed scientific literature concerning the complex nature of vector competence. T he remaining chapters will examine the vector competence of Cx. p. quinquefasciatus females for WNV and expands on the accepted view of factors that influence vector compet ence by also examining the influence of WNVbinding midgut proteins and the i mmune response of mosquitoes to WNV infection. 39

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CHAPTER 2 EFFECTS OF WEST NILE VIRUS DOSE AND EXTRINSIC INCUBATION TEMPERATURE ON TEMPOR AL PROGRESSION OF VECTOR COMPETENCE IN CULEX PIPIENS QUINQUEFASCIATUS SAY (DIPTERA: CULICIDAE) West Nile virus (WNV, family Flaviviridae genus Flavivirus ) is cycled between wild birds and ornithophilic mosquitoes in the genus Culex (Hayes 1989, Day 2005). Culex pipiens quinquefasciatus Say have been found infected with WNV in the field (Rutledge et al. 2003, Godsey et al. 2005), is a compet ent laboratory vector of WNV (Sardelis et al. 2001, Goddard et al. 2002), and is consid ered an important WN V vector in the United States. Vector competence is influenced by both ex trinsic and intrinsic factors (Hardy et al. 1983). Extrinsic factors in clude extrinsic incubation temper ature (EIT) (Hardy et al. 1983, Dohm et al. 2002) and virus dose (Krame r et al. 1981). Biol ogical factors that influence vector competence include mosquito species (Goddard et al. 2002) and virus strain (Moudy et al. 2007), and can be different for different mosquito populations (Richards et al. 2009). Extrin sic and intrinsic factors may also influence the extrinsic incubation period (EIP) and affect vector competence (Hardy et al. 1983, Dohm et al. 2002, Reisen et al. 2006, Kilpatrick et al. 2008). The EIP begins when a virus is ingested with a blood meal. The virus infe cts mosquito midgut epithelial cells, disseminates out of the midgut, and the EI P ends when the mosquito is capable of transmitting the virus to a susceptible host. Culex pipiens quinquefasciatus given a high WNV dose in the laboratory showed higher infection and dissemination rates co mpared to mosquitoes given a low WNV dose (Sardelis et al. 2001, Richards et al. 20 07a). There is also a positive relationship between extrinsic incubation temperatur e (EIT) and WNV vector competence for Cx. p. 40

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pipiens L. (Dohm et al. 2002), Culex tarsalis Coquillet (Reisen et al. 2006), and Cx. p. quinquefasciatus (Richards et al. 2007a) with increasing vector competence associated with higher EITs. Dissemination of WNV to ti ssues outside the midgut of Cx. p. quinquefasciatus has been found as early as three days post-infe ction (dpi) (Girard et al. 2004), and at four dpi (Kilpatrick et al. 2008) depending on vari ous factors including mosquito species, mosquito population, viral dose, and EIT. The objective of this study was to determine how viral dose and EIT affect temporal changes in vector competence, here represented by WNV infection and dissemination in Cx. p. quinquefasciatus Materials and Methods Mosquitoes Culex pipiens quinquefasciatus (F >45 ) collected from Gainesville, FL (CPQG) were maintained at 28 C and 70% RH on a 14:10 L:D cycle as described previously (Richards et al. 2007a). Larvae were mainta ined on a slurry containing 20 g/L of 1:1 liver powder: brewers yeast. Approximately 120 fourto sixday old female mosquitoes were placed into one-liter cardboard cart ons (Dade Paper Company, Miami, FL) with mesh screening for the duration of the expe riment. Adult mosquitoes were fed 20% sugar and water ad libitum Mosquito Infection Mosquitoes were blood fed as descri bed elsewhere under biosafety-level 3 conditions (BSL-3; Richards et al. 2007a) wit h the exception that the virus used was freshly propagated in African gr een monkey kidney cells (Vero) cell culture (Anderson et al. 2010). The Florida WNV isolat e (WN-FL03p2-3) (D oumbouya 2007, Genbank accession no. DQ983578) used was passaged once in baby hamster kidney cells and 41

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four times in African green monkey kidney (V ero) cells. Mosquitoes were allowed to feed for 30 min on cotton pledgets soaked with a high or low dose of WNV mixed with citrated bovine blood (Hemostat, Dixon, CA) that had been warmed (35 C) for 10 min. Virus doses used were within the range of viremias commonly found in WNV infected birds in Florida (Komar et al. 2003). Two aliquots (0.1 mL each) of the heated blood were placed into separate tubes of 1 mL of BA-1 diluent prior to mosquito feeding and stored at -80 C for viral titer analysis. Subsequent to feeding, mosquitoes were immobilized with cold and 110 fully engorged s pecimens per dose were transferred to cages, provided 20% sugar solution ad libitum and maintained in incubators at 28 C or 25 C for the duration of the experiment. Whole bodies of five freshly fed mosquitoes were each placed in separate tubes containi ng 1 mL BA-1 diluent with two 4.5 mm zincplated beads and stored at -80 C until tested for virus titer (Richards et al. 2007a). Mosquito Processing At the end of each IP, 4, 6, 8, and 12 dpi, the bodies and legs (combined) of approximately 20 mosquitoes were placed in to separate tubes containing 1 mL BA-1 diluent and two 4.5 mm zinc plated beads us ing previously described sterile techniques until trituration followed by nucleic acid extr action (Richards et al. 2009). The amount of WNV RNA was determined using quantitative real-time Taqm an reverse transcription polymerase chain reaction (qRT-PCR) and a standard curve based upon plaque assay as previously described (Lanciotti et al. 2000; Richards et al. 2007a). Infection rate was the number of WNV-positive bodies divided by the total number of mosquitoes tested. Dissemination rate was the number of WNVpositive leg samples divided by the number of mo squitoes with infected bodies. 42

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Statistics Fishers exact tests ( = 0.05) were used to determine dose, incubation period (IP), and EIT effects on infection and disseminat ion (SAS Institute, 2002). Data were log (x + 1)-transformed and analysis of variance (ANOVA) ( = 0.05) used to determine virus dose, IP, and EIT effects on body and leg titers. The factor dissemination status was used to test the effect of dissemi nation compared to non-dissemination on total body virus titer. Significant differences were evaluated us ing Duncans multiple range test ( = 0.05) (SAS In stitute, 2002). Results Blood Meals and Freshly Fed Mosquitoes The high-dose (7.0 0.1 log 10 equivalent WNV log 10 plaque-forming unit (pfu)/mL) blood meal contained a significantly higher titer than the low-dose (5.9 0.1 log 10 pfu/mL) (F = 52.48; df = 1, 3; P = 0.019). The bodies of fres hly fed mosquitoes provided the high dose contained significantly more WNV (5.5 0.1 log 10 pfu/mL of mosquito homogenate) than low dose mosquitoes (4.2 0.1 log 10 pfu/mL of mosquito homogenate) ( F = 91.03; df = 1, 9; P = 0.001). Infection Rates Table 2-1 shows the temporal progression of infection rates, dissemination rates, body titers, and leg titers at different EITs and doses. As IP did not influence infection rates at each dose or EIT (all P > 0.05), infection rates acro ss IPs were combined. The effect of high virus dose on infection rate was observed at both EITs. Infection rates were higher for mosquitoes at 25 C fed the high dose (76/80 = 90%) compared to low dose mosquitoes (63/75 = 84%) (P = 0.033). Mosquitoes at 28 C also had higher infection rates when given the high dose (70/71 = 99%) compared to the low dose 43

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(69/77 =90%) ( P = 0.035). However, ther e was little influence of EIT on infection rates. Infection rates did not differ between the lo w doses at 25 C (76/80 = 95%) and 28 C (70/71 = 99%) ( P = 0.371) or between the high doses at 25 C (63/75 = 85%) and 28 C (69/77 = 90%) ( P = 0.345). Dissemination Rates There were more disseminated infections at later Ips at both high ( P = 0.001) and low ( P = 0.006) doses at 28 C and at the high dose ( P = 0.001) at 25 C (Table 2-1). Dissemination rates did not differ between Ips for the low dose at 25 C ( P = 0.450). However, there were more disseminated infect ions at 8 dpi for mosquitoes given the high dose ( P = 0.001) and at 12 dpi for the low dose ( P = 0.005). There were also more disseminated infections at 8 dpi for mosquitoes at 28 C ( P = 0.001) and at 12 dpi for mosquitoes at 25 C ( P = 0.004). Body and Leg Titer The ANOVA showed that body titer was signi ficantly different between Ips, doses, EITs, and dissemination status (T able 2-2). The absence of si gnificant IP*dose, IP*EIT, and EIT*dose interactions showed that diffe rences between the IPs were the same at both doses and EITs. The diffe rences between EITs were the same for both doses. Body titer increased with increasing dose, EI T, and IP as expected for virus replication in mosquito tissues. The lowest body titers were in the low dose group at 25 C at the earliest time points of 4 and 6 dpi (Table 21) consistent with the least permissive conditions. Leg titers were significantly different between IPs, but not between doses and EITs (Table 2-2). Leg titers were lower at 8 dpi for both doses at 28 C compared to 25 C and there were significantly lowe r leg titers at the high dose and the high EIT at 4 dpi. 44

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Discussion Previous studies have shown the influence of EIT and dose on WNV infection in Cx. p. quinquefasciatus (Richards et al. 2007a) and t he temporal progression of dissemination and transmission rates (Dohm et al. 2002, Kilpatrick et al. 2008). The present study also revealed that low EIT and low dose together can influence dissemination. At the low dose mosquitoes maintained at 25 C produced only one disseminated infection (Table 1). These obser vations suggest that the midgut infection (MIB) and escape barriers (MEB) were infl uenced by EIT and dose. The MEB in Cx. p. quinquefasciatus is an important factor in WNV transmission (Girard et al 2004). Dose, EIT, and mosquito age influenced the MEB in Cx. p. quinquefasciatus infected with both St. Louis encephalitis virus (Richards et al 2009) and WNV (Richards et al. 2010). Although IP did not influence infe ction in the current study, t here were significant affects on dissemination, particularly under the more permissive c onditions of high dose and high EIT. At the later IPs, there were more dissemination consistent wit h the greater probability of virions to escapi ng from the midgut and replicat ing with more time to do so. Viral dissemination at 4 dpi (28 C) in mosquitoes fed the high dose may be due to virus leaking from the mosquito midgut into the hemocoel or to rapid dissemination at higher temperatures. This observation has also been observed elsewhere (Dohm et al. 2002, Kilpatrick et al. 2008). Either cause is likely influenced by the virus dose since the earliest disseminated infections occurred in only the mosquitoes fed the high dose, regardless of EIT. Although there were temporal changes in body titer, these changes were not dependent on dose and EIT in th is study. There were hi gher total body titers in mosquitoes with disseminated infections compared to mosquitoes with non45

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disseminated infections likely, the result of only the midgut containing virus in nondisseminated infections while both the mi dgut and other tissues contain virus in disseminated infections. The absence of a significant EIT*dissemination status interaction showed that higher body titers in mosquitoes with disseminated infections compared to non-disseminated infections occu rred at both EITs. T herefore the higher body titers at 28 C were not due to t he differences between nonand disseminated infections and were most likely due to the ef fect of temperature on virus replication. The significant two way interactions between di ssemination status with dose and with IP show that the effect of dissemination stat us on titer changed with dose and with IP. This was due to the higher body titer in mo squitoes with non-disseminated infections compared to disseminated infections observed at 8 dpi in mosquitoes fed the low dose and held at 28 C. West Nile virus replicated to a higher titer in the midgut alone in nondisseminated mosquitoes compared to replicati on in both the midgut and other tissues in those with disseminated infections under th is condition. Therefore mosquitoes with disseminated infections do not always have higher total body titers than those with only midgut infections as some apparently replic ate more virus in the midgut alone than other mosquitoes do that have both midgut and disseminated infections. This study showed there are environmental conditions w here mosquitoes with a MEB contain more virus in the midgut compared to mosquitoes without a MEB. The relationship between the MEB and virus replication in the midgut requires further study. Dissemination status did not affect any of the two-way interacti ons between the other factors as shown by the lack of significance for the three-way intera ctions. The three-way interaction between 46

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IP, dose, and EIT was significant showi ng that the EIT*dose interaction changed depending on IP. Body titer increased with increasing dose, EIT, and IP as expected for virus replication in mosquito tissues. The lowest body titers were in the low dose group at 25 C at the earliest time poi nts of 4 and 6 dpi (Table 2-1) consistent with the least permissive conditions. Virus replication outside of the midgut was characterized by analyzing WNV in legs. The cause of similar leg titers between doses and EITs is unknown and requires further study. The significant IP*EIT interact ion showed that differences in leg titers between mosquitoes at different IPs were not the same at different EITs. The IP*dose and EIT*dose interactions were not signifi cant, because both the differences between Ips were the same at both doses, and both EIT and dose did not have any influence on leg titer. The three-way interaction between dose, EIT, and IP could not be calculated for leg titers as mosquitoes given the lo w dose at 25 C did not show disseminated infections at most IPs. T he analyses of WNV in legs supports the hypothesis that once virus escapes the midgut, infection of other tissues, like the leg, depends more on IP and the time allowed for replication rat her than initial dose or temperature. Knowledge of the temporal progression of infection and dissemination and the influence of environmental factors at different time points during infection are critical to understanding pathogen transmission and epidemiology. The occurrence of IP-, EITand dose-dependent progression of WNV infection in Cx. p. quinquefasciatus tissues, including effects on the MIB and MEB indicate that these factors influence vector competence under the conditions employed in th is study. Further studies are needed 47

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48 that use more factors and a wid er range of levels for each fa ctor to expand the range of environments. This will provide information that further elucidates critical events that may be overlooked in studies that focus on one or a few factors at only one or a few time points.

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Table 2-1. The mean West Nile virus (WNV) titers (log 10 pfu/mL) SE in bodies and legs of Culex pipiens quinquefasciatus (CPQG) mosquitoes initially given a high (7.0 0.1 log 10 pfu/mL) or low (5.9 0.1 log 10 pfu/mL) virus dose and incubated at 28 C or 25 C for per iods of 4, 6, 8, and 12 dpi. Means with the same letters are not significantly different for each body part. Incubation period (days) Dose No. tested Body titer (no. disseminated infections) Body titer (no. non-disseminated infections) No. infected (%) Leg titer No. disseminated (%) Extrinsic incubation temperature = 28 C 4 Low 20 5.3 0.1 (19) defg 19 (95) 0 (0) 6 Low 20 6.3 (1) abcde 5.2 0.4 (16) efgh 17 (85) 2.3 abc 1 (6) 8 Low 20 5.2 0.4 (12) fgh 5.8 0.2 (8) bcdefg 20 (100) 1.7 0.6 abc 7 (35) 12 Low 17 6.8 0.3 (7) ab 6.4 0.3 (6) abcd 13 (76) 4.6 0.3 a 7 (54) 4 High 19 6.3 (1) abcde 5.8 0.2 (18) bcdefg 19 (95) 0.5 c 1 (5) 6 High 20 6.6 0.1 (5) abc 6.4 0.1 (15) abcd 20 (100) 4.5 0.4 a 5 (25) 8 High 20 6.7 0.1 (18) abc 6.4 0.1 (2) abcd 20 (100) 3.7 0.4 ab 18 (90) 12 High 11 7.2 0.2 (9) a 6.0 0.1 (2) abcdef 11 (100) 5.0 0.2 a 8 (73) Extrinsic incubation temperature = 25 C 4 Low 20 3.3 0.3 (16) i 16 (80) 0 (0) 6 Low 20 4.0 0.1 (19) hi 19 (95) 0 (0) 8 Low 20 5.5 (1) cdefg 4.6 0.1 (15) gh 16 (80) 3.4 ab 1 (7) 12 Low 15 5.0 0.2 (12) efgh 12 (80) 0 (0) 4 High 20 6.8 (1) ab 4.7 0.1 (16) gh 17 (85) 5.0 a 1 (6) 6 High 20 6.3 0.6 (2) abcde 5.0 0.1 (18) fgh 20 (100) 3.7 1.3 ab 2 (10) 8 High 20 5.7 0.2 (4) bcdefg 5.3 0.1 (15) defg 19 (95) 3.6 0.5 ab 5 (26) 12 High 20 6.1 0.3 (9) abcdef 5.4 0.1 (11) cdefg 20 (100) 4.3 0.3 ab 10 (50) 49

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50 Table 2-2. Analysis of Variance results of Culex pipiens quinquefasciatus (CPQG) body and leg titer (log 10 pfu WNV/mL) differences for incubation periods (IPs), dose, extrinsic incubation temperatures (EITs), and dissemination status. Source df (num, denom) F P Body titer IP 3, 279 31.02 0.001 Dose 1, 279 117.87 0.001 EIT 1, 279 201.54 0.001 Dissemination Status 1, 279 4.03 0.046 IP*Dose 3, 279 2.90 0.036 IP*EIT 3, 279 2.26 0.082 EIT*Dose 1, 279 1.14 0.286 IP*Dissemination Status 3, 279 3.08 0.028 Dose*Dissemination Status 1, 279 6.77 0.010 EIT*Dissemination Status 1, 279 1.73 0.190 IP*EIT*Dose 3, 279 3.12 0.027 IP*Dose*Dissemination Stat us 2, 279 1.78 0.171 EIT*Dose*Dissemination Status 1, 279 2.56 0.111 IP* EIT*Dose*Dissemination Status 3, 279 1.47 0.223 Leg titer IP 3, 67 7.45 0.001 Dose 1, 67 2.20 0.144 EIT 1, 67 3.85 0.055 IP*Dose 3, 67 2.02 0.142 IP*EIT 2, 67 2.97 0.039 EIT*Dose 1, 67 1.59 0.213

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CHAPTER 3 CULEX PIPIENS QUINQUEFASCIATUS SAY (DIPTERA: CULICIDAE) MIDGUT ESCAPE BARRIER IS INFLUENCED BY EXTRINSIC INCUBATION TEMPERATURE AND INCUBATION PERIOD Vector competence is defined as the ability of arthropod vectors to become infected with and transmit pathogens (i.e. arbovir uses) (Kramer et al. 1981, Hardy et al. 1983, DeFoiart et al. 1987, Tabachnick 199 4). Barriers to pathogen infection and transmission in mosquitoes incl ude: 1) midgut infection barri ers (MIB), 2) midgut escape barriers (MEB), 3) salivary gland infection ba rriers, and 4) salivary gland escape barriers (Kramer et al. 1981, Tabachnick 1994, Mellor 2000, Black et al. 2002) The extent of these barriers depends on the affect of biological and environmental factors in arbovirus-vector systems (Hardy et al. 1983). The mosquito midgut is considered the first formidable barrier to vector competence in a mosquito. When an arbovirus is ingested with a blood meal, the virus must infect and multiply within epithelial cells located in the posterior midgut (Whitfield et al. 1973). To be able to observe the initiati on of viral infection in the midgut using the numbers of mosquitoes generally used in ex periments requires a high enough viral titer to overcome a threshold of infection (C hamberlain and Sudia 1961, Hardy et al. 1983, Kramer et al. 1983) and ingestion of low titers of virus may result in lower infection in some mosquitoes (Chamberlain and Sudia 1961, Kramer et al. 1983, Richards et al. 2007a). This is partly due to the lower numbers of virions associated with low titers and the associated lower probability of virions infe cting the midgut (Lord et al. 2006). After infecting midgut cells, arboviruses must es cape from the midgut cells and disseminate into the hemolymph (Kramer et al. 1981, Hard y et al. 1983, Kramer et al. 1983). The details of the mechanism of virus entry and escape from midgut cells are unclear. For 51

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example, the pore size of the basal lamina is much smaller than many virus particles (Hardy et al. 1983), and there may be an ac tive transport mechanism involved (Mellor 2000). Both MIBs and MEBs can also be in fluenced by environmental factors such as virus dose, (Kramer et al. 1981, Richards et al. 2007a, 2009, 2010, Anderson et al. 2010), mosquito age (Richards et al. 2009, 2010), and extrinsic incubation temperature (EIT) (Kramer et al. 1983, Dohm et al. 2002, Anderson et al. 2010). The influence of genetic factors was apparent in the variation in vector competence for WNV of different Culex pipiens quinquefasciatus Say colonies (Richards et al. 2009, 2010). Several studies have shown that there are interactions between many of the different factors that influence vector competenc e, i.e., the effects of tem perature on vector competence can influence the effects of virus dose and mosquito age (Richards et al. 2009, 2010). Mosquito infection and escape barriers to arboviruses play major roles in contributing to vector compet ence. The factors contributing to the different barriers likely differ between vector-virus systems. For example, an individual mosquito or a mosquito species that is competent for one vi rus may be refractory to variants of the same virus and/or different viruses (Houk et al. 1986). Different populations of the same mosquito species may also show di fferential vector competence under the same environmental conditions (Tabachnick et al. 1985) due to genetic differences contributing to vector com petence (Bosio et al. 1998, 2000, Beerntsen et al. 2000). West Nile virus (WNV, family Flaviviridae genus Flavivirus) is found on every continent except Antarcti ca (Kramer et al. 2008). Culex pipiens quinquefaciatus is considered an important vector of WNV in the southern United States (Goddard et al. 2002, Blackmore et al. 2003, Rutledge et al 2003, Vitek et al. 2008) and its vector 52

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competence differs from population to population (Reisen et al. 2008, Vanlandingham et al. 2008, Richards et al. 2010). Both genet ic and environmental factors contribute to Cx. p. quinquefasciatus vector competence for WNV. Culex pipiens quinquefasciatus from Gainesville, FL (CPQG) were more competent for WNV compared to Cx. p. quinquefasciatus from Vero Beach, FL (CPQV2007) al though this relationship is further influenced by mosquito age, EIT, and init ial virus dose (Richards et al. 2010). The objective of this study was to exami ne the influence of temperature and length of the incubation period on MEBs to WN V in a newly established colony of Cx. p. quinquefasciatus from Vero Beach, FL (CPQV) in 2008. Materials and Methods Mosquitoes Culex pipiens quinquefasciatus from a colony started orig inally from mosquitoes collected in Indian River County, Vero Beach, FL in 2008 (F 3 ), herein named CPQV colony, were maintained at 28 C and 70-75% RH on a 14:10 L:D cycle as previously described (Richards et al. 2007a). Approxim ately 100 female mosquitoes from CPQV were placed into six 1 L cardboard cages and the sugar solution was removed from mosquito cages 24 h prior to blood feeding. Mosquito Infection Fresh virus preparation was used following the methods of Richards et al. (2009) under biosafety level 3 (BSL-3) conditions. West Nile virus (strain WN-FL03p2-3, Genbank accession no. DQ983578) was mixed with citronated bovine blood (Hemostat, Dixon, CA) and heated for 10 mi n at 35 C. Two 0.1 mL a liquots of this virus-blood mixture were placed into 0.9 mL of BA-1 diluent and stored at -80 C to determine blood meal titer. Mosquitoes were fed blood meals for 30 min. at 28 C using cotton pledgets. 53

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Mosquitoes were immobilized using cold an d fully engorged mosquitoes were held at EITs of 28 C or 25 C for IPs of 4, 8, 12 days post-infection (dpi). Mosquito Processing Mosquitoes were processed using methods described elsewhere (Richards et al. 2007a, 2009). Briefly, bodies and legs were separated from mosquitoes, placed into separate tubes containing 0.9 mL BA-1 diluen t. Mosquito samples were homogenized, nucleic acids were extracted, and quantitat ive real-time Taqman reverse transcription polymerase chain reaction (qRT-PCR) was perfo rmed to determine infection rate (% of WNV-positive bodies) or dissemination rate (% of WNV-positive legs that also had infected bodies). Statistical Analyses Significant differences in infection or dissemination rates on different days between mosquitoes fed WNV and held at eit her 28 C or 25 C were determined using Fishers exact tests ( = 0.05) due to a small number of disseminated infections (SAS Institute, 2002). Scatter plots were used to determine if body and leg titer data were normally distributed. The data were log (x + 1) transformed prior to analysis to provide a normal distribution. Anal ysis of variance (ANOVA, = 0.05) was used to determine significant differences in body or leg tite r on different days between mosquitoes fed WNV (SAS Institute 2002). If titer means were significantly different, Duncans multiple range test was used to compare the m eans (SAS Institute 2002). The factor, dissemination status, was used to test the effect of di ssemination compared to nondissemination on whole body virus titer (Anderson et al. 2010). Because means comparisons for terms that interact cannot be calculated by SAS, each treatment was coded using a dummy variable so that m eans could be compared (Richards et al. 54

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2009). Single factor ANOVA with means compar isons were calculated for body and leg titer separately to enable the iden tification of different means. Results Blood Meal Titer and Freshly Fed Mosquitoes West Nile virus blood meal titer was (mean standard error) 6.9 0.1 log 10 plaque-forming equivalent units (pfu)/mL WNV. The whole bodies, including legs, of freshly fed mosquitoes contained 5.4 0.1 log 10 pfu/mL of mosquito homogenate. West Nile Virus Infection and Dissemin ation Rates Between Incubation Periods and Extrinsic Incubation Temperatures Infection rates were not diffe rent between IP s or EITs ( P > 0.05). Greater than 90% of the mosquitoes had infected bodies at all IPs and at both EITs tested (Table 31). However, dissemination rate was significantly different between IPs and EITs. There were more disseminated infections at later IPs for mosquitoes held at both 25 C ( P = 0.001, p = 0.014) and 28 C ( P = 0.001, p = 0.036). Dissemi nation rates were also higher for mosquitoes 8 ( P = 0.001, p = 0.017) and 12 days post-infection (dpi) ( P = 0.015, p = 0.036) for mosquitoes held at the hi gher EIT. Dissemination rates did not differ between EITs for mosquitoes 4 dpi ( P = 0.026, p = 0.057). The Effect of Incubation Period and Extr insic Incubation Temperature on the West Nile Virus Titer in Bodies and Legs The ANOVA showed that body titer was signi ficantly different between IPs, EITs, and dissemination status (DS) (Table 3-2) In general, body titer was higher for mosquitoes held for 12 dpi compared to mo squitoes held for 8 or 4 dpi and for mosquitoes held at 28 C compared to mo squitoes held at 25 C. The significant dissemination status (DS) effe ct was observed in the higher body titers in mosquitoes with disseminated infections co mpared to mosquitoes with nondisseminated infections. 55

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The significant two-way interaction between IP*EIT shows that the effect of EIT on body titer changed with IP. In mosquitoes held at 28 C, body titer decreased in mosquitoes with non-disseminated infections over time and remained approximately the same in mosquitoes with disseminated infections. In mosquitoes held at 25 C, the body titer increased in mosquitoes with non-disseminated infections over time and increased from 8 to 12 dpi in mosquitoes with disseminated infections. The absence of a significant EIT*DS and IP*DS interaction showed that higher body titers in mosquitoes with disseminated infections compared to those wit h non-disseminated infections occurred at both EITs at all IPs. The absence of a si gnificant three-way interaction IP*EIT*DS indicated that higher body titers in mosquitoes with disseminated infections compared to mosquitoes with non-disseminated infections was the same for both EITs at each IP. Leg titers did not differ significantly between IPs ( F = 0.320, df = 2, 48, P = 0.725) or EITs ( F = 0.02, df = 1, 48, P = 0.885). The interaction between IP*EIT was also not significant ( F = 1.60, df = 2, 48, P = 0.215), indicating that l eg titer was the same for both EITs at each IP. Discussion The MEB constitutes an important barri er to transmission in mosquitoes (Chamberlain and Sudia 1961, Kramer et al. 1981, Hardy et al. 1983, Black et al. 2002, Richards et al. 2007, 2009, 2010). When fed an infectious blood meal containing 6.9 0.1 log 10 pfu/mL WNV, greater than 90% of th e CPQV colony mosquitoes became infected with WNV regardless of IP (4, 8, and 12 dpi) and EIT (28 C and 25 C) (Table 3-1). Therefore, at this dose of virus, the CPQV colony does not have a significant MIB. In a previous study, greater than 85% of Cx. p. quinquefasciatus established from field collections from Gainesville, FL, herein CPQG colony, fed an equivalent 7.0 0.1 log 10 56

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pfu/mL WNV-containing blood meal and held at 28 C or 25 C became infected 4, 8, and 12 dpi (Anderson et al. 2010). In addition, the CPQG colony fed 7.1 0.1 log 10 pfu/mL WNV and held at 28 C all become in fected 13 dpi (Smartt et al. 2010). These results indicate that CPQV and CPQG colony mosquitoes have consistent rates of infection if given similar initial virus doses (6.9 0.1 log 10 pfu/mL-7.1 0.1 log 10 pfu/mL). At 4 and 8 dpi at 28 C there were mo re mosquitoes with non-disseminated infections (83%, 68%; respecti vely) than mosquitoes with disseminated infections (18%, 32%; respectively). At 12 dpi, there were mo re mosquitoes with disseminated infections compared to non-disseminated infections (60% 40%; respectively). The CPQG colony mosquitoes also had more non-disseminated infections at 4 dpi (95%) than disseminated (5%) (Anderson et al. 2010). At 8 dpi, however, there were more CPQG colony mosquitoes with disseminated infect ions (90%) compared to mosquitoes with non-disseminated infections ( 10%). At 12 dpi, there were also more disseminated infections (82%) than non-di sseminated infections (18%). The higher percentage of non-disseminated infections than disseminated in fections in CPQV colony mosquitoes at 4 and 8 dpi compared to CPQG colony mos quitoes at 4 and 8 dpi represents a MEB that is influenced by IP and potentially represents a difference in vector competence in these two colonies of mosquitoes. Theref ore the MEB is influenced by other factors whose characterization is beyond the sc ope of these two studies, i.e., genetic differences between the colonies. Culex pipiens quinquefasciatus (CPQV) colony body titers in mosquitoes with disseminated infections at 28 C were not di fferent between IPs, while mosquitoes with non-disseminated infections showed lower body titers 8 and 12 dpi compared to non57

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disseminated infections at 4 dpi (Table 3-1), indicating that virus levels are declining possibly due to apoptosis of midgut cells as seen in Cx. p. quinquefasciatus salivary glands (Girard et al. 2005). These findings we re similar to body titers for CPQG colony mosquitoes held under similar conditions (Anderson et al. 2010). Culex pipiens quinquefasciatus (CPQG) colony body titers fo r mosquitoes with disseminated infections were similar 4 and 8 dpi and in creased 12 dpi, while mosquitoes with nondisseminated infection showed similar body tite rs at all IPs (Anderson et al. 2010). These results suggest that in these coloni es under these conditions the virus must replicate in the midgut to a high enough titer (5.9 0.1 log 10 pfu/mL) in order to disseminate out of the midgut and that mosquitoes that do no t reach this level will not develop disseminated infections. This hypothesis is consistent wit h the observation that dissemination is less likely at early IPs sinc e time is required to reach the appropriate virus titer. The differences between the colonies could be the result of differences in the time course of virus replication dynamics in each colony and/or differences in the levels of virus needed to escape from the midgut in each colony. Culex pipiens quinquefasciatus (CPQV) colony body titers for mosquitoes held at 25 C with disseminated infections were higher 4 and 12 dpi compared to 8 dpi, which could be due to the small sample sizes observed in the disseminated mosquitoes at all time points in the 25 C treatment group. CPQV colony body titers for mosquitoes held at 25 C with non-disseminated infections increased from 4 to 8 dpi and from 8 to 12 dpi, although titers were never above (5.9 0.1 log 10 pfu/mL. The observations at 25 C are consistent with the hypothesis that the virus must rep licate to a high enough titer to disseminate and that this is dependent on EIT. 58

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The percentage of disseminated CPQV colony individuals was higher for mosquitoes held at 28 C compared to mo squitoes held at 25 C for all IPs. Dissemination rate was higher for both 25 C and 28 C at 8 and 12 dpi compared to 4 dpi. These results are comparable to other results (Richards et al. 2007a, Anderson et al. 2010, Smartt et al. 2010) that have found that dissemi nation rate can be lower depending on conditions such as initial viru s dose, EIT, and IP and may depend on the colony of Cx. p. quinquefasciatus tested. Other studies with Cx. p. quinquefasciatus have found higher rates of WNV dissemination (Dohm et al. 2002, Vanlandingham et al. 2007, Kilpatrick et al.2008) fu rther supporting the hypothesis that different vector populations of mosquitoes have different vector competences (Tabachnick 1994, Black et al. 2002, Reisen et al. 2008). There was no difference in the leg titers in the CPQV colony at different EIT for all IPs. This further supports the hypothesis t hat there is a limit to the titer of WNV in the legs which is consistent with other st udies (Richards et al. 2009, 2010, Anderson et al. 2010). This may be due to small numbers of mosquito legs or due to a small amount of material present in leg tissues. Results from this study suggest that the CPQV colony differs in its WNV vector competence from the CPQG colony. The CP QV colony has a MEB that differs when mosquitoes are held at different EITs and ov er time post infection as evidenced by differences in dissemination rates and body tite rs of those with disse minated infections vs non-disseminated infections (Table 3-1). T hese differences can be further examined to determine if there are differences in infection (midguts alone) and dissemination rates 59

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60 (legs) in the different Cx. p. quinquefasciatus populations and to tease out the mechanisms involved in the MEB in these mosquitoes.

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61 Table 3-1. The mean West Nile virus (WNV) titers (log 10 pfu/mL) SE in bodies and legs of Culex pipiens quinquefasciatus (CPQV) mosquitoes initiall y given a WNV (6.9 0.1 log 10 pfu/mL) virus dose and held at 28 C or 25 C for extrinsic incubat ion periods of 4, 8, and 12 days post-infect ion. Means with the same letters are not significantly different for each body part. Incubation period (days) No. tested Body titer (no. disseminated infections) Body titer (no. non-disseminated infections) No. infected (%) Leg titer No. disseminated (%) Extrinsic incubation temperature = 28 C 4 40 6.2 0.2 (7) ab 6.1 0.1 (33) ab 40 (100) 2.8 0.6 a 7(18) 8 40 6.1 0.1 (12) ab 5.9 0.1 (25) abcd 37 (93) 3.5 0.4 a 12(32) 12 40 6.0 0.1 (24) abc 5.4 0.1 (16) bcde 39 (98) 4.5 0.3 a 17(44) Extrinsic incubation temperature = 25 C 4 40 6.3 (1) ab 4.8 0.1 (39) e 40 (100) 4.3 a 1 (3) 8 40 4.9 0.2 (2) de 5.2 0.1 (38) cde 38 (95) 3.4 0.3 a 2 (5) 12 40 6.5 0.3 (8) a 5.9 0.1 (32) abc 40 (100) 3.3 0.7 a 10 (25)

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Table 3-2. Analysis of Variance results of Culex pipiens quinquefasciatus (CPQV) body and leg titer (log 10 pfu West Nile virus/mL) di fferences for IP, EIT, and dissemination status. P-values in bold are significant. Source df (num, denom) F P Body titer IP 2, 234 4.98 0.008 EIT 1, 234 4.47 0.036 Dissemination Status 1, 234 9.14 0.003 IP*EIT 2, 234 10.34 <0.001 IP*Dissemination Status 2, 234 1.88 0.156 EIT*Dissemination Stat us 1, 234 0.43 0.511 IP*EIT*Dissemination St atus 2, 234 2.85 0.060 Leg titer IP 2, 48 0.32 0.725 EIT 1, 48 0.02 0.885 IP*EIT 2, 48 1.60 0.215 62

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CHAPTER 4 BLOOD DIGESTION IN CULEX PIPIENS QUINQUEFASCIATUS SAY (DIPTERA: CULICIDAE) AFTER INFECTION WITH WEST NILE VIRUS Digestion of vertebrate blood in female mo squitoes involves proteolytic enzymes, such as trypsin and chymotrypsin, which clea ve proteins used in vitellogenesis and egg development (Clements 1992). After imbibi ng a blood meal, the peritrophic matrix (PM) forms around the meal to protect the midgut epithelium eit her from mechanical damage or pathogens (Clements 1992). The PM is fully formed by 18 h pbm in Culex pipiens quinquefasciatus Say (Okuda et al. 2002). During formation of the PM, levels of trypsin and chymotrypsin start increasing in the posterior midgut 6 hours post-blood meal (hpbm), peak at 36 h, and decline through 72 hpbm (Okuda et al. 2002) upon completion of the gonotrophic cycle when eggs are mature and can be oviposited (Okuda et al. 2002, Garc ia-Rejon et al. 2008). After a mosquito feeds on an infectious host, virions enter the mosquito midgut and infect posterior midgut epithelial cells. Th is process must occur rapidly in order for the virions to avoid excretion or inactiva tion by digestive enzymes (Chamberlain and Sudia 1961). Once virions have infected mi dgut epithelial cells, virus titer becomes undetectable because viral replication occurs intracellularly during the eclipse phase (McLean 1955). Following the eclipse phase, virions disseminate into the hemocoel and must infect and escape the salivary glands in the saliva in order to be transmitted to a susceptible host (Hardy et al. 1983). It is known that proteolytic digestive enzymes, such as trypsin, in the midgut interact with arboviruses including La Crosse virus (LACV, family Bunyaviridae genus Bunyavirus ) (Ludwig et al. 1989) and dengue virus (DENV, family Flaviviridae genus Flavivirus ) (Molina-Cruz et al. 2005). In Aedes aegypti (L.), trypsin increases DENV 63

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replication in the midgut and dissemination out of the midgut into the hemocoel (MolinaCruz et al. 2005). Virions also influence the formation of the PM. The PM begins forming earlier (1 hpbm) and is thicker at 6 and 12 hpbm in Ae. aegypti fed a DENV-infected blood meal compared to mosquitoes given an uninfec ted blood meal (Suwanmanee et al. 2009). Dengue virus presence resulted in modifications of the PM struct ure, suggesting that virus presence in the blood meal influences physiological processes involved in PM formation. West Nile virus (WNV, family Flaviviridae genus Flavivirus ) is of veterinary and public health importance and is now found on ever y continent except Antarctica, making it the most widespread arbovirus in the world (Kramer et al. 2008). Culex pipiens quinquefasciatus is a competent vector of WNV in the laboratory (Sardelis et al. 2001) and infected mosquitoes have been collected from the field (Godd ard et al. 2002, Rutledge et al. 2003). T he presence of WNV in Cx. p. quinquefasciatus causes morphological changes (e.g. apoptosis, cell death) (Girard et al. 2005) as well as gene expression changes in the mosqui to midgut (Smartt et al. 2009). Genes that are upregulated (expressed at higher levels) in the presence of virus include those involved in PM formation, nutrient uptake, metabolism, and stress and immune responses (Sanders et al. 2005). Since aspects of mosquito physiology in volved in digestion are altered in the presence of virus, this study investigated the extent to which digestion rate in Cx. p. quinquefasciatus changes after infection with WNV. Potential effects of digestion rate 64

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on mosquito physiology and the gonotrophic cycle are discussed with regard to the epidemiology of WNV. Materials and Methods Mosquitoes A colony of Cx. p. quinquefasciatus (F > 45) originally st arted from mosquitoes collected in Gainesville, FL was used for ex periments. Approximat ely 100 four to six day old female mosquitoes were placed in to each of six separate one-liter cardboard cartons (Dade Paper Company, Miami, FL ) with mesh screen covering the top. Mosquitoes were provided both 20% sugar and water ad libitum via cotton pledgets. Sugar was removed 24 h prior to blood feeding to ensure mosq uitoes would blood feed. Mosquitoes were held at 28 C at 75% humidity in a 14:10 L: D cycle for the duration of the experiment. Blood Meal Preparation Three groups of blood fed mosquitoes were co mpared in this study: 1) no virus, 2) high WNV dose (mean standard error) (6.2 0.1 log 10 plaque-forming units (pfu) WNV/mL (mean standard error)), and 3) low WNV dose (5.3 0.1 log 10 pfu WNV/mL). Blood meal preparation is de scribed elsewhere (Richards et al. 2007a) with the exception that WNV (strain WN -FL03p2-3, accession number DQ983578) was propagated freshly in Vero cells. For the mosquitoes not giv en virus, 1.0 mL cell culture supernatant from uninfected Vero cells was a dded to blood meals to substitute for the effect of diluting blood meals with cells and media. Two cotton pledgets each containing 3 mL bovine blood in citrate (Hem ostat, Dixon, CA) with or without virus were heated for 10 min at 35 C, placed on top of the screen on each cart on, and mosquitoes were allowed to feed for 30 min. Two cartons of mos quitoes were fed per blood 65

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treatment. Two 0.1 mL aliquots of the heated WNV infected blood for each treatment were placed into separate tubes containing 0. 9 mL of BA-1 diluent and stored at -80 C for later viral titer analysis. All the mosquit oes in each treatment were chilled at -20 C for 45 sec and fully engorged mosquitoes se parated and transferred to new cartons for the duration of the experiment. Unfed or partially fed mosquitoes were discarded. Five freshly fed fully engorged mosquitoes from both the high and low WNV dose treatments were collected in individual tubes containi ng 0.9 mL BA-1 diluent and stored at -80 C for determination of viral ti ter. The remaining engorged mosquitoes (~150 per group) were placed into separate cartons by treatment, maintained in an incubator held at 28 C, 75% humidity, and provided 20% sugar and water on cotton pledgets ad libitum. Digestion Rate and Vector Competence Twenty mosquitoes per group were collect ed daily from 1-6 days post-blood meal (dpbm) and characterized for blood meal diges tion using the Sella scale (Table 4-1) (Sella 1920, Detinova 1962, Smith 1966). The scale is a visual measure of blood meal digestion and egg development in relation to the abdominal sternites and tergites. The scale was developed for Anopheles spp. (Sella 1920) but has also been used to score digestion rate in Cx. p. quinquefasciatus (Smith 1966). Fully engorged (digestion rate = 2) mosquitoes were used as a starting poi nt in this experiment (Table 4-1). Mosquito Processing The same 20 mosquitoes scored for digestion rate were tested for WNV infection and dissemination, as well as for body and l eg titer. The body (including the head, thorax, wings, and abdomen) and legs of each mosquito were separated with forceps using previously established sterile techniques (Richards et al. 2007a) and placed into separate tubes containing 0.9 mL of BA-1 diluent and two 4. 5 mm zinc-plated beads per 66

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tube. Body and leg samples were homogenized, nucleic acids extracted, and the amount of viral ribonucleic acid was dete rmined using quantitative real-time TaqMan reverse transcription polymerase chain reaction (qRT-PCR) based on a standard curve derived by plaque assay as previously descr ibed (Lanciotti et al. 2000, Richards et al. 2007a). Infection rate was calculated as: (the number of virus-positive bodies divided by the total number of mosquitoes tested) *100. Dissemination rate was calculated as: (the number of mosquitoes with virus-positive legs (six per tube) divided by the number of mosquitoes with virus-positive bodies) *100 (Richards et al. 2007a, 2009). Statistical Analysis Digestion rate data were not normally di stributed, thus non-parametric KruskalWallis tests followed by Bonferroni m eans comparisons were used to determine differences in digestion rates between t he blood meal treatment s at each day postblood meal (dpbm) (SAS 9.1, SAS Institute, Inc., Cary, NC). Virus titers of bodies and legs were norma lized by log (x + 1) transformation and analysis of variance (ANOVA) was used to determine if titers were dependent ( P < 0.05) on dose, day pbm, and/or the interaction between these factors. If significant differences were observed, then treatm ents were coded with dummy variables (Richards et al. 2009) and means were compared using Duncans multiple range test ( = 0.05) (SAS 9.1, SAS Inst itute, Inc., Cary, NC). Multivariate ANOVA was carried out separately for the means of body and leg titer to identify significant differences between treatments for each body part. Fishers exact tests ( = 0.05) were used to determine dose or day pbm effects on infection and dissemination. 67

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Results Virus Titer of Blood Meals and Freshly Fed Mosquitoes The high dose (6.2 0.1 log 10 plaque-forming units WNV/mL) blood meal had a significantly higher titer than the low dose (5.3 0.1 log 10 pfu WNV/mL) ( F = 27.54; df = 1, 3; P = 0.034). Whole bodies of freshly fed mosquitoes provided the high dose had significantly greater WNV titers (5.7 0.1 log 10 pfu WNV/mL homogenate) compared to mosquitoes provided the low dose (4.7 0.1 log 10 pfu WNV/mL homogenate) ( F = 140.45; df = 1, 9; P = 0.001). Blood Digestion Rates in Uninfected Versus West Nile Virus-Infected Mosquitoes Table 4-2 shows digestion rates for mo squitoes provided different blood meal treatments at 1-6 days pbm. At 2 dpbm, mosquitoes gi ven the high virus dose (6.2 0.2 Sella digestion rate) and low dose (6.3 0.2 Sella digestion rate) did not show a significant difference in digestion rate. Howe ver, at 2 dpbm mosquitoes given either the high or low WNV dose digested blood significantly faster co mpared to mosquitoes that were not given infected blood (high dose: H = 7.29, df = 1, P = 0.007; low dose: H = 8.43, df = 1, P = 0.004). Three to six dpbm, the digestion rate was similar for mosquitoes in all treatment groups (> 90% of mosquitoes had fully digested blood meals). Effects of Virus Dose on Early Infection Whole bodies of freshly fed mosquitoes (0 dpbm) given the high dose had body titers of 5.7 0.1 log 10 pfu WNV/mL which decreased to 2.9 0.1 log 10 pfu WNV/mL at 1 dpbm (Table 4-3, Fig. 4-1). After 2 dpbm mosquitoes in the high dose group showed significantly higher WNV body titers (4.7 0.1 log 10 pfu WNV/mL) compared to 1 dpbm indicating that WNV replication was occurring in midgut epithelial cells between 1 and 2 68

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dpbm. There was no difference in WNV body titer for mosquitoes sampled 3-6 dpbm (Table 4-3). Whole bodies of freshly fed mosquitoes gi ven the low dose had body titers of 4.7 0.1 log 10 pfu WNV/mL, and titers decreased from 1 to 2 dpbm (3.2 0.2 log 10 pfu WNV/mL), similar to what was observed wit h body titer in mosquitoes given the high dose (Table 4-3, Fig. 4-1). At 3 dpbm, mosquitoes showed an increase in body titer (4.8 0.1 log 10 pfu WNV/mL) compared to body titers at 1 and 2 dpbm showing evidence for virus replication. There was no significant difference in WNV body titer 4-6 dpbm (Table 4-3). Effects of Virus Dose on Body and Leg Titers Body titer was different between dpbm wit h body titers generally higher at later days pbm (Table 4-4). Body titers were higher in mosquitoes given the high dose. The dose* dpbm interaction was significant, indicati ng that the differences in body titer were not the same between days pbm (Fig. 4-1). Leg titer did not differ between virus doses or dpbm (Table 4-3) nor was there a significant dose*dpbm interaction. Effects of Virus Dose on Inf ection and Dissemination Rates Infection and dissemination rates we re not different between virus dose treatments on any dpbm ( P > 0.05). However, infection rates were lower at earlier days pbm for mosquitoes given the low dose ( P = 0.048), but were the same for mosquitoes given the high dose on all dpbm ( P = 1.000). Dissemination rates between dpbm were similar for mosquitoes giv en either virus dose (low, P = 0.217; high, P = 0.417). Discussion The digestion rate in Cx. p. quinquefasciatus in both WNV treatments was significantly faster digestion rates than that in mosquitoes not fed virus, indicating that 69

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blood digestion is altered by the presence of WNV in the blood meal. By 3 dpbm, >90% of mosquitoes had completed digestion in all three treatment groups. This finding is consistent with the 3 day gonotrophic cycle observed for Cx. p. quinquefasciatus given uninfected blood meals (Okuda et al 2002, Garcia-Rejon et al. 2008). This study showed that virus entry into midgut epithelial cells influences mosquito digestion of the blood meal as reported elsewhere (Mellor 2000). The faster digestion rate in mosquitoes exposed to infected blood ensures that virions enter the posterior midgut cells sooner and avoid excretion and/or inactivation by digestive enzymes (Chamberlain and Sudia 1961). West Nile virus presence in the midgut cells of Cx. p. quinquefasciatus may influence blood digestion to fac ilitate entry into the cells or intracellular replication. Faster digestion in virus-infected mosquitoes likely is the result of accompanying alterations in physiological processes involved in blood digestion. The PM of Ae. aegypti begins forming 1 hour post-blood meal ( hpbm) in mosquitoes given a DENVinfected blood meal compared to 6 hpbm in mosquitoes gi ven an uninfected blood meal (Suwanmanee et al. 2009). The same study found a thicker PM in DENV-infected mosquitoes compared to uninfected mosquitoes at 6 and 12 hpbm (Suwanmanee et al. 2009). These findings showed that the presence of virus in the blood meal may lead to changes in the time of PM deposition and structur e of the PM that likely further influence mosquito physiology. The current study demonstrated that digestion rate is influenced by the presence of virus in the blood meal a nd this could also be related to early PM formation as observed with Ae. aegypti and DENV (Suwanmanee et al. 2009) or viral interaction with digestive enzymes such as trypsin (Molina-Cruz et al. 2005). The 70

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affects of WNV on the action of digestive enzymes and on the formation of the PM requires further investigation. The findings that the eclipse phase was sh ifted from 1 dpbm in mosquitoes given the high dose meal to 2 dpbm in mosquit oes given the low viral dose blood meal indicate that WNV replication in mosquito midgut cells may be dose-dependent. The findings that body titers are higher at late r dpbm in mosquitoes given the high WNV dose support previous studies showing Cx. p. quinquefasciatus is a competent vector of WNV under the most permissive conditions of high dose and later days pbm (Anderson et al. 2010). Culex pipiens quinquefasciatus provided either high or low doses of WNV had titers in the legs that reached an infection limit unrelated to dose and dpbm as reported previously (Richards et al. 2009, Anderson et al. 2010). The rates of infection and dissemination observed here are simila r to those reported elsewhere for Cx. p. quinquefasciatus under similar conditions (Anderson et al. 2010). This study showed that there is a rela tionship between the presence of WNV in a blood meal and digestion rate in Cx. p. quinquefasciatus More importantly, the data show that presence of WNV in the blood meal can influence digestion rate. The faster digestion observed with WNV-infected blood may accelerate the gonotrophic cycle, thereby enabling infected mosquitoes to ta ke another blood meal sooner. This could potentially have an effect on t he epidemiology of WNV by s hortening the blood feeding intervals and thereby the effect of increas ing the number of trans missions. Further studies are needed to address changes in the digestion rate of Cx. p. quinquefasciatus and the impact of WNV infection on proteolytic enzymes that are secreted during blood meal digestion. 71

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Table 4-1. The description of the Sella scale stages of blood digestion and egg development in the mosquito abdomen adapted by Smith 1966. Digestion rating Blood description Egg development 1 Empty None 2 Fully engorged; blood in 6 or more segments None 3 Blood dark; blood in 4-5 segments Apparent 4 Blood dark; blood in 3 segments Apparent 5 Blood dark; blood in 2 segments Apparent 6 Blood dark; blood in line on ventral side of the Abdomen Almost developed 7 Blood absent Full Table 4-2. The digestion rate (mean standard error) (possible rate range 3-7) for mosquitoes given a blood meal with no virus or high or low West Nile virus dose. Blood digestion was rated for mosquitoes 1-6 days post-blood meal (dpbm). Treatment groups with are significantly different from each other. Day post-blood meal No. Tested Sella digestion rating No virus 1 20 3.7 0.1 2 20 5.3 0.2* 3 20 7.0 0.0 4 20 7.0 0.0 5 20 7.0 0.0 6 20 7.0 0.0 High WNV dose 1 20 4.1 0.3 2 19 6.2 0.2* 3 20 7.0 0.0 4 20 6.9 0.1 5 20 7.0 0.0 6 20 6.9 .1 Low WNV dose 1 20 3.8 0.2 2 18 6.3 0.2* 3 20 7.0 0.0 4 20 7.0 0.0 5 20 7.0 0.0 6 20 7.0 0.0 72

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Table 4-3. The West Nile virus titers ( pfu/mL) (mean standard error) in bodies and legs of Culex pipiens quinquefasciatus initially given a high (6.2 0.1 log 10 pfu/mL) or low (5.3 0.1 log 10 pfu/mL) virus dose and held at 28 C for 6 days post-blood meal (dpbm). Whole mosquit oes (bodies + legs) are tested 0 day pbm. The number infected (No. infected) represents mosquitoes that tested positive for the presence of WNV by quantitative Reverse TranscriptionPolymerace Chain Reaction (qRT-PCR). Means with the same letters are not significantly different fo r titer of each body part. Day post-blood meal No. tested Body titer No. infected (%) Leg titer No. disseminated (%) High dose 0 5 5.7 0.1 abc 5 (100) 1 20 2.9 0.09 f 20 (100) 0.9 a 1 (5) 2 19 4.7 0.1 d 19 (100) 0 3 20 6.0 0.1 ab 20 (100) 0 4 20 6.1 0.1 a 20 (100) 4.1 0.8 a 2 (10) 5 20 6.4 0.07 a 20 (100) 1.6 a 1 (5) 6 20 6.2 0.07 a 20 (100) 1.7 0.4 a 6 (30) Low dose 0 5 4.7 0.1 d 5 (100) 1 20 4.1 0.1 e 19 (95) 2.6 1.0 a 3 (16) 2 18 3.2 0.2 f 18 (100) 1 (6) 3 20 4.7 0.2 d 20 (100) 0 4 20 5.5 0.2 bc 16 (80) 5.3 a 1 (6) 5 20 5.8 0.1 abc 18 (90) 2.7 1.5 a 2 (11) 6 20 5.4 0.2 c 20 (100) 0 Table 4-4. Analysis of Variance results of Culex pipiens quinquefasciatus body and leg West Nile virus titer (log 10 pfu/mL) differences. Significant P -values in bold. Source df (num, denom) F P Body titer Day pbm 5, 228 130.35 0.001 Dose 1, 228 60.77 0.001 Day pbm*Dose 5, 228 22.83 0.001 Leg titer Day pbm 4, 13 1.99 0.215 Dose 1, 13 1.68 0.242 Day pbm*Dose 2, 13 0.05 0.956 73

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2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 0123456 High Low D ay Post-Blood Meal P West Nile Virus Titer (logsFU / mL) abc d e f d f ab d a d a abc a c West Nile Virus Titer ( lo g 10 p fu/mL ) Day post-blood meal Fig. 4-1. The West Nile virus body titer (log 10 pfu/mL) (mean standard error) for mosquitoes given a high or low WNV dose and held at 28 C for 6 days postblood meal. Means with the same lette rs are not significantly different. 74

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CHAPTER 5 THE EFFECT OF WEST NILE VIRUS INFECTION ON THE MIDGUT GENE EXPRESSION OF CULEX PIPIENS QUINQUEFASCIATUS SAY (DIPTERA: CULICIDAE) West Nile virus (WNV, family Flaviviridae genus Flavivirus ) is an important threat to humans and animals as it continues to cause morbidity and mortality in the United States (CDC 2007) since its introduction into New York in 1999 (Lanciotti et al. 1999). The virus is maintained in an enzootic transmission cycle between birds and ornothophilic mosquitoes in the genus Culex (Hayes 1989). Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, Culex tarsalis Coquillett, and Culex nigripalpus Theobald are all considered impo rtant vectors of WNV in the United States (Turell et al. 2000, Sardelis et al. 2001, Goddard et al. 2002, Blackmore et al. 2003, Rutledge et al. 2003). Culex pipiens quinquefaciatus vector competence for WNV varies between populations of mosquitoes (Richards et al. 2009, 2010). Competence of a mosquito for a virus is influenced by both internal and exte rnal factors (Hardy et al. 1983). Viruses ingested with a blood meal infect midgut epithelial cells as part of biological transmission. The first barri er that the virus must ov ercome is midgut infection (Chamberlain and Sudia 1961, Hardy et al. 1983). The barrier may be physical or due to the inactivation of virus by digestion enzymes (Hardy et al. 1983). Barriers to infection may be influenced by several diffe rent genes (Tabachnick 1994, Beerntsen et al. 2000). For example, two quantitative trait loci (QTL) influence a midgut infection barrier (MIB) to dengue virus-2 (DENV-2) in Aedes aegytpi (L.) (Bosio et al. 1998, 2000) and these two QTL have been shown to be aff iliated with vector competence (MercadoCuriel et al. 2008). 75

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Midgut gene expression can be influenced by the presence of virus in the blood meal (Sanders et al. 2005, Xi et al. 2008, Sm artt et al. 2009). Changes include altered levels of chitin-binding proteins, vesicl e transporters, and components of the innate immune pathways (Sanders et al. 2005). Fluorescent differential display analyses showed several differentially expressed midgut genes in Cx. p. quinquefasciatus exposed to WNV compared to mosquitoes gi ven an uninfected blood meal (Smartt et al. 2009). Several cDNAs (26) were altered in the presence of WNV. Temporal gene expression studies of one of the c DNAs with high similarity to a Cx. p. quinquefasciatus leucine-rich repeat-containi ng protein-like gene (LRR) s howed that message levels change in Cx. p. quinquefaciatus midguts that have been exposed to WNV compared to mosquitoes given uninfected blood meals (Sma rtt et al. 2009). There were increases in LRR message after infection which corresponded to incubation periods in which WNV midgut titer was lowest, potentially implicating LRR in an immune response to WNV. The objective of the experiments described here was to investigate gene expression changes after WNV exposure in Cx. p. quinquefasciatus In this study, two genes were characterized that were up-regulated in the Cx. p. quinquefasciatus midgut after exposure to WNV. Results from th is study will contribute to the general understanding of the molecular interactions between the mosquito midgut and WNV. Materials and Methods Virus Florida WNV isolate (W N-FL03p2-3) (GenBank a ccession number DQ983578) was passaged once in baby hamster kidney cells and four times in African green monkey kidney (Vero) cells prior to use. Th is strain shows sequence similarity to the NY99 genotype of WNV (Davis et al 2005, Chisenhall and Mores 2009). 76

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Mosquitoes Culex pipiens quinquefasciatus from two different coloni es were used. The first colony was established in 1995 from Gaine sville, FL and will be referred to as CPQG. This colony was used for two separate experim ents: one to charac terize expression of the two genes (F >62 ; experiment one) and one to characterize colony differences in gene expression (F >64 ; experiment two). The second co lony was established in 2008 from Vero Beach, FL (F 19 ) and will be referred to as CPQV. Mosquitoes were reared at 28C and 70% RH under at 14:10 L:D cycle using standard methods (Richards et al. 2007a). Adult mosquitoes were provided 20% sugar solution and water ad libitum Approximately 100 four to six day old mosquit oes were transferred to five 1 L cardboard cages with mesh screening and sugar was removed from cages 24 h prior to each experiment. Sequence Analyses Two differentially expressed PCR amplified products of interest (G1A1 and G43A2) were selected from a fluorescent di fferential display analysis and cloned using TA cloning into the pCR2.1 cloning vector (TA cloning kit, Invi trogen, Carlsbad, CA; Smartt et al. 2009). Clones were prepared and sequenced following methods described by Smartt et al. (2009). BLAST and Vector Base analyses were used to find similarity between cloned sequences and sequences in GenBank using the published genome sequences of Ae. aegypti (Nene et al. 2007) and Cx. p. quinquefasciatus (Arnesburger et al. 2010). Mosquito Infection Two separate mosquito infections were performed, herein called experiment one and experiment two. For bot h experiments, one group was gi ven a WNV-infected blood 77

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meal and the other group was given a blood meal without virus. Virus was propagated and blood meals were prepared using previously described methods (Richards et al. 2007a, Anderson et al. 2010). Two 0.1 mL ali quots of WNV-infected blood were placed into 1.0 mL BA-1 dilute with two copper bea ds and stored at -80C until processing. Briefly, mosquitoes were allowed to feed for capproximately 45 min on cotton pledgets soaked with defibri nated bovine blood (Hemostat, Dixon, CA). Subsequent to feeding, mosquitoes were anesthetized wit h cold, fully engorged specimens transferred to new cages, and mosquitoes were provided 20% sugar solution as previously described (Richards et al. 2007a). Mosquito Midgut Dissection a nd Semi-Quantitative RT-PCR To characterize the temporal gene expression (experiment one), midguts (N = 20 per treatment) were dissected from CPQG mos quitoes that were given WNV-infected and no virus blood meals at 3, 6, and 9 hour s post-blood meal (hpbm) and 1 8 dpbm. Midgut RNA was extracted and semi-quantit ative reverse transcription polymerase charin reaction (RT-PCR) performed as prev iously described (Smartt et al. 2009). Integrity of the ribonucleic acid (RNA ) was determined using gel electrophoresis following standard procedures. The following pr imer sets were used to characterize gene expression (Integrated DNA Technologies, Coralville, IA): CQ G1A1 forward primer, 5-ACG AAG AGG GGA CTC ATC TGG GGG-3, CQ G1A1 reverse primer, 5GGC AGC CAA TCG TCC CTT TTC TCC-3; CQ G43A2 forward primer 5-CAG CTT GCT TTG CCG TTT TGT-3, CQ G43A2 reve rse primer 5-GCC ACC AGC GCT CAG TTC C-3. The CQ G1A1 primer set generated a 460 base pair (bp) product and the G43A2 primer set generated a 296 bp product. 78

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To characterize colony differences in G1A1 gene expression (experiment two), midguts (N = 20 per treatment) were dissect ed from CPQG and CPQV mosquitoes that were given WNV-infected and no virus blood m eals at 3, 6 and 9 hours pbm, and 1 10 days pbm. The following primer set was used to characterize gene expression: CQ G1A1 forward primer, 5-CCG GAC AGG GA T TCA ACA AAG -3, CQ G1A1 reverse primer, 5-TCT CAC GTA GTC AAT CTG GAA GG-3 which produced a 410 bp product. Quantitative Reverse Transcription Polymerase Chain Reaction The quantity of WNV RNA in blood meal and pooled midgut samples was determined using established methods (Ric hards et al. 2007a, 2009, 2010). Standard curves were previously generated based on 10fold serial dilutions of WNV determined by plaque assay (Richards et al. 2007a). Results Sequence Analysis The effect of WNV infection on the midgut gene expression of Cx. p. quinquefasciatus was studied using fluorescent differ ential display analysis (Smartt et al. 2009). One cDNA that was up-regulated in the presence of WNV, G1A1, contained an insert that was 1400 bp in length. Sequenc e analysis of this cDNA resulted in a putative translation product of 311 amino acid s that was incomplete at the 5 end. BLAST searches of the protein p database with the putative trans lation product of G1A1 (GenBank accession no. JF907422) shows that it is 81% identical to a Gramnegative bacteria-binding protein (GNBP) in Cx. p. quinquefasciatus (GenBank accession no. XM_001845915.1; Mauc eli et al. 2007, Atkinson et al. 2009; Fig. 5-1). The other cDNA that was characteriz ed (G43A2), contained an insert that was 280 bp (GenBank accession no. JF707421). Sequence analysis resulted in a putative 79

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translation product of 100 amino acids that was complete. BLAST searches of the protein p database with the putat ive translation product of G43A2 shows that it is 100% identical to defensin-A in Cx. p. quinquefasciatus (GenBank accession no. XM_001842893.1, Lowenberger et al. 1999; Fig. 5-2). Virus Titers of Blood Meals The virus titer of the blood meal for experiment one was 4.85 0.2 (mean standard error) log 10 plaque forming units equivalents (pfu )/mL. The viru s titer of the blood meal for experiment two was 5.81 0.02 log 10 pfu/mL. Results from the CPQG colony cannot be directly compared between experiments due to significantly lower WNV titer in blood meals for experiment one compared to experiment two ( F = 9.21, df = 3, 1, P = 0.009; Anderson et al. 2010). Experiment One G1A1 temporal gene expression in midguts of Culex ppiens quinqufasciatus fed meals containing West Nile virus or no virus Integrity of t he midgut RNA was checked using ge l electrophoresis (Fig. 5-3a, b). To determine the temporal expression of CQ GNBP, semi-quantitative RT-PCR was performed on CPQG mosquito midgut RNA (3, 6, and 9 h, and 1-8 dpbm) using G1A1 primers with mosquitoes given either a WNV-infected blood meal or mosquitoes given an uninfected blood meal. The G1A1 PCR produc t is present in midguts of mosquitoes given WNV at each time point. However ex pression was higher 1-3 h and 8 dpbm (Fig. 5-4a). G1A1 was expressed in mosqui toes provided the uninfected blood meals through day 4, was up-regulated 5 dpbm and decreased 6-8 dpbm (Fig. 5-4b). 80

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G43A2 temporal gene expression in midguts of Culex pipiens quinqufasciatus fed meals containing WNV or no virus Expression using semi-quantitative RT-PCR was performed for the putative defensin gene on the same CPQG mosquito midgut RNA as G1A1 using the G43A2 primer set. G43A2 PCR produc t was expressed at all incubation periods (3, 6, 9 h, and 1-8 d pbm) regardless of the presence or absence of WNV in the blood meal (Fig. 5-5a, b). Virus titers of mosquito midguts The same midgut samples were analyz ed for WNV titer using q-RT-PCR to determine if pooled mosquito midgut samples from experiment one had significantly different titers between time point s with rates increasing 5-8 dpbm ( F = 10.47. df = 10, 20, P = 0.001) (Fig 5-6). G1A1 gene expres sion increased 1-3 dpbm while WNV titer dropped 1 and 2 dpbm and increased only slightly 3 dpbm. G43A2 expression did not impact WNV titers in this colony under these conditions. Experiment Two G1A1 temporal gene expression in midguts of Culex pipiens quinquefasciatus Gainesville and Culex pipiens quinquefasciatus Vero Beach fed meals containing West Nile virus or no virus Integrity of the midgut m RNA was checked using semi-quantitative RT-PCR to amplify a Cx. p. quinquefasciatus actin cDNA (Fig. 5-7). In experiment 1, G1A1 expression was up-regulated upon WNV exposure in mosquitoes (Fig. 5-4), perhaps in association with antiviral responses. The ex pression of this gene was characterized in two colonies (CPQG originally from Gaine sville, FL and CPQV originally from Vero Beach, FL) that differ in their vector co mpetence for WNV. Semi-quantitative RT-PCR analyses of RNA from midguts showed that G1 A1 gene expression varied in the CPQG 81

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midgut over time in mosquitoes exposed to WNV compared to mosquitoes given an uninfected blood meal (Fig. 5-8a, b). G1A1 was expressed c onstantly in the midgut of the CPQV colony regardless of the presenc e of WNV, but was expressed at higher levels in mosquitoes given WNV (Fig. 5-8c, d). G43A2 temporal gene expression in midguts of Culex pipiens quinquefasciatus Gainesville and Culex pipiens quinquefasciatus Vero fed meals containing West Nile virus or no virus Expression using semi-quantitative RT-PCR was performed for the putative defensin gene on the same CPQG and CPQV colony mosquito midgut RNA as G1A1 using the G43A2 primer set. G43A2 PCR products were present at all time points in both colonies regardless of infection status (Fig. 5-9a-d). Virus titers of mosquito midguts To determine if G43A2 or G1A1 expressi on was correlated with the WNV titer in the two colonies, WNV titer analysis usi ng q-RT-PCR was performed on the same pooled midgut samples. The WNV titer from pooled mosquito midguts was significantly different between the different colonies (CPGQ vs CPQV), wit h the CPQG colony generally reaching a stable midgut titer st arting 4 dpbm and the CPQV colony reaching stable midgut titers starting 3 dpbm (F = 13.78, df = 1, 49, P = 0.001) (Fig. 5-10). West Nile virus midgut titers were significantly different between IPs with higher titers occurring at later IPs in both t he CPQG and the CPQV colonies ( F = 51.67, df = 12, 49, P = 0.001). The colony*IP interaction was si gnificant, indicating that the difference observed in WNV titer in the midgut was not the same for each colony at each IP ( F = 4.47, df = 10, 12, 49, P = 0.001). 82

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Discussion The gene named G1A1 showed altered ex pression in the mosquito midgut following exposure of Cx. p. quinquefasciatus to WNV. This gene shares high sequence identity to the family of Gram-negat ive bacteria binding proteins (GNBPs). Gram-negative bacteria binding proteins are one of the pattern re cognition receptors that can activate the Toll pathway in Drosophila melanogaster Meigen and Ae. aegypti (Kim et al. 2000) in response to Gram-positive bacteria, fungi, Plasmodium (Magalhaes et a. 2010), and Gram-negative bacteria (Warr et al. 2008). There are three subfamilies of GNBP. Subfamily B is specific to mos quitoes (Christophides et al. 2002). There are six members of the GNBP gene family (GNBPA1, GNBPA2, GNBPB1, GNBPB2, GNBPB3, GNBPB4) in Anopheles gambiae Giles all of which are implicated in some aspects of immune response (Waterhouse et al. 2007). Anopheles gambiae GNBP has a -1,3 glycan binding domain (Lee et al. 1996) and activates the mosquitos Toll pathway as part of an immune response (Kim et al. 2000) to fungi, Gram-positive bacteria, Plasmodium (Magalhaes et al. 2010), and Gramnegative bacteria (Warr et al. 2008). The similarity of CQ G1A1 to An. gambiae GNBP-B (81% identity, accession no. XM_312118.3, Mongin et al. 2004) is indicative that this gene could be involved in an immune response pathway such as the Toll-like pathway in response to bacteria (Magalhaes et al. 2010). However, this has not yet been shown for viruses. The increase in G1A1 message following WNV in gestion, suggests, for the first time, potential association with an antiviral response. The sequence identity to Ae. aegypti and Cx. p. quinquefasciatus GNBP was significant but the functional characterization in these mosquitoes has yet to be performed. 83

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The Toll pathway has been shown to be involved in DENV infection in Ae. aegypti (Xi et al. 2008) and WNV in Cx. p. quinquefasciatus (Smartt et al 2009). When Cx. p. quinquefasciatus was infected with WNV, G1A1 was expressed at higher levels 1 3 dpbm (Fig. 5-4a) compared to mosquitoes given an uninfected blood meal (Fig. 54b). This expression pattern also corres ponds to the WNV titer in the midgut tissue which drops 1 and 2 dpbm and then begins to increase 3 dpbm due to viral replication (Fig. 5-6). Hence the expression of G1A1 was higher when WNV was low, consistent with the hypothesis that G1A1 expression wa s associated with an antiviral response in the infected mosquitoes. Additionally, the dr op and rise in WNV titer correlates to the eclipse phase when virus titer drops to lower levels (McLean 1955, Mellor 2000), which implicates G1A1 expression associated with the eclipse phase in these mosquitoes. Whether or not the eclipse phase is also correlated to an immune response to virus in the mosquito (such as by the Toll pathway) or the eclipse phase needs to be further evaluated. G1A1 is also expressed in mosquitoes provided a WNV blood meal at higher levels 8 dpbm which does not correlate with a reduction in the WNV titer in the midgut. As this is late in infection (i.e. after the virus has escaped the midgut (Girard et al. 2004, Anderson et al. 2010), it is likely that increased expression in G1A1 is related exclusively to WNV infection and there ar e other possible unknown factors influencing its expression. This also likely explains the increased G1A1 expression in mosquitoes given an uninfected blood meal 5 dpbm which may correlate with the presence of bacteria in either blood or sugar meals. Similar gene expression changes were correlated with a decrease in WNV titer when the gene expression of G12A2 (a putative LRR) was examined in WNV infected Cx. p. quinquefasciatus compared to uninfected 84

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mosquitoes (Smartt et al. 2009). Mosqui to midgut WNV titer showed a decrease 6 dpbm which correlated with an increase in G12A2 expression (Smartt et al. 2009). A second up-regulated cDNA G43A2, examined by sequence analysis was similar to Ae. aegypti defensin-C (63% similarity, accession no. AA D40116.1, AA D40116.2, Lowenberger et al. 1999). Defensins are one of the antimicrobial peptides that result when the Imd pathway is activated in Ae. aegypti in response to Grampositive bacteria, fungi, and Plasmodium (Magalhaes et al. 2010) There are three isoforms in the defensin (def) gene family in Ae. aegypti (def-A, def-B, and def-C) (Lowenberger et al. 1999). Defe nsin-A and B are highly expre ssed in the fat body while def-C is expressed at higher levels in the midgut of Ae. aegypti (Lowenberger et al. 1999). The similarity of G43A2 to Ae. aegypti def-C suggests that this gene could be involved in an antimicrobial response as par t of the Imd pathway. Defensin protein levels have been found to increase with in fection of both Gram-negative and Grampositive bacteria (Magalhaes et al. 2010), as well as nematodes (Bartholomay 2004), and malarial parasites (Dimopoulos et al. 1997) suggesting that defensin may function as part of a general immune response (Bart holomay 2004). When part of the Imd pathway in Ae. aegypti is inhibited by injection of dsRNA to either the relish gene (Rel2) or the Rel2 effecter molecule (def-A) and then mosquitoes are challenged with Grampositive and Gram-negative bacteria, mos quitoes with Rel2 knocked-out had lower survival rates compared to mosquitoes with def-A knocked-out (Magalhaes et al. 2010). These findings, combined with the finding that G43A2 is expressed in both WNVinfected and uninfected midguts at all ti me points further suggests a generalized immune function for defensins in mos quitoes. It is odd that def-C in Cx. p. 85

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quinquefasciatus occurs in the midgut, since it is localized to the fat body of Ae. aegypti (Lowenberger et al. 1999), however other tissues were not examined during this experiment. The finding that G43A2 is present in mosquitoes given uninfected blood meals suggests that Cx. p. quinquefaciatus def acts as an antimicrobial agent perhaps in response to bacteria that are introduced duri ng the feeding process. It suggests also that G43A2 may be present before pathogen challenge and this finding should be investigated further. Because of the altered expression of these midgut genes in response to WNV infection, G1A1 and G43A2 expression was compared in two different colonies of Cx. p. quinquefasciatus (CPQG and CPQV) with known differences in competence for WNV. There were no population differences in t he expression of G43A2 (Fig. 5-9a-d). However, G1A1 expression differed between the two colonies. G1A1 was expressed in the CPQG colony at all time pointes regardl ess of infection stat us, although expression was not consistant between time points (312 hours and 1-10 dpi) (Fig. 5-8a, b). G1A1 was expressed in the CPQV colony at all time points regardless of infection status, although expression appeared much higher in mosquitoes given WNV (Fig. 5-8c, d). Expression seemed to be consistent acro ss time points in both WNV+ and WNVmosquitoes. These two colonies are k nown to have different dissemination rates (CPQG > CPQV), and there appears to be a mi dgut escape barrier (MEB) in the CPQV colony. The enhanced expression of G1A1 (GNBP) in CPQV infected with WNV may contribute to the MEB obser ved in the CPQV colony. The titer of WNV in the midgut was not si gnificantly different on days 4-8 pbm in the CPQG colony and was not significantly diffe rent 3-8 dpbm in the CPQV colony. If 86

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the WNV midgut titers are compared between co lonies qualitatively, it seems that the midgut titer is different in the CPQG col ony 4-8 dpbm compared to the CPQV colony, perhaps indicating a different MEB in CPQG as virus begins disseminating out of the midgut 4 dpbm (Girard et al. 2004, Anderson et al. 2010). The consistent increase in G1A1 expression in the CPQV colony fo llowing WNV exposure may contribute to the MEB observed in the CPQV colony and ce rtainly bears further investigation. Using fluorescent differential display analysi s, three genes, i.e. G12A2 (Smartt et al. 2009), G1A1, and G43A2 in two immune response pathways (Toll and Imd) have been characterized with respect to WNV infection in Cx. p. quinquefasciatus G1A1 (GNBP) expression in WNV infected mosquit oes seems to increase with a decrease in WNV titer in the mosquito midgut indicating GNBP involvement in antiviral immune system and could play a role in the eclip se phase of the mosquito. G43A2 ( Cx. p. quinquefasciatus def-C) expression is consistent in both WNV infected and uninfected mosquitoes, indicating that it is present before pathogen challenge and that def-C may play a more general role in immuni ty than previously postulated in Drosophila Because these cDNAs were expressed in the same differential display analysis, there is indication that more than one immune pathway might be involved with viral immune responses in mosquitoes. 87

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88 Fig. 5-1. Multiple protein sequenc e alignments of a fragment of the Culex pipiens quinquefasciatus putative Gram-negative bacteri a binding (GNBP) protein with GNBP proteins from other insect s. Aligned protein sequences include the G1A1 putative translation produc t (311 amino acids; CQ G1A1 GNBP from Cx. p. quinquefasciatus ; (CQ GNBP, accession no. XP_ 001845967.1, Atkinson et al. 2009), 1,3 gluc an recognition Protein-4 from Bombyx mori (BM b1,3 glucan rec 4,; accession no. NP_001159614.1, Pauchet et al. 2009), GNBP from Ae. aegypti (Ae GNBP, accession no. XP_001659797.1, Nene et al. 2007), and GNBP subgroup B from Anopheles gambiae (An GNBP-B, accession no. XM_312118.3, M ongin et al. 2004). Represents amino acids that are the same as in the CQ G1A1 partial protein sequence, the numbering represent the amino acid number, the dots are included to maximize spacing.

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89 ------------------------------------------LNHRDNSFVEDGVLYLKP 18 G1A1 partial protein VDRKAFCPGELLFEDNFDTLDLDRTG---------------S.................. 105 Q GNBP AP-VTVCSGQLIFADDFVDFDLEKWQHENTLAGGGNWEFQYYN.N.T...TNN.L..IR. 88 M b1,3 glucan rec P-4 VYRKTFCSGDLIFEDNFDKLNLEKWEHEHTLGGGGNWEFQYY..N.K..Y..N.I..IR. 104 e GNBP VKRSAFCPGDLIFEDNFDRLDLERWQHEVTLAGGGNWEFQYY..S.R....K..IFFIR. 87 n GNBP-B TFIGLEPGGEEYLKTGKLDINGGDPGNFCTNPAWDGCVRTGTPESILNPVKSARIRTAHS 78 G1A1 partial protein ............................................................ 165 Q GNBP SLTSDQF.-SAF.HS.R.N.E..A.ADR....Q.Y..E.V...TN....I.......VN. 147 M b1,3 glucan rec P-4 .LLAD.T.-..F.TS.T.NL...S.YDS.........E......NP...I....L..LK. 163 e GNBP .LLAD.T.-..F.SS.N.NVH..T.YD.....SNY..E.Q.S.TNY...I....V..VN. 146 n GNBP-B FNFKYGKLEIRAKLPTGDWMWPALWLMPRTNQYGTWPASGEIDLMEARCNVDYRDEEGTH 138 G1A1 partial protein ............................................................ 225 Q GNBP .S.Q...V.V...M.S...L...I....AY.K............V.S.G.KNMF-LN.L. 206 M b1,3 glucan rec P-4 ...................L........KL.......T........S.G.L...VAD... 223 e GNBP ...R...V.....I.....L........KI.......S........S.G.L..S-VN.NQ 205 n GNBP-B LGVEQVLSTLHFGPNAWTNAYDTSTAPKNSASGQGFNKDFHRYQLEWTPEFMKFSVDGEH 198 G1A1 partial protein ............................................................ 285 Q GNBP I.TQEAG....Y..FPGLSGWERAHWVRRNSA.--YDTN..........D.IS.RI.DSE 264 M b1,3 glucan rec P-4 I.....G........PSL.GFE....A....P.E...N............Y......D.E 283 e GNBP ....H.GT......QWDL.G.EMA..V...PK......G.............R....D.Q 265 n GNBP-B ILQVD---GNFWQRGNFDERAPGTRNPWVSGTKMAPFDQEFFIILNLAIGGTNGYFPDEK 255 G1A1 partial protein .....---.................................................... 342 Q GNBP .GR.APGN.G..EY.G.NN.-..IH...RY.S.......K.YL.I...V.....F...G322 M b1,3 glucan rec P-4 T.V..---..........Q.....P...I..G......E..HV.M...V.........PP 340 e GNBP VM..E---....EL.R....R..VQ....T.G.........Y..M...V.....F...VP 322 n GNBP-B -VVN-TKPKPWSNQSPVGPAMTSFWEKRDDWLPTWNLDINDGKDAAFQIDYVRIWAL. 311 G1A1 partial protein -...-.................................................... 397 Q GNBP -.K.-PI....W.N..T--.A.D..NGQGG.......NV...Q..SL.V....V... 375 M b1,3 glucan rec P-4 -AT.KDN....T.G..T--.RGG..SAKE......K.EE..S.E.SL.V....V... 394 e GNBP PA..ANGN.....N..T--.LRD..LG.S......K.QE..SAE.S..V....V... 377 n GNBP-B CQ C B A A CQ C B A A CQ C B A A CQ C B A A CQ C B A A CQ C B A A

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MNSLGTACFAVLCLFAIVSTGNGFPQESA DRVQSFANTLF 40 G43A2 pro ..............................Q..Y...... 40 CQ def-A .RT.TVV..VA...S..FT...AL.E.L..D.R.Y..S.. 40 Ae def-2 .RT.IVV..VA...S..FT..SAL.E.L..D.R.Y..S.. 40 Ae def-C1 .RT.IVV..VA...S..FT..SAL.G.L..D.RPY..S.. 40 Ae def-C2 DELPEQSYQAAAENLRLKRATCDLLSGLGVNDSACAAHCI 80 G43A2 pro ........................................ 80 CQ def-A .....E.....V..F............F..G......... 80 Ae def-2 .....E.....V..F............F..G......... 80 Ae def-C1 .....E.....V..F............F..G......... 80 Ae def-C2 ARGNRGGYCNSKKVCVCRN. 100 G43A2 pro ................... 99 CQ def-A ..R.......A........ 99 Ae def-2 ..R.......A........ 99 Ae def-C1 ..R.......A........ 99 Ae def-C2 Fig. 5-2. Multiple protein sequence ali gnments of the complete sequence of the Culex pipiens quinquefasciatus putative defensin,G43A2 (100 amino acids) with defensin proteins from other mosquit oes. Protein sequences represent the defensin-A ( Cx. p. quinquefasciatus ; accession no. XP_001842945.1), defensin-2 ( Ae. aegypti ; accession no. XP_001657288, Nene et al. 2007), defensin isoform C1 ( Ae. aegypti ; accession no. AAD40116.2, Lowenberger et al. 1999) and defensin isoform C2 ( Ae. aegypti ; accession no. AAD40116.2, Lowenberger et al. 1999). Represents amino acids t hat are the same as in the CQ G43A2 complete protein sequence, the numbering represent th e amino acid number. 90

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CPQG WNV100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d 100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d CPQG WNV+ B A Fig. 5-3. The RNA int egrity of Culex pipiens quinquefas ciatus midguts by gel electrophor esis. A). C P QG given virus (WN V +), B). CPQG no virus (WNV-). 100 bp= DNA molecular weight marker; bp, base pairs; h, hours; d, days. 91

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A 100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d B CPQG WNV+ CPQG WNV100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d Fig. 5-4. G1A1 gene expression analysis Culex pipiens quinquefas ciatus midguts by semi-quantitative RT-PCR. A). CPQG gi ven virus (WN V +), B). CPQG no virus (WNV-). 100 bp= DNA molecular weight marker; bp, base pairs; h, hours; d, days. 92

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100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d A B CPQG WNV+ CPQG WNV100 bp 3 h 6 h 9 h 1 d 2d 3d 4d 5d 6d 7d 8d Fig. 5-5. G43A2 gene express i on analys is in Culex pipiens quinquefasciatus ( C PQG) midguts by semi-quantitative RT-PCR. A). CPQG given virus (WN V +), B). CPQG given no virus (WNV-). 100 bp= DNA molecular weight marker; bp, base pairs; h, hours; d, days. 93

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Fig. 5-6. Experiment one: Culex pipiens quinquefasciatus (CPQG) midgut West Nile virus titer at different incubation periods after infection. There is a significant difference in WNV titer in the mosquito midgut tissues at different incubation periods after infection (F = 10.47, df = 20, 10, P = <0.001). Initial virus dose was 4.85 0.2 log 10 pfu/mL WNV. Means with the same letters are not significantly different by the Duncan mult iple range test (Richards et al. 2009, 2010). H, hours; d, days. 3 3.5 4 4.5 5 5.5 6 3 h6 h9 h1 d2 d3 d4 d5 d6 d7 d8 d Incubation Period Post-Blood Meal E C BC CD E DE ABC CD C AB A M i d g u t W N V T i t e r (Log 10 pfu/mL) 94

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Fig. 5-7. Experiment two: The RNA integrity of Culex pipiens quinquefasciatus (CPQG and CPQV) midguts by semi-quantitative RT-PCR using CQ actin primers. A). CPQG given virus (WNV+), B). CPQG no vi rus (WNV-), C) CPQV given virus (WNV+), D) CPQV given no virus (W NV-). 100 bp= DNA molecular weight marker; bp, base pairs; h, hours; d, days. 95

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3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp A CPQG WNV+ 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp B CPQG WNVCPQV WNV+ 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp C 3 h 6 h D 100 bp 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d CPQV WNV96

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Fig. 5-8. G1A1 gene expre ssion analysis in midgut tiss ue from two populations of Culex pipiens quinquefasciatus (CPQG and CPQV) by semi-quantitative RT-PCR. A). CPQG given WNV (WNV+), B). CPQG given no virus (WNV-), C). CPQV given WNV (WNV+), D). CPQV gi ven no WNV (WNV-). 100 bp= DNA molecular weight marker; bp, base pairs; h, hours; d, days. 97

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. 100 bp 3 h 6 h 9 h 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d CPQG WNV+ CPQG WNVCPQV WNV+ CPQV WNV3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp A B 3 h 6 h 9 h 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 3 h 6 h 9 h 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp 100 bp C D 98

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Fig. 5-9. G43A2 gene expr ession analysis in midgut tiss ue from two populations of Culex pipiens quinquefasciatus (CPQG and CPQV) by semi-quantitative RTPCR. A). CPQG given WNV (WNV+), B). CPQG given no virus (WNV-), C). CPQV given WNV (WNV+), D). CPQV given no WNV (WNV-). 100 bp= DNA molecular weight marker; bp, ba se pairs; h, hours; d, days. 99

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100 C P Q G W N V + 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 100 bp A 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d B 100 bp C P Q G W N V 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 3 h 6 h 9 h 6 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d C P Q V W N V C P Q V W N V + 100 bp 100 bp D C

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101 0 1 2 3 4 5 6 7 8 6 h9 h12 h 1 d2 d3 d4 d5 d6 d7 d 8 d 9 d10 d Series1 Serie Fig. 5-10. Experiment two, Culex pipiens quinquefasciatus (CPQG and CPQV) midgut West Nile virus titers at different incubation periods after infection. Means with the same letters are not significantly different by the Duncan multiple range test (Richards et al. 2009, 2010). Letters that are in bold represent midgut samples that were only tested one time. H, hour; d, day. s2 B C D K E F G J K G H I H I F G H I I J K E F G H E F E F G E D D C D C D D D C A B C D A A B A B C D C D Midgut WNV Titer (Log 10 PFU/mL) Time Point Post-Blood Meal CPQG CPQV A B C D A B C A B C D

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CHAPTER 6 WEST NILE VIRUS-BINDING PROTEINS IN THE MIDGUT OF CULEX PIPIENS QUINQUEFASCIATUS SAY AND CULEX NIGRIPALPUS THEOBALD (DIPTERA: CULICIDAE) Arboviruses must infect and replicate within mosquito tissues in order for biological transmission to occur. Virions are ingest ed with an infectious blood meal and must attach to receptors located on the posterior midgut epithelial cells (Hardy et al. 1983). The receptor interacts with virus attachment proteins (VAPs) (Ren et al. 2007, SalasBenito et al. 2007), then penetra tes and enters the cell (Hardy et al. 1983). Once in the cell, the virus will uncoat and undergo replication (Hardy et al. 1983, Mellor et al. 2000). Because the midgut is the first point of cont act for arbovirus virions, it represents a formidable barrier to infection and subsequent transmission (Kramer et al. 1981). One hypothesis for the existence of the midgut infe ction barrier (MIB) is the lack of cellular receptors on midgut epithelial cells (K ramer et al. 1981, Hardy et al. 1983). Several putative flavivirus receptors have been identified in C6/36 Aedes albopictus Skuse cell culture [dengue virus (D ENV); Lourdes-Munoz et al. 1998, Mendoza et al. 2002, Reyes-del Valle et al. 2004, MercadoCurial et al. 2006, 2008, Salas-Benito et al. 2007, West Nile viru s (WNV), DENV, Japanese encephalitis virus (JEV); Chu et al. 2005, Ren et al. 2007]. The putative receptors that bind WNV on C6/36 cells include proteins that are 140, 95, 70, and 55 kilo Dalton (kDa) in apparent molecular weight (Chu et al. 2005). The binding of the 95 and 70 kDa proteins was shown to be specific under stringent condition s of high salt and detergent washing (Chu et al. 2005). In addition, antibodies against the 95 and 70 kDa proteins blocked WNV, DENV, and JEV entry into C6/36 cells wher eas antibodies against the 140 and 55 kDa proteins did not prevent entry of these flaviviruses into C6 /36 cells (Chu et al. 2005). 102

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The 140 and 55 kDa proteins may not serve as a functional receptor, although they may associate with the receptor complex in so me manner (Chu et al. 2005). There is an additional 74 kDa protein that interacts with WNV, DENV, and JEV (Ren et al. 2007). While Ae. albopictus is considered to be a competent vector of WNV (Sardelis et al. 2002), Culex pipiens quinquefasciatus Say and Culex nigripalpus Theobald are both considered to be important vectors of WNV in the southeastern United States (Blackmore et al. 2003, Rutledge et al. 2003, Vitek et al. 2008). The objective of this study was to det ermine the apparent molecular weight of proteins that bind to WNV in the midguts of Cx. p. quinquefasciatus and Cx. nigripalpus This is the first study to examine WNV mi dgut binding proteins in the midguts of principle vectors of WNV. Materials and Methods Mosquitoes and Midgut Dissections Colonies of Cx. p. quinquefasciatus started from collections in Gainesville, FL (F >45 ) (CPQG), Cx. p. quinquefasciatus originally obtained from Vero Beach, FL (F >17 ) (CPQV), and Cx. nigripalpus (F >113 ) (CNG) started from mo squitoes collected in Gainesville, FL were maintained as de scribed previously (Knight and Nayar 1999, Richards et al. 2007a). Midguts from approx imately, 200 fourto sixday old mosquitoes were dissected in phosphate bu ffered saline (PBS) containing 1% Halt Protease Inhibitor Cocktail (Pierce, Rockf ord, IL) and placed into 1.5 mL tubes containing 0.2 mL PBS containing 1% Halt Protease Inhibitor Co cktail and stored at 80C. Midguts were homogenized by hand using a plastic pestle. 103

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Protein Quantification and 1-D Electrophoresis Proteins were quantified using the BCA Protein Quantification kit (Pierce, Rockford, IL). Approximately 0.06 mg/L of homogenized proteins were mixed with 0.025 mL NuPage LDS Sample Buffer (Inv itrogen, Carlsbad, CA ), heated at 70C for 10 min, and electrophor esed using the XCell SureLock Mini cell (Inv itrogen, Carlsbad, CA) onto a NuPage 3 8% Tris-Acetate gel (Invitrogen, Carl sbad, CA). HiMark Pre-Stained Protein Standard (Invitrogen, Carlsbad, CA ) was used to determine if electrophoresis had occurred and the molecu lar weight of the midgut proteins (Invitrogen, Carlsbad, CA). Two gels were run simultaneously. The first gel was stained using SimplyBlue Safe Stain followi ng manufacturers instructions (Invitrogen, Carlsbad, CA). Proteins fr om the second gel were trans ferred to a nitrocellulose membrane using the XCell II Blot Module (Invitrogen, Carlsbad, CA) for further processing. Virus Overlay Binding Protein Assay (VOBPA) The nitrocellulose membrane was blocked for 1 h at room temperature (RT) in blocking solution made of 5% skim milk in Tris buffered saline (TBS) (100 Mm Tris-HCl, 0.15% NaCl). The membrane was washed at room temperature fo r 10 min three times in TTBS (TBS with 0.2% Tween 20). Freshly propagated WNV (strain WN-FL03p2-3, Genbank accession no. DQ983578) was mixed 1: 1 with cell culture media (Medium 199 with Earles salts, 10% fetal bovine serum, 2% penicillin and streptomycin, and 0.2% mycostatin). The membrane was incubated overnight at 28C. Membranes were washed as before and then incu bated overnight at 25C in a 1:100 dilution of WNV monoclonal antibody (CDC cat # H5-46). Membranes were washed as before and then incubated at RT for 2 h in a 1:1000 diluti on of secondary antibody conjugated to 104

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horseradish peroxidase. The VIP kit (Vector VIP Peroxidae Substrate Kit, Vector Laboratories, Burlingame, CA) was used to detect secondary antibody following manufacturers instructions. Results Polyacrylamide gel electrophoresis showed that there were at least 15 midgut proteins in both Cx. p. quinquefasciatus colonies with no discernable differences (Fig. 61A), ten of which bound WNV after a VOBPA (F ig. 6-1B). The proteins that bound WNV ranged in size from 31 kDa to >460 kDa. Polyacrylamide gel electrophoresis of Cx. nigripalpus midgut proteins revealed at least 21 mi dgut proteins (Fig. 6-1A), eight of which bound WNV after VOBPA (Fig. 6-1B). The Cx. nigripalpus midgut proteins that bound WNV also ranged in size from 31 kDa to >460 kDa. Discussion This is the first report of Culex spp. midgut proteins that bind to WNV. In general, mosquito proteins that bind flaviviruses via VOBPA (i.e. WNV, DENV, and JEV) range from about 95 to approximately 40 kDa in apparent molecular weight (de LourdesMunoz et al. 1998, Mendoza et al. 2002, Reyes del Valle et al. 2004, Mercado-Curiel et al. 2006, 2008, Salas-Benito et al. 2007). Resu lts of this study show four protein bands per colony (CPQG vs. CPQV) and specie s (CPQ vs. CNG) with apparent molecular weights in this range (Fig. 6-1B). Evidence has shown that the virus attachment protein (VAP) envelope glycoprotein of different flaviviruses are similar to one another which may suggest that mosquito-flavivirus interactions might also be similar (Ren et al. 2007). The interaction between the VAP and the putative ce llular receptor may contribute to host range, tissue tropism, and viral pathogenicity (Chu et al. 2005) West Nile virus, DENV, and JEV use 105

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similar sized proteins to enter C6/36 cells (Chu et al. 2005, Ren et al. 2007), although future studies need to be conducted to determi ne if the same relationship occurs in mosquito tissues. The next steps in this study is to identif y the proteins of appar ent molecular weight similar to previously described proteins us ing liquid chromatography (LC) followed by two rounds of mass spectrophotometry (MS). Identification of pr otein superfamilies and families will allow the determination of propos ed structure and function of the proteins. Further experiments will use RNA-interference technology to knock-down genes that are associated with putative WN V-binding proteins to determine if these proteins are involved with virus entry into Culex spp. midgut epithelial cells. 106

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Fig. 6-1. Culex pipiens quinquefasciatus (CPQG, CPQV) and Culex nigripalpus (CNG) midgut protein analysis by 1-D electr ophoresis and virus overlay binding protein assay (VOBPA). A). Crude mosquito midgut proteins run by Sodium Dodecyl Sulfate Polyacrylamide Ge l Electrophoresis (SDS-PAGE) and stained with SimplyBlue Safe Stain, B). VOBPA of midgut proteins using WNV. M; HiMark Prestained Protein Standard. 107

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CHAPTER 7 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS The primary goal of this dissertation was to explore the interactions between the Culex pipiens quinquefasciatus Say midgut and West Nile viru s (WNV). As extrinsic factors (outside of the mosqui to) can influence intrinsic fa ctors (within the mosquito), this dissertation explored the influence of bot h on vector competence. Fig. 7-1 shows how the extrinsic and intrinsic factors exam ined through this dissertation research may interact to influence Cx. p. quinquefasciatus mosquito physiology and vector competence. The mosquito midgut plays a critical role in the infection process because it is involved in blood digestion (Clements 1992) and is the first point of contact for arboviruses such as WNV (Hardy et al. 1983) The interaction of the mosquito and virus is also affected by temperat ure, dose, age, colony, and ot her extrinsic factors which may impact the midgut environment but does c ontribute to modifications in vector competence as shown in Chapters 2 and 3 of this dissertation. Experimental results showed that the Culex pipiens quinquefasciatus Gainesville (CPQG) colony differed in its WNV vector competence from the Culex pipiens quinquefasciatus Vero (CPQV) colony. The difference can be partially attributed to the presence of a MEB that is influenced by different factors in the CPQG (73% dissemination, 12 dpi, 28 C) colony compared to the CPQV colony (44% dissemination, 12 dpi, 28 C). These re sults suggest that MEBs are dynamic and possibly have different mechanisms contribu ting to MEBs. These vector competence differences can be further examined to determi ne if there are gene or protein expression differences in the midguts of mosquitoes in t hese two different colonies. Correlation of 108

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expression difference with colony variation in vector competence would help elucidate genes and proteins involved in WNV vector com petence. This is only the first step that provides tools to further investigate how widespread any such genes are in the species and whether phenotypic variation in the specie s is influenced by these genes. Further work would then be needed to assess non-genetic effects on the phenotype caused by the genes. As virions enter the midgut with a blood meal, several genes and proteins must be present in order for the virus to infect the mosquito and eventually be transmitted to a new host. Midgut genes are translated into proteins involved in peritrophic matrix formation around the blood meal (Smartt et al 1998). Midgut genes are also translated into digestive enzymes such as trypsin and chymotrypsin necessary to digest blood meals (Noriega and Wells 1999, Brackney et al. 2008). The process of blood meal digestion is well understood, however as show n by work in Chapter 5, digestion rate was altered in the midgut of mosquitoes infected with WNV, indicating a need to reevaluate this basic physiol ogical process and its importanc e in vector competence. The faster digestion observed with WNV-infe cted blood may accelerate the gonotrophic cycle, thereby enabling infected mosquitoes to take another blood meal sooner potentially affecting epidemiological cycles in nature. These results could partially explain the high levels of WNV transmissi on (CDC 2007) throughout the United States compared to other arboviruses and may differ for other mosquito-virus interactions. Further studies are needed to address changes in the digestion rate of Cx. p. quinquefasciatus and the impact of WNV infection on proteolytic enzymes that are secreted during blood meal digestion and whether this is present in other mosquito-virus 109

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systems. The effect of blood meal digesti on on infection and dissemination rates should be further examined in the tw o colonies studied here. A dditional studies on blood digestion are needed to examine digestive en zymes at both the protein and transcript level. Other factors such as virus dose, mosquito age, and EIT, among others need to be evaluated as they are expected to influence blood digestion. Midgut genes also encode virus binding prot eins and receptors that allow virus entry into the midgut epithelial cells (Chu et al. 2005). A process that if changed can prevent a mosquito from being a competent vector. Chapter 6 shows that although there are pathogen specific bind ing proteins and these vary by mosquito species, WNV appears to bind to ten general proteins that appear common in two Culex spp. It has been postulated that mosquito proteins in teract with virus proteins in order to enter cells (Ren et al. 2007, Salas-Benito et al. 2007). The number of midgut proteins that bind to WNV could be related to the r eason why >62 species of mosquitoes in 11 genera (Brault 2009) can become infected with WNV. This is supported by other studies that found that approximately four Aedes aegypti (L.) proteins bind to dengue virus Serotype 2 (DENV-2). These studies represent a first step to identifying Cx. p. quinquefasciatus midgut proteins that allow WNV entry into cells. Further studies are needed to deduce if these proteins represent cellular receptors and if these protei ns are involved in vector competence. Midgut genes are also translated into protein components of the innate immune pathway (Christophides et al. 2002, Xi et al. 2008). The immune response to WNV 110

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infection in the mosquito can limit the am ount of viral replication in the midgut of mosquitoes (Xi et al. 2008). Two midgut genes, G1A1 and G43A2, whose expression changed in the presence of WNV, were characterized in Cx. p. quinquefasciatus and found to belong to two mosquito immune response pathways [Toll and i mmune deficiency (Imd)]. The fact that these cDNAs were expressed in the same di fferential display analysis indicates that more than one immune response pathway is likely involved with viral immune response in mosquitoes. To determine the actual role these genes play in the mosquito midgut these genes will need to be silenced and mid gut genes need to be examined over the course of WNV infection. Due to the finding that the CPQG and CP QV colonies showed different vector competence for WNV, further characteri zation of gene expression in the different Cx. p. quinquefasciatus colonies was performed. These co lonies have a known difference in the presence and possibly the mechani sms contributing to the MEB in Cx. p. quinquefasciatus Dissemination rate is greater in the CPQG compared to the CPQV under the same permissive conditions of 28 C, similar virus doses, and at all IPs tested (Anderson et al. 2010, unpubl.). The putativ e GNBP gene (G1A1) in the CPQG colony showed variable expression regardless of WN V infection. The GNBP cDNA in the CPQV colony was expressed at all time poi nts tested, but was expressed at a higher level in mosquitoes that were exposed to WNV. Defensin-1 (G43A2) was present at all time points regardless of the presence of WNV and regardless of the colony tested. The finding that these two colonies expre ssed one midgut gene differently could help explain the disparity in vector competence that has been observed in these colonies. 111

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The GNBP gene could be up-regulated in the mi dgut of CPQV mosquitoes as a means to keep the virus restricted to the midgut und er the conditions of this experiment. The development of high throughput sequencing will allow genes of these colonies to be compared to elucidate differences. Another physiological process affected by the presence of WNV is the eclipse phase as the virus enters midgut cells as obser ved through this research. This process is dose-dependent in the CPQG colony. The eclipse phase of CPQG given a high dose of virus (5.8-6.2 log 10 pfu/mL) occured earlier (1 dpi) compared with mosquitoes given a low dose of virus (4.9-5.3 log 10 pfu/mL) where the eclipse phase occurred over a two day period (1 and 2 dpi). The timing of the eclipse phase is also colony specific, occurring earlier in CPQV (12 hours post-infection) compared to CP QG (1 dpi). This suggests that the speed of entry into midgut cells might activate immune response pathways sooner, which might be involved with the MEB difference observed in the different Cx. p. quinquefasciatus colonies. This is supported by the lower body titers observed in mosquitoes with non-disseminated infections in CPQV compared to the CPQG colonies. There may be more cellula r receptors in midguts of CPQV, allowing more virions to enter and the eclipse phase to occur faster. Virus replication may be occurring earlier in CPQV compared to CP QG midguts. Further studies should be conducted to determine if this is mosquito-v irus specific and influenced by virus dose, EIT, and digestion rate. Four aspects of Cx. p. quinquefasciatus vector competence for WNV were investigated during the course of this proj ect: the impact of environmental factors on infection, dissemination, body and leg ti ter in two different colonies of Cx. p. 112

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quinquefacsciatus ; the impact of WNV on blood digesti on and the relationship to the WNV eclipse phase on Cx. p. quinquefasciatus the immune system function in the midguts of mosquitoes, and the midgut pr oteins that bind to WNV in both Cx. p. quinquefasciatus and Cx. nigripalpus. Although a gene that contributes to vector competence has not been elucidated, these data increase our knowledge and understanding of Cx. p. quinquefasciatus vector competence for WNV and show that the virus and the midgut interact closely to produce an infection that will lead to dissemination and transmission. 113

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Fig. 7-1. This figure shows the potentia l interaction between in trinsic (within the mosquito) factors associated with vector competence. Although preliminary, the preceding research suggests that these factors interact within the Culex pipiens quinquefasciatus midgut to produce a competent vector of West Nile virus. Mosquito midgut genes produce proteins that are involved in the function of blood digestion in the form of digestive enzymes, immune response genes, and receptors that allow WNV entry into the midgut epithelial cells. All of these intrinsic factors can be influenced by extrinsic factors such as temperature. 114

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BIOGRAPHICAL SKETCH Sheri Lizbeth Anderson was born in Denver, CO. She enrolled at Colorado State University in the Fall of 1996 and received he r Bachelor of Science degree in biological science in May 2000. In August 2001, she enro lled at Colorado State University and began her work under the supervision of Dr Donald Nash. Due to unfortunate circumstances, in the spring of 2002, she moved to the supervision of Dr. Janice Moore. In the summer of 2003, she became employ eed as a technician at the Arthropod-Borne and Infectious Disease Laboratory at Colorado State University. She graduated with her Master of Science degree in zoology in the summer of 2006. She became a University of Florida Alumni Award student in the Fall of 2006 under the supervision of Dr. Walter Tabachnick. She performed her research at the Florida Medical Entomology Laboratory under the supervivion of Dr. Chel sea Smartt beginning in the summer of 2007. 129