• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Literature review
 Materials and methods
 Results
 Discussion
 Bibliography
 Biographical sketch














Group Title: Purification, partial characterization, and serology of the capsid and cylindrical inclusion proteins of four isolates of watermelon mosaic virus /
Title: Purification, partial characterization, and serology of the capsid and cylindrical inclusion proteins of four isolates of watermelon mosaic virus
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 Material Information
Title: Purification, partial characterization, and serology of the capsid and cylindrical inclusion proteins of four isolates of watermelon mosaic virus
Physical Description: xi, 96 leaves : ill. ; 28 cm.
Language: English
Creator: Baum, Robert H., 1942- ( Dissertant )
Purcifull, Dan E. ( Thesis advisor )
Edwardson, J. R. ( Reviewer )
Hiebert, E. ( Reviewer )
Pring, D. R. ( Reviewer )
Roberts, D. A. ( Reviewer )
Stall, Robert E. ( Reviewer )
Vasil, I. K. ( Reviewer )
Fry, Jack L. ( Degree grantor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 1980
Copyright Date: 1980
 Subjects
Subject: Mosaic diseases   ( lcsh )
Watermelons -- Diseases and pests   ( lcsh )
Watermelon mosaic virus
Plant Pathology thesis Ph. D
Dissertations, Academic -- Plant Pathology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida.
Bibliography: Bibliography: leaves 89-95.
Statement of Responsibility: by Robert H. Baum.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00099242
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000014237
oclc - 06327738
notis - AAB7436

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
    List of Figures
        Page vii
        Page viii
    Abstract
        Page ix
        Page x
        Page xi
    Introduction
        Page 1
        Page 2
        Page 3
    Literature review
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
    Materials and methods
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
    Results
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
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        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
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        Page 79
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        Page 82
        Page 83
        Page 84
        Page 85
    Discussion
        Page 86
        Page 87
        Page 88
    Bibliography
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
    Biographical sketch
        Page 96
        Page 97
        Page 98
        Page 99
Full Text











PURIFICATION, PARTIAL CHARACTERIZATION, AND SEROLOGY OF
THE CAPSID AND CYLINDRICAL INCLUSION PROTEINS OF
FOUR ISOLATES OF WATERMELON MOSAIC VIRUS



















BY

ROBERT H. BAUM


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREIMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA














ACKNOWLEDGEMENTS


I wish to express my deepest appreciation to my wife, Diane, who

supported and championed me throughout this study and whose patience,

love, and understanding made light the burden of this experience. I

also wish to thank her for the excellent technical advice and expertise

which as an immunologist she was able to offer.

I wish to thank Dr. Dan Purcifull, chairman of my supervisory

committee, for his support and counsel throughout this study and

particularly for his advice on the writing of this dissertation.

Appreciation is extended to other members of my supervisory

committee, Drs. Ernest Hiebert, John R. Edwardson, Daryl R. Pring,

Daniel A. Roberts, Robert E. Stall, and Indra K. Vasil for their help-

ful suggestions during the research and their constructive criticism

of the manuscript. Dr. Purcifull and Dr. Hiebert deserve special

thanks for their untiring efforts to instill in me a need for rigor

and objectivity in research. Dr. F. W. Zettler deserves an A-plus for

the excellent virology courses which he organized and for his ability

to convey to his students his own genuine excitement about virology. I

want to sincerely thank Mr. Richard G. Christie for providing an out-

standing example of whack a scientist should be and also for his attempts

to teach this somewhat colorblind student how to recognize inclusions

in the light microscope. I also thank Richard Christie and Dr.

Edwardson for allowing me to cite some of their unpublished data on

Moroccan inclusion structure in this manuscript. The technical aid










and optimism of Mr. W. E. Crawford is appreciated. I appreciate the

support of former graduate students in Plant Pathology and in particular

those in Plant Virology who never hesitated to pass on to others

knowledge which they had acquired. These are Dr. Albersio Lima, Dr.

Francisco Morales, Dr. David Thornbury, and Ms. Diana Zurawski. In

this same vein, I thank Dr. Mary Conde and Dr. Prem Chourey for discussions,

advice, and friendship. Finally, I am thankful for again being able to

use the excellent typing skills of Ms. Donna Gillis in the final

preparation of this manuscript.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . . . . . . . . .. . . . ii

LIST OF TABLES. . . . . . . . . .. . . . . vi

LIST OF FIGURES . . . . . . . . ... ...... vii

ABSTRACT. . . . . . . . . ... . . . . .. ix

INTRODUCTION. . . . . . . . . ... . . .... 1

LITERATURE REVIEW . . . . . . . . ... . . . 4

WTMV- and WMV-2 Defined . . . . . . . . .. 4
Transmission and General Characteristics. . . . . 5
Differential Systemic and Local Lesion Hosts for
WMV-1 and WMV-2 . . . . . . . . . . . 8
Purification of WMV Isolates. . . . . . . . .. 10
Inclusions ofWMV . . . . . . . . . . .. 13
Serological Relationships of WMV Isolates . . . ... 16

MATERIALS AND METHODS . . . . . . . . .. . . 20

Source of Virus Isolates. . . . . . . . . ... 20
Maintenance and Propagation of Virus Isolates . ..... 20
Virus and Inclusion Purification. . . . . . . ... 21
Purification of Cylindrical Inclusions for Peptide Mapping. 25
Cleavage of WMV Capsid and Cylindrical Inclusion Proteins . 27
Polyacrylamide Gel Electrophoresis of Viral and
Inclusion Proteins . . . . . . . ... 28
Gel Electrophoresis of Cyanogen Bromide Cleaved Viral
Capsid Proteins. . . . . . . . . 28
Serology. . . . . . . . . ... ....... 30
Cross-Absorption of Antisera. . . . . . . . ... 31
Serological Tests . . . . . . . . . . 32
Fractionation of Gamma Globulin for ELISA . . . ... 33
Conjugation of Alkaline Phosphatase with
Gamma Globulin . . . . . . . ... .. 34
Preparation of ELISA Plates . . . . . . . .. 35

RESULTS . . . . . . . . ... . . . . . 39

Purification and Properties of Watermelon Mosaic
Virus and Inclusions . . . . . . . ... 39
Infectivity of Purified Viruses . . . . . . ... 48
Particle Length Determination of WMV-M. . . . . ... 51










Molecular Weight Determination. . . . . . . ... 51
Capsid Protein Digests by Cyanogen Bromide. . . . ... 58
Cylindrical Inclusion Digests. ... . . . . . . 58
Serology. . . . . . . . . ... ....... 58
Enzyme-Linked ImmunosorbentAssay (ELISA) . . . . 75

DISCUSSION. . . . . . . . . ... . . . . 86

LITERATURE CITED. . . . . . . . . ... . . . 89

BIOGRAPHICAL SKETCH . . . . . . . . ... . . . 96















LIST OF TABLES

Table

1 Geographical distribution of watermelon mosaic viruses. . 6

2 Aphid transmissibility of watermelon mosaic viruses . .. 7

3 Serological reactions with W'IV virus antisera ...... .63

4 Serological reactions with WNV inclusion antisera .... .65

5 ELISA serology of WMV isolates. . . . . . . ... 84















LIST OF FIGURES


Figure

1 Scheme for determining optimum concentration of coating
y-globulin and enzyme labelled y-globulin . . . . 38

2 Flow diagram outlining the procedure for purification of
W-MV-2 using n-butanol. . . . . . . . .. 41

3 Flow diagram outlining purification procedure of WMV
isolates and their cylindrical inclusions using
chloroform and carbon tetrachloride . . . . ... 43

4 Second stage in purification of the cylindrical inclusions
of WMV. . . . . . . . . . . . . 45

5 Absorption spectra of purified preparations of WMV
isolates and WMV cytoplasmic inclusions . . . ... '47

6 Electrophoretic analysis of purified undegraded WMV
capsid and cylindrical inclusion protein subunits in
an 8% polyacrylamide gel. . . . . . . . .. 50

7 Histogram of WMV-M particle lengths from a purified
preparation to show particle length from 500 to 900 nm. . 53

8 Electrophoretic analysis of an 8% polyacrylamide gel
of purified WMV isolates stored at 4 C for three
weeks or longer . . . . . . . . .. . . 55

9 Electrophoretic analysis of an 8% polyacrylamide gel of
purified MNV isolates stored at 4 C for three weeks or
longer. . . . . . . . . ... . . .... 57

10 Analysis of cyanogen bromide cleaved WMV capsid proteins
in a 12.5% polyacrylamide gel . . . . . . .. 60

11 Electrophoretic analysis of WMV cylindrical inclusion
proteins partially digested with StaphyZococcus xrieus
V-8 protease. . . . . . . . ... .... . 62

12 Reciprocal SDS-double immunodiffusion tests between
WMV-1, WMV-2, and WMV-M with antisera obtained during
the first four months after initial injection of
immunogen . . . . . . . . ... . . . 68


vii










13 Double immunodiffusion serology of three WMV-M antisera
showing heterologous reactions with WMV-1 isolates. ... 70

14 Intragel cross-absorption tests with WMV-1 Jordan and
WMV-1 Florida using SDS double immunodiffusion tests. . 72

15 Reciprocal SDS double immunodiffusion tests between WMV-2
and BCMV, BlCMV, and SoyMV and heterologous reactions
between WMV-2 antiserum and DMV and PVY . . . ... 74

16 Reciprocal SDS double immunodiffusion tests of WMV
cylindrical inclusions. . . . . ... . .. . 77

17 Heterologous reactivity of WMV-M inclusion antiserum with
other potyviruses by SDS double immunodifffusion
serology. . . . . . . . . ... ..... 79

18 Intragel absorption of WMV-M inclusion antiserum with
inclusions of WMV-1, WMV-2, and WMV-M in an SDS
immunodiffusion medium. . . . . . . . . . 81

19 Intragel absorption of WMV-1 inclusion antiserum with
WMV-1 Jordan inclusions and WMV-1 Florida inclusions
in an SDS double immunodiffusion medium . . . ... 83


viii










Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


PURIFICATION, PARTIAL CHARACTERIZATION, AND SEROLOGY OF
THE CAPSID AND CYLINDRICAL INCLUSION PROTEINS OF
FOUR ISOLATES OF WATERMELON MOSAIC VIRUS

By

Robert H. Baum

March, 1980


Chairman: Dan E. Purcifull
Major Department: Plant Pathology


Watermelon mosaic viruses (WMV), which are members of the potyvirus

group, have been classified into two distinct types, WMV-1 and WMV-2, on

the basis of host range, serology of the capsid proteins and morphology

of virus-induced inclusions. Several isolates, however, are at variance

with the two general types on the basis of host range or serology of

the capsid proteins. The purpose of this study was to further clarify

the distinctiveness of both WMV-1 and WMV-2 and an isolate from Morocco

(WMV-M) by the use of serology and peptide maps of the capsid and

cylindrical inclusion proteins.

Virus isolates used extensively in this study were WMV-M, WMV-l

and WMV-2 from Florida, and WMV-l from Jordan. Viruses were increased

in pumpkin, Cucurbita pepo L. "Small Sugar," and purified by clarification

of sap with a mixture of chloroform and carbon tetrachloride followed by

concentration with polyethylene glycol and fractionation on either

cesium chloride or cesium sulfate isopycnic density gradients. Cyto-

plasmic cylindrical inclusions were purified using the same clarification

procedure followed by fractionation on sucrose step gradients or

preparative polyacrylamide gels.










Antisera to both formaldehyde fixed and unfixed virus and to

purified inclusions were produced in New Zealand white rabbits. Sera

collected following immunization up to approximately one year were

checked for immunochemical specificity.

The serological relationships of selected potyviruses were

determined by enzyme linked immunosorbent assay and sodium dodecyl

sulfate (SDS) immunodiffusion. In reciprocal SDS immunodiffusion tests,

heterologous reactions were obtained between WMV-2 and: bean common

mosaic virus (BCMV), blackeye cowpea mosaic virus (BICMV), and potato

virus Y (PVY), but not WMV-1 (Jordan or Florida), WMV-M, or papaya

ringspot virus (PRSV). WMV-1 Florida and WMV-1 Jordan, hereafter

collectively referred to as WMV-1, were shown to be serologically

identical by intragel absorption in SDS immunodiffusion tests. WNV-1

reacted heterologously with PRSV infected sap but not in reciprocal

immunodiffusion tests with WMV-2, WMV-M, BCMV, BlCMV, or PVY. Sera

from early bleedings of WMV-M did not react with WMV-1 or WMV-2, but

bleedings after four months did give reactions of partial identity with

WMV-1. This was confirmed by intragel absorption and ELISA.

Antisera specific for the cylindrical inclusions of WMV-M, WMV-1,

and WMV-2 in SDS immunodiffusion tests with sap and purified inclusions

were obtained. Antisera to WMV-2 inclusions reacted heterologously

with BCMV, dasheen mosaic virus (DMV), soybean mosaic virus (Soy.MV),

and lettuce mosaic virus (LMV). WMV-M inclusion antiserum reacted

heterologously with WMV-1, WMV-2, DMV, and PRSV and with some isolates

of LMV. The WMV-1 inclusion antiserum did not react with either WMV-M

or WNM-2 inclusions.









Analysis of peptide fragments after cleavage of capsid proteins

with cyanogen bromide (CNBr) showed distinct patterns for WV-1, WMV-2,

and WMV-M. StaphylZoccus aureus V-8 protease digests of the cylindrical

inclusions of WMV-1, WMV-2, and WMV-M were analyzed by disc poly-

acrylamide gel electrophoresis. While there were some similarities

between patterns, significant differences existed.

This study provides additional evidence that WMV-1 and WMV-2 are

distinct viruses on the basis of inclusion serology and analysis of the

peptides formed by partial cleavage of capsid and cylindrical inclusion

proteins. Evidence is also provided that WMV-M is a distinct virus.














INTRODUCTION


Watermelon mosaic viruses, which are members of the potyvirus

group (Brandes, 1964; Harrison et a.., 1971), cause economically

important diseases of cucurbits throughout the world (van Regenmortel,

1971). The watermelon mosaic viruses have flexuous anisometric particles

700-800 nm long, induce cylindrical inclusions in their hosts, and are

transmitted in a nonpersistent manner by aphids (van Regenmortel, 1971;

Edwardson, 1974a).

Symptoms induced by WIMV are highly variable, depending on the

virus strain, the host, and on environmental conditions. Symptoms range

from mild mottling to severe shoestring distortion and blistered ;leaves

(Anderson, 1954; Webb and Scott, 1965; Greber, 1969; Milne et aZ., 1969;

Bakker, 1971; Thomas, 1971a; van Regenmortel, 1971; Webb, 1971; Demski

and Chalkley, 1974; Fischer and Lockhart, 1974; Horvath st aJ., 1975).

Due to variability, this characteristic cannot be readily used to

distinguish watermelon mosaic from other viral diseases of cucurbits.

Serological tests have been used to distinguish six potyviruses that

infect cucurbits (Purcifull and Hiebert, 1979). Other viruses that

infect cucurbits are distinguished from WMV on the basis of the host

range (Lindberg et aZ., 1956; Grogan et al., 1959), mode of transmission

(Greber, 1969), absence of cylindrical inclusions in the cytoplasm of

infected plants (Christie and Edwardson, 1977), physical properties

(Lindberg et a,., 1956), serology, and virus morphology (van Regenmortel

et ai., 1962).









Watermelon mosaic virus was first described by Anderson (1954)

and it was first purified by van Regenmortel (1961). The North American

isolates of the virus were divided into watermelon mosaic virus 1 (WMV-1)

and watermelon mosaic virus 2 (WMV-2) on the basis of the host range

(Webb and Scott, 1965). WMV-1 isolates were limited to the Cucurbitaceae,

whereas WMV-2 had a wider host range covering some 17 plant families

(van Regenmortel, 1971). WMV-1 and MV-2 were reported to be serologi-

cally distinct (Webb and Scott, 1965) and reciprocal tests indicated

that neither could cross-protect against a challenge by the other virus

(Webb and Scott, 1965). It was concluded, therefore, that WMV-1 and

WiV-2 are distinct viruses. The serological results have been con-

firmed (Purcifull and Hiebert, 1979; Greber, 1978; Baum et al., 1979).

However, Milne and Grogan (1969) reported a close serological relation-

ship between WMV-l and WMV-2. On the basis of serology, cross-protection,

indicator hosts, and host range, Milne and Grogan concluded that their

WMV-1 and WMV-2 isolates were strains of the sane virus. Webb and

Scott (1965) suggested the possibility of a third virus or virus group

because they were unable to get a positive serological reaction between

WMV-1 or WMV-2 and van Regenmortel's South African WMV antisera. The

presence of a third member of the watermelon mosaic virus subgroup

was also suggested by Schmelzer (1969) and by Horvath et ac. (1975)

based on differences in host range and symptoms and by Purcifull and

Hiebert (1979) based on serological experiments. They were

unable to detect a serological relationship between a

Moroccan isolate of WMV (WMV-M) (Fischer and Lockhart, 1974)

and antisera to Florida isolates of WMV-I and WMV-2.





3



Although antisera to the cylindrical inclusions have been useful

in studying relationships of other potyviruses (Hiebert et al., 1971;

Purcifull et al., 1973; McDonald and Hiebert, 1975), the serological

relationships of WHV-induced inclusions have not been studied previously.

The objectivesof this dissertation were to: (i) study the sero-

logical relationship of WMV-M to Florida isolates of WMV-1 and WMV-2 and

to a Jordan isolate of WMV-1, (ii) test the use of WMV cylindrical

inclusion antisera in determining relationships among isolates of WMV,

and (iii) analyze the relationships among WMV isolates by comparing

peptide maps of capsid and cylindrical inclusion proteins.














LITERATURE REVIEW


Watermelon mosaic virus was described by Anderson (1951a, 1951b,

1954), who recognized that it was distinct from other cucurbit viruses.

WMV was first noted to be distinct from cucumber mosaic virus (CMV) in

spite of what appeared to be partial cross-protection (Anderson, 1951a).

These distinguishing factors were: a synergistic effect resulting in a

more severe disease when both viruses were present in cucumber

(Cucurmis sativus L.), increased numbers of primary lesions induced by

CMV on watermelon (Citrullus lanatus [Thunb.] Matsun. and Naki) when

inoculation and infection occurred with both viruses, and no evidence

that CMV predominated over WMV (Anderson, 1951a).

The symptoms observed by Anderson on systemically infected leaves

of cucurbits were mild chlorosis, mottle, green vein banding, raised

blisters, distortion, and shoestringing. These have generally been

the symptoms observed on WMV infected cucurbits, but fruit distortion,

stunting, and mottling are often noted--especially on plants infected

early in the season (Thomas, 1971b; Demski and Chalkley, 1974; Fischer

and Lockhart, 1974).

WMV-1 and WMV-2 Defined


Webb and Scott (1965) divided WiN into two distinct viruses,

partly on the host range of North American isolates. WMV-1, as defined

by Webb and Scott (1965) infects only the Cucurbitaceae while WMV-2

can infect plants in 21 additional families (Edwardson, 1974b; Molnar

and Schmelzer, 1964; Webb and Scott, 1965; Greber, 1969; Provvidenti

4










and Schroeder, 1970; Webb, 1971). WMV-2 usually produces milder leaf

symptoms with less distortion and blistering than WMV-1 (Webb and

Scott, 1965). Both viruses have a worldwide distribution (Table 1),

though WMV-1 may have been recently introduced into Europe (Horvath

et al., 1975) and Australia (Greber, 1978) from the western hemisphere.


Transmission and General Characteristics

Seed transmission of WMV was not demonstrated in a test using

several hundred seeds from infected Summer Crookneck Squash (Cucurbita

pepc. var. melopepo Alef.) and cantaloupe (Cucumis melo L.) (Anderson,

1951b). Seed transmission of WMV has not been detected by others using

different isolates of WMV and other cucurbit hosts (Anderson, 1951b;

Grogan et al., 1959; Greber, 1969; Thomas, 1971a; Fischer and Lockhart,

1974; Bhargava, 1977). WMV may therefore be distinguished from viruses

which are seed-borne in cucurbits. These include squash mosaic virus

(SqMV) (Campbell, 1971), and melon necrotic spot virus (MNSV)

(Gonzalez-Garza et at., 1978).

WMIV-1 and WMV-2 have been transmitted in a nonpersistent manner

by several aphid species (Table 2). Aphis gossypii Glover transmits

WMV, single aphids being capable of transmitting WMV to about 50% of

test plants after an acquisition period of 18-36 seconds (Anderson,

1951b). Thomas (1971a) was unable to transmit WMV-2 from infected to

healthy plants of Buttercup Squash (Cucurbita maxima Duch.) using A.

gossypii or Myzus persicae (Sulzer). Bakker (1971) also reported the

nontransmissibility of a Kenyan isolate of WMV by Aphis fabae.

The physical properties observed by Anderson (1954) have been

confirmed for both WMV-1 and WMV-2. The physical properties are: a










Table 1. Geographical
viruses.


Australia

Canada
Chile
Cuba
Czechoslovakia
Eastern Europe


France
Germany
Guadeloupe
Hungary
India


Iran

Iraq
Israel
Italy

Japan
Kenya
Mexico

Morocco
New Zealand
South Africa
United States
Arizona
California

Florida

Georgia
Hawaii


Massachusetts
New York

U.S.S.R.
Venezuela

Yugoslavia


WMV-1
WMV-2
WMV-2
WMV-2
WMV-1
WMV-2
WMV-1
WMV-2
WMV-?
WMV-2
WMV-1
WMV-1
WMV-2
WMV-1
WMV-1
WMV-2
WMV-1
WMV-2
WMV-2
WMV-2


WMV-2
WMV-Kenya
WMV-1
WMV-2
WMV-Morocco
WMV-2
WMV- SA*

WMV-2

WMV-2
WMV-I
WMV-2
WKH- 2

WMV-1
WMV-2
WMV-Kauai**
WMV- 2
WMV-1 I
WMV- 2
WMV-2

WMV-2
WMV-2


**WMV-SA = South African isolate
WMV-Kauai isolates from Kauai were


distribution of watermelon mosaic


Greber, 1978
Greber, 1978
Gates and Bronskill, 1976
Auger et al., 1974
Schmelzer, 1969
Schmelzer and Milicic, 1966
Molnar and Schmelzer, 1964
Molnar and Schmelzer, 1964
Molnar and Schmelzer, 1964
Arteaga et al., 1976
Hein, 1977
Quiot et al., 1971
Horvath et al., 1975
Bhargava, 1977
Ghosh and Mukhopadhyay, 1979
Ghosh and Mukhopadhyay, 1979
Ebrahim-Nesbat, 1974
Ebrahim-Nesbat, 1974
Shawkat and Fegla, 1979
Cohen and Nitzany, 1963
Ragozzino and Stefanis, 1977
Ragozzino and Stefanis, 1977
Inouye, 1964
Bakker, 1971
Milne and Grogan, 1969
Milne and Grogan, 1969
Fischer and Lockhart, 1974
Thomas, 1971a
van Regenmortel, 1961

Nelson and Tuttle, 1969
Milne and Grogan, 1969
Milne and Grogan, 1969
Adlerz, 1969
Adlerz, 1969
Demski, 1968
Shanmugasundaram et al., 1969
Shanmugasundaram et al., 1969
Shanmugasundaram et al., 1969
Komm and Agrios, 1978
Prowidenti and Schroeder, 1970
Providenti and Schroeder, 1970
Schmelzer and Milicic, 1966
Lastra, 1968
Lastra, 1968
Stakic and Nikolic, 1966


distinct from WMV-1 and WMV-2.

















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dilution end-point of approximately 10 to 10 a thermal inactivation

point of about 55 C to 65 C, and a longevity in vitro of about 6 to 20

days (van Regenmortel et al., 1962; Webb and Scott, 1965; Milne and Grogan,

1969; Greber, 1978).


Differential Systemic and Local Lesion Hosts for
WMV-1 and WMV-2


Pinpoint, brown-bordered, local, circular, paper-white necrotic

lesions with minute dark brown centers developed on cotyledons and leaves

of muskmelon (Cuoumis melo L. var. reticulatus Naud.) selection P.I.

180280) inoculated 4-6 days earlier with two isolates of WMV (Webb,

1963). Some plants that developed these local lesions remained free of

virus in secondary leaves whereas others died after developing stem and

top necrosis. Muskmelon cotyledons inoculated with other isolates of WMV

developed a systemic mottle but not local lesions. In reciprocal tests,

no cross-protection occurred with local-lesion and systemic mottle

isolates (Webb, 1963), suggesting that WMV might consist of at least

two unrelated viruses. Further study showed that isolates that induced

local lesions on muskmelon were also restricted to the Cucurbitaceae;

these isolates were hereafter classified as belonging to the WMV-1

group. Those isolates which induced a systemic mottle in muskmelon

also had wider host ranges, including certain species in the families

Leguminosae, Chenopodiaceae, and Euphorbiaceae, and were designated

members of the WMV-2 group (Webb and Scott, 1965). The WMV-2 isolates

were maintained free of WMV-1 by culturing the former in non-cucurbitaceous

hosts (Webb, 1965).

Toba (1962) used Chenopodium amaranticolor Coste and Reyn. in a

host range study of WMV isolates in Hawaii. All of his isolates were










restricted to the Cucurbitaceae and did not form local lesions on C.

;naranticolor. Cohen and Nitzany (1963) studied cucurbit viruses in

Israel. Based on its ability to induce lesions in C. nmaranticolor,

to infect legumes, and on its physical properties, the melon mosaic

virus they reported was of the WMV-2 type. Molnar and Schmelzer (1964)

in an extensive study of the host range of two Eastern European isolates

of WMV, found that several members of the Chenopodiaceae formed local

lesions in response to inoculation with one of these strains but not

with the other. Both strains, however, infected many families outside

the Cucurbitaceae, indicating that not all WMV-2 isolates will form

local lesions on C. amaranticolor. This inability of some isolates of

WMV to induce local lesions on C. vnaranticolor led to the identification

of a virus which was latent in cucurbits and named cucurbit latent virus

(CLV) by Webb and Bohn (1961). CLV formed local lesions in C.

amaranticolor and had a host range similar to WMV-2. Lack of cross-

protection in tests involving CLV infections challenged by several other

viruses (SqMV), CMV, tobacco ringspot virus [TRSV] and WMV) indicated

that CLV was unrelated to these other viruses. Separation of WMV

into WMV-1 and WMV-2 was not recognized in 1961 and Webb and Bohn did

not indicate what isolate of WMV was used in the cross-protection tests.

That CLV was probably synonymous with WMV-2 was suggested by studies

in which it was impossible to separate or differentiate WMV-2 from

CLV (Milbrath and Nelson, 1968; Demski, 1968; Milne et al., 1969). All

of Milne and Grogan's WMV-2 isolates formed local lesions on C.

cnaranticolor and both WMV-2 and CLV were systemic in the malvaceous

plant Lavatera trinestris L. (Milne et at., 1969).









Separation of WMV-1 from WMV-2 was achieved by using Luffa

acutangula Roxb., which was found to be susceptible to WMV-1, but immune

to the WMV-2 isolates tested (Webb, 1965). Symptoms induced by

inoculation of cotyledons with WMV-1 were expressed as mild to severe

chlorotic spots with stunting of leaves and runners. This use of L.

acutangula to separate WI--1 from WMV-2 has been questioned by Milne

et al. (1969), who found it to be susceptible to 16 of 46 isolates

of WMV-2 tested. Several other workers have since used Luffa to

separate WMV-1 from WMV-2 without difficulty (Greber, 1969; Quiot

et al., 1971; Arteaga et al., 1976; Purcifull and Hiebert, 1979).

Bhargava (1977) found that certain cultivars of Luffa aeutangula

were susceptible to two different isolates of WMV-2. These two

isolates, however, were primarily limited to the Cucurbitaceae. Many

species outside the Cucurbitaceae were tested by Bhargava, but only

Vigna sinensis Savi ex Hasski var. Black Turtle was susceptible to

both isolates and Zinnia elegans Jacq. was a symptomless carrier of one

of the isolates. By the use of reciprocal tests, Webb (1971) was unable

to achieve infection with 17 isolates of WMV-2 in L. acutangula by

approach grafting to watermelon or cantaloupe.


Purification of WMV Isolates


Several schemes have been developed or modified for purifying

WMV. van Regenmortel (1961) developed a purification procedure for

several South African isolates of WMV, testing different methods for

clarifying and concentrating infective plant sap. Freezing or treat-

ment of sap with ethanol greatly reduced infectivity and antigenicity.

The use of n-butanol or a mixture of c.loroform and n-butanol was less










effective than chloroform alone for clarification of sap in phosphate

buffer. Three cycles of differential centrifugation were superior in

removing host material as compared with perosmosis, pervaporation,

salting out with ammonium sulfate or acid precipitation with 10% acetic

acid. Following the removal of most host material, the virus was

further purified by zone electrophoresis (van Regenmortel, 1960). Even

though this method was superior to DEAE chromatography, aggregation

sometimes occurred. Preparations of WMV obtained by this method were

satisfactory for the production of antisera but were too aggregated for

determination of particle lengths. An alternative method was developed

(van Regenmortel et al., 1962), in which the leaf sap was clarified

with chloroform in sodium citrate buffer followed by three cycles of

differential centrifugation. The concentrated, clarified virus suspen-

sion was filtered through a column consisting of 4% granulated agar in

sodium citrate buffer. This method removed all visible impurities and

resulted in a nonaggregated virus preparation suitable for particle

length determination, as well as for the production of antisera.

Webb and Scott (1965) clarified infective sap in sodium phosphate

buffer by centrifugation at 5000 g followed by two cycles of differential

centrifugation. The resuspended virus had been concentrated thirty

times based on an infectivity dilution end point of 10-6 after purifica-

tion of WMV-1. They were successful in purifying WMV-1 and producing

an antiserum against it, but were unable to purify sufficient virus for

the production of an antiserum against WMV-2.

Milne and Grogan (1969) used potassium phosphate buffer to

homogenize pumpkin (Cuczurita epo L. 'Small Sugar') leaves infected










with either WMV-1 or WMV-2. The resulting sap was clarified using

n-butanol followed by two cycles of differential centrifugation. The

virus was further purified by zone electrophoresis (van Regenmortel,

1960). Neither the WMV-1 nor WMV-2 preparations reacted with an anti-

serum made against concentrated healthy pumpkin sap. Antisera were

made against the purified WMV-1 and WMV-2. The virus was severely

aggregated after zone electrophoresis, and leaf dips (Hitchborn and

Hills, 1965) were used in normal length determinations of virus

particles.

Purcifull and Hiebert (1979) clarified WMV-1 sap from infected

Small Sugar Pumpkin with a chloroform-carbon tetrachloride mixture in

phosphate buffer. Butanol was used as the organic solvent for clarifying

sap from WMV-2 infected pumpkin leaves. The virus was precipitated

from the aqueous phase with polyethylene glycol and subjected to

equilibrium density gradient centrifugation in CsC1 (p = 1.28 g/ml).

The virus-containing zone was diluted with buffer and subjected to one

cycle of differential centrifugation. These purified virus preparations

were used for determinations of the molecular weights of the capsid

proteins and for the production of antisera. The molecular weights of

undegraded capsid proteins of WMV-1 and WMV-2 were about 36,500 daltons.

Several other workers have used simplified or shortened pro-

cedures to purify WMV-2. Thomas (1971a) and Auger et al. (1974) used

modifications of Milne and Grogan's purification scheme using n-butanol

clarification to purify WMV. Bakker (1971) and Bhargava (1977) used

chloroform clarification to purify isolates of WMV. Fischer and

Lockhart (1974) modified the procedure of Damirdagh and Shepherd










(1970) to partially purify a Moroccan isolate of WMV. Thomas (1971a)

used the purified WMV-2 to produce an antiserum. None of these workers

used zone electrophoresis or cesium chloride equilibrium density gradient

centrifugation in their purification schemes.


Inclusions of WMV

Potyviruses induce the formation of distinctive cylindrical

inclusions in the cytoplasm of infected host cells (Edwardson, 1966;

Purcifull and Edwardson, 1967; Hiebert et al., 1971; Edwardson, 1974a).

Cylindrical inclusions are composed of protein (Shepard, 1968) whose

monomeric subunits have estimated molecular weights of approximately

67,000 to 70,000 daltons (Hiebert and McDonald, 1973) and are immuno-

logically distinct from the viral capsid protein (Shepard and Shalla,

1969; Purcifull et al., 1973; McDonald and Hiebert, 1975) and host

proteins (Purcifull et al., 1973). Edwardson (1966) proposed that the

presence of cytoplasmic cylindrical inclusions was diagnostic for

infection by members of the potyvirus group. Differences in morphology

of the inclusions as determined by ultrastructural studies (Edwardson

ec al., 1968) led to the separation of potyviruses into three sub-

divisions (Edwardson, 1974a). Viruses in Subdivision I, to which WMV-1

belongs, induce tubular inclusions attached to the central portion of

the cylindrical inclusion. In cross section, the inclusions appear

as scrolls, while in longitudinal section they appear as tubes. Sub-

division II viruses form laminated aggregate inclusions attached to

the central portion of the cylindrical inclusion. The laminated

aggregates are usually observed in negatively stained preparations

as roughly triangular or rectangular plates appressed together for part










or all of their length. Viruses in Subdivision III, to which WMV-2

belongs, induce both tubes and laminated aggregates in their host

cells.

Martelli and Russo (1976) found several isolates of WMV which

infected C. waaranticolor and C. quinoa. They therefore classified

these isolates as WMV-2 since their host ranges extended outside the

Cucurbitaceae. These isolates, however, induced tubular inclusions.

(as reported for Subdivision I types) and amorphous cytoplasmic in-

clusions, as reported for WMV-1 (Edwardson, 1974; Christie and Edward-

son, 1977). Purcifull and Hiebert (1979) tested two of Martelli and

Russo's isolates and found that they were serologically identical to

WMV-1 but not to WMV-2, gave a systemic reaction when inoculated to

Luffa acutanrula, and did not infect either Nicotiana benthcniana

(Christie and Crawford, 1978) or Pisum sativuw. Hence the question

of the reliability of using the Chenopodiaceae for typing WMV isolates

is probably more relevant than considering Martelli and Russo's

isolates as exceptions to Edwardson's scheme for separating potyviruses

on the basis of inclusion morphology.

Other types of inclusions are associated with certain potyviruses

(Edwardson, 1974a; Christie and Edwardson, 1977). In addition to

tubular inclusions, WMV-1 induces amorphous inclusions similar to those

induced by papaya ringspot virus (Edwardson, 1974a). A thin plate-like

nuclear inclusion has been detected in cells infected with WMV-2, by

both light and electron microscopy (Christie and Edwardson, 1977).

These nuclear inclusions have not been observed in plants infected

with WMV-1 isolates (Edwardson, 1974a; Christie and Edwardson, 1977).










Many virus induced inclusions can be observed and identified with

the light microscope after relatively simple staining techniques (Christie,

1967; Christie and Edwardson, 1977). These procedures allow for the

rapid screening of infected tissue at minimal time and expense. Often,

accurate identification to potyvirus subgroup or even to a specific

virus is possible. The cytoplasmic and nuclear inclusions induced by

WMV isolates can be detected by these techniques (Christie and Edward-

son, 1977).

The serological properties and relationships of some potyvirus

cylindrical inclusions have been investigated (Hiebert et al., 1971;

Purcifull ec aL., 1973; Batchelor, 1974; McDonald and Hiebert, 1975;

Purcifull and Batchelor, 1977; Lima, 1978; Zurawski, 1979). Antisera

produced against partially purified inclusions of tobacco etch virus

(TEV) and potato virus Y (PVY) gave strong homologous reactions but did

not cross react with each other (Hiebert et al., 1971).

Five potyviruses (TEV, PVY, turnip mosaic virus, bidens mottle

virus, and pepper mottle virus) were found to induce cylindical inclusions

that were serologically distinct (although some were related) and the

propagative hosts did not affect the antigenic specificity of the

inclusions (Purcifull et al., 1973). Antigenic differences between

strains of turnip mosaic virus (TuMV) (McDonald and Hiebert, 1975) were

detected in the capsid proteins but not the cylindrical inclusions even

though one of the three strains studied had distinctly different

laminated aggregates. This work supported the concept that cylindrical

inclusions are coded for by the viral nucleic acid and that serological

studies of inclusions could be useful in determining the taxonomic

relationship between potyviruses.










Direct evidence for the hypothesis that cylindrical and some

nuclear inclusions are products of the potyvirus genome come from in

vitro translation of pepper mottle virus (PeMV) and TEV RNAs (Dougherty,

1979). In these studies, molecular weight determinations utilizing

SDS polyacrylamide gel electrophoresis and serology indicate cylindrical

inclusion and capsid proteins are synthesized in vitro from PeMV and

TEV RNA.


Serological Relationships of WMV Isolates


There have been several points of controversy concerning serological

relationships among WMV isolates. Specifically, there have been dis-

agreements about the serological relationships between WMV-1 and WMV-2

(Webb and Scott, 1965; Milne and Grogan, 1969; Purcifull and Hiebert,

1979), and about the serological relationship of papaya ringspot virus

to WMV-1 and WMV-2 (Milne and Grogan, 1969; Purcifull and Hiebert,

1979). There also have been indications that at least one serotype

distinct from either WMV-1 or WMV-2 may exist (Webb and Scott, 1965;

Purcifull and Hiebert, 1979). Complicating interpretation of the

various results is the use of different virus isolates for the prepar-

ation and testing of antisera, and the use of various types of

serological tests.

Webb and Scott (1965) divided WIV into two groups (WMV-1 and

WMV-2), partly on the basis of serological differences between the two

types. Milne and Grogan (1969), however, reported that WMV-1 and

WMV-2 were serologically very closely related and they concluded that

WMV-1 and WMV-2 should be considered as strains of the same virus.

Several subsequent workers have been unable to find a close serological










relationship between WMV-1 and WMV-2 (Bakker, 1971; Greber, 1978;

Purcifull and Hiebert, 1979). Purcifull and Hiebert (1979) produced

antisera to Florida isolates of WMV-1 and WMV-2. In SDS double

immunodiffusion tests, the WMV-1 antiserum gave a positive reaction

only with isolates of WMV-1. Likewise, the kMV-2 antiserum reacted

only with WMV-2 isolates. Martelli and Russo (1976) found several

isolates of WMV from the Mediterranean region which were limited in

host range to the Cucurbitaceae and Chenopodiaceae. Purcifull and

Hiebert (1979) tested two of these Mediterranean isolates and found

that they reacted with WMV-1 antiserum but not with WMV-2 antiserum.

In addition to serological differences between WMV-1 and WMV-2,

there has been conflicting evidence on the serological relationship

between WMV-1 or WMV-2 and several African isolates of WMV. Some of

this evidence has led to the suggestion that 1WV may consist of three

or more serologically distinct viruses. Webb and Scott (1965) suggested

the presence of a third serologically distinct virus in the WMV group

when they were unable to obtain positive serological reactions in tests

with an antiserum to a South African isolate of WMV and antigens of

North American WMV-1 and WMV-2 isolates. Lastra (1968) obtained

positive reactions in microprecipitin tests between Venezuelan isolates

of WMV-1 and WMV-2 and antiserum obtained from Grogan and thought to have

been made against WMV-1. He was, however, unable to get a positive

reaction using the same isolates and antisera specific for the South

African isolates. Bakker (1971) found that a Kenyan isolate of WMV,

which was limited in host range to the Cucurbitaceae and Chenopodiaceae,

reacted with Milne's WMV-2 antiserum and van Regenmortel's South African










WMV antiserum but not with Milne's WMV-1 antiserum. Milne and Grogan

(1969), however, state that their WMV-1 and WMV-2 isolates gave

positive reactions with the South African WMV antiserum. Purcifull

and Hiebert (1979) got negative reactions between WMV-M and antisera

specific for Florida isolates of WMV-1 or WMV-2. They were unable to

produce an antiserum to WMV-M and therefore considered the possibility

of WMV-M representing a third serotype as tentative. Schmelzer (1969)

suggested the possibility of a third type of WMV based on host range

differences and symptom expression of the South African isolates but

did not present serological evidence.

Serological relationships between WMV and other potyviruses have

been established though some confusion has resulted due to lack of

agreement as to whether WMV-1 and WMV-2 are serologically distinct.

Milne and Grogan (1969) found WMV-1 and WMV-2 to be closely related to

papaya ringspot virus, while Purcifull and Hiebert (1979) obtained

reactions of identity between papaya ringspot virus and WMV-1 antiserum

but did not get a reaction with WMV-2 antiserum. Purcifull and Hiebert

also showed in reciprocal immunodiffusion tests that soybean mosaic

virus was closely related to but distinct from WMV-2. Van Regenmortel

et al. (1962) demonstrated a serological relationship using an antiserum

specific for the South African isolate of WMV and both BYMV and potato

virus Y (PVY). Several other potyviruses which have been reported to

infect cucurbits, viz. LMV, TuMV, and the severe strain of BYMV, were

found to be serologically distinct from WMV-1 and WMV-2 (Florida

isolates) in reciprocal gel immunodiffusion tests performed by Purcifull

and Hiebert (1979). No cross-reactions were observed in reciprocal










tests between WMV-1 and soybean mosaic virus. Blackeye cowpea mosaic

virus (B1CMV) was shown in reciprocal immunodiffusion tests to be related

to but distinct from WMV-2. No cross-reactions were observed in

reciprocal immunodiffusion tests between BlCMV and WMV-1 (Lima et al.,

1979).

Milne and Grogan (1969) obtained precipitin band when detergent-

treated, partially purified papaya ringspot virus (PRSV) was tested with

either WMV-1 or WMV-2 antisera. They observed no detectable serological

difference between PRSV and WMV-1 or WMV-2 antigens created in the same

manner. Purcifull and Hiebert (1979) did not get a detectable reaction

in gel immunodiffusion tests with PRSV in sap and WMV-2 antiserum but

PRSV gave a reaction of serological identity when compared with the

Florida isolate of WMV-1.














MATERIALS AND METHODS


Source of Virus Isolates


The isolates were subcultures of those used in a previous study

(Purcifull and Hiebert, 1979). The Florida strain of WMV-1 (WMV-1

Florida) was obtained originally from W. C. Adlerz. The Florida strain

of WMV-2 was isolated from watermelon in Alachua County by D. E.

Purcifull. The Jordanian isolate of NMV (WMV-1 Jordan) was obtained

from G. Martelli, and the Moroccan isolate of WhV (WMV-M) was received

from B. Lockhart.


Maintenance and Propagation of Virus Isolates


Watermelon mosaic viruses, WMV-1, WMV-2, and WMV-M were propagated

in Cuuobita pepo L. var. Small Sugar Pumpkin. WMV-2 was also prop-

agated in Nicotiana benthamiana Domin. All watermelon mosaic virus

isolates were maintained in separate screened cages. Powdery mildew

was a serious problem on pumpkin, and it was controlled by weekly

sprayings with Dinocap. Frogeye spot, caused by Carcosvora nicotianae

Ell. and Ev., was controlled on N. benthamiana by spraying with Benomyl

when symptoms appeared. Pesticides were used at concentrations

recommended by their manufacturers.

Five to six seeds of pumpkin were sown per six inch pot. Nicotiana

benthxr.iana were seeded in Jiffy pots consisting of peat moss and were transferred

to six inch pots after 30 days Seedlings were germinated in a greenhouse from










which known virus-infected plants were excluded. About one week after

planting and just prior to virus inoculation, pumpkin seedlings were

transferred either to screened cages or greenhouse benches. Pumpkin

seedlings in the cotyledonary stage were mechanically inoculated with

either WMV-1, WMV-2, or WMV-M. Inocula were prepared by grinding

infected pumpkin leaf tissue in water with carborundum. One month old

seedlings of N. benthamiana were mechanically inoculated as above with

WMV-2 infected pumpkin tissue. After inoculation, seedlings were

routinely fertilized every two weeks with a 20-20-20 soluble fertilizer

until harvested.


Virus and Inclusion Purification

Extracts from plants infected with WMV-2 were clarified either with

n-butanol or with a combination of chloroform (CHC13) and carbon tetra-

chloride (CC14) (Lima, 1978; Lima et al., 1979). WMV-1 and WMV-M were

unstable in n-butanol and, therefore, extracts containing them were

clarified only with the CHC13-CC14 combination (Lima, 1978). System-

ically infected pumpkin leaves were harvested 21 to 45 days after

inoculation, whereas infected N. benthamiana leaves were harvested 30 to

90 days after the plants were inoculated. The leaves were kept in

plastic bags up to four days at 4 C before purification of the virus.


n-Butanol Clarification Method


One hundred to 700 g of leaf tissue were homogenized in a blender

with two parts (w/v) of a buffer (homogenization buffer) consisting of

0.5 M potassium phosphate, 0.01 M Na2EDTA, and 0.5% Na2SO3 (pH of the

mi :ure was 7.5 to 7.7). The homogenate was filtered through two leaves










of cheesecloth and centrifuged at 13,200 g (max) for 10 min. The

supernatant, containing virus, was decanted from the pellet, which

contained inclusions. The inclusions were purified from the pellets

as described below. The supernatant was stirred while n-butanol (8 ml

per 100 ml supernatant) was added slowly. The mixture was stirred for

4 hr at 4 C. The coagulated material was removed by a low speed centri-

fugation at 13,200 g for 10 min. The virus was precipitated (Hebert,

1963) from the aqueous phase by adding 8 g of polyethylene glycol MW

6000 (PEG) per 100 ml of supernatant at 4 C with stirring until the PEG

dissolved, followed by centrifugation at 10,400 g for 10 min. The

pellets were resuspended in 0.02 M potassium phosphate, 0.01 M Na2EDTA,

pH 8.2 (virus buffer) and subjected to equilibrium density gradient

centrifugation in CsC1 (p = 1.28 g/ml of virus buffer) in a Beckman

SW 50.1 rotor at 150,000 g (max) for 13 to 18 hr. The virus zone,

located 8 to 11 mm from the bottom of the tube, was collected dropwise

through a hole punched in the bottom of the tube. The collected zone

was then diluted with three volumes of virus buffer. The virus

preparation was further clarified by a low speed centrifugation at

12,000 g and then precipitated as before with PEG. The final pellet

was resuspended in a buffer consisting of 0.02 M Tris, 0.01 M Na2EDTA,

pH 8.0 to 8.2. The virus concentration was determined spectrophoto-

metrically using an extinction coefficient of 2.4 per cm for a 0.1%

solution at 261 nm (Purcifull, 1966). The optical density readings

at 260 and 280 nm were corrected for light scattering by least squares

linear regression analysis of readings taken at 320, 330, 340, 350,

and 360 nm.










Cylindrical Inclusion Purification

The first 13200 g pellets from the n-butanol purification method were

resuspended in 100 to 300 ml of the homogenization buffer and emulsified

in a blender with an organic solvent consisting of a 1:1 solution (v/v)

of chloroform (CHCl3) and carbon tetrachloride (CC14). The ratio of

organic solvent to homogenization buffer was 1:3 (v/v). The emulsion

was broken by centrifugation at 4,080 g for 5 min. The aqueous phase

consisting of inclusions was decanted and filtered through glass wool

and the inclusions were precipitated by centrifugation at 16,300 g for

10 min. The inclusion pellets were resuspended in 0.05 M potassium

phosphate buffer, pH 8.2, containing 0.5% 2-mercaptoethanol (2-ME).

Triton X-100 (TX-100) solution was added to the inclusion preparation

to yield a final concentration of 5% TX-100. This was stirred for one

hr at 4 C. The inclusions were pelleted by centrifugation at 17,300 g

(max) for 15 min and resuspended in 0.02 M potassium phosphate buffer

containing 0.1% 2-ME, pH 8.2 (inclusion buffer). The resuspended

inclusions were layered on a fresh sucrose step gradient consisting

of 6 ml of 80% sucrose, 10 ml of 60% sucrose, and 10 to 12 ml of 50%

sucrose. All sucrose stock solutions were made up in 0.02 M potassium

phosphate buffer, pH 8.2. Inclusions were centrifuged on the fresh

sucrose gradients at 21,000 rpm for 1 hr in a Beckman SW 25.1 rotor.

Inclusions were found layered on top of the 80% sucrose and were

collected dropwise. The inclusion fraction was diluted with 3 volumes

of inclusion buffer and precipitated by centrifugation at 17,300 g

(max) for 15 min. The pellet was resuspended in 0.02 M Tris-HCl, pH

8.2, and the inclusion yield was estimated spectrophotometrically

after being dissolved in 1% sodium dodecyl sulfate (SDS) (Hiebert et al.,










1971). Inclusions were examined in a Philips 200 electron microscope

after negative staining with 2% aqueous uranyl acetate, ammonium

molybdate, or 1% phosphotungstate. The inclusion preparations were

either used immediately for immunization of rabbits or stored by one

of the following methods: freezing with 0.02 M Tris-HCl buffer, pH

8.2 at -20 C, freeze drying in Tris buffer followed by storage at

-20 C, solubilization by dissociation in either 1% SDS or in the

Weber-Osborn dissociation buffer (Hiebert and McDonald, 1973), followed

by freezing at -20 C. Inclusion preparations were tested for proteolytic

degradation and contamination with viral capsid and host proteins by

analytical polyacrylamide gel electrophoresis (PAGE) and by serology

against the corresponding virus antisera.


Chloroform-Carbon Tetrachloride Clarification Method


One hundred to 700 g of leaf tissue were homogenized in a blender

for 1 min with 2 parts (w/v) of the homogenization buffer. The

homogenate was then emulsified by adding to 3 parts of the homogenate,

1 part- (v/v) of a 1:1 solution of CHC13 and CC1I and blending at

high speed for 2 min. Several hundred milliliters of shaved ice were added

during emulsification. The emulsion was broken by centrifugation at

480 g for 5 min. The aqueous phase was decanted, filtered through

glass wool and centrifuged at 12,100 g for 15 min. The supernatant

containing virus was decanted from the pellet containing inclusions.

The supernatant was made 1% (v/v) in TX-100. The mixture was

stirred for 1 hr and centrifuged at 12,100 g for 10 min. The virus

was precipitated from the supernatant with PEG as described previously.

The remainder of the virus purification was essentially the same as










that described for the n-butanol purification scheme except that an

additional PEG precipitation was often required prior to density gradient

centrifugation. When Cs2SO4 was substituted for CsCI during density

gradient centrifugation of WMV-M the amount used was 10.5 g dissolved

in 27 ml of virus buffer and adjusted to a final pH of 8.2 with 1 M KOH.

Purification of the inclusion pellets was identical to the pro-

cedure described previously for the n-butanol purification scheme

beginning with the solubilization step employing TX-100.


Purification of Cylindrical Inclusions for Peptide Mapping


The procedure required 150-200 g of pumpkin leaves showing strong

mosaic and distortion symptoms and free of powdery mildew. The tissue

was homogenized at 4 C in 3 parts of homogenization buffer and 1 part

(v/v) of a 1:1 chloroform-carbon tetrachloride mixture (see virus purifi-

cation). The emulsion was broken by centrifuging at 480 g for 5 min.

The supernatant was filtered through glass wool and the pellet resuspended

in about 250 ml of homogenization buffer and homogenized a second time

at 4 C. The emulsion was broken as before and the filtered supernatant

was combined with the first supernatant and centrifuged at 12,100 g for

20 min. The pellets were resuspended with the aid of a glass tissue

homogenizer, in 36 ml of a pH 8.2, 0.02 M potassium phosphate buffer

containing 0.1% 2-mercaptoethanol (final concentrationP Fourmilliliters of

20% TX-100 (v/v) were then added and the mixture was stirred at 4 C for

90 min. After centrifugation at 27,000 g (max) for 15 min, the green

supernatant was discarded and the light green pellet was resuspended

and washed in buffer two or three times to remove all green pigments.

Occasionally a second exposure to 2% TX-100 was required. After several










washes, the gray inclusion pellet was resuspended in 4 ml of buffer and

homogenized in the micro-homogenizer of a Sorvall Omnimixer at high

speed for 2 min at 4 C. The inclusions were centrifuged at 270 g for

5 min. The supernatant was decanted and a soft pellet consisting

primarily of inclusions was removed by gently resuspending it in buffer

and aspirating. A hard white pellet was discarded. Inclusion protein

concentrations of the supernatant and resuspended soft pellet were

determined spectrophotometrically (Hiebert et al., 1971). The partially

purified preparations were further purified by preparative gel electro-

phoresis using 8% Weber-Osborn gels run in an Ortec 4217 cell with

preparative well former (Ortec Inc., Oak Ridge, Tn.). After electro-

phoresis for 3 to 4 hr, the gels were removed, and immediately

wrapped in clear plastic wrap and chilled overnight at 4 C. The opaque

inclusion band was visible about 2/3 the distance (60 mm) from the bottom

of the gel. The band could be further identified by staining with

Coomassie brilliant blue. The opaque band was cut out by pressing

down on a taut wire held in a coping saw. The band was then cut into

slices approximately 1 mm thick and eluted in 5 to 7 ml of water over-

night at 4 C. The eluate was then removed and saved and the bands

again eluted in water for about S hr at room temperature.

The eluates were pooled and small fragments of polyacrylamide

gel were removed by passing the eluates through a 0.045 pm pore size

membrane filter. The eluates were then dialyzed exhaustively against

0.02 M Tris-HCl, pH 8.0 or water. Concentrations of inclusion proteins

were determined spectrophotometrically prior to pooling and again before

freeze drying. The inclusion samples were tested serologically for

antigenic specificity by double immunodiffusion tests. The dissociated










inclusions were also examined on Weber-Osborn (1969) or Laemmli (1970)

analytical polyacrylamide gels to test for the presence of host or viral

capsid protein contaminants. After freeze drying the inclusion proteins

were stored at -20 C.


Cleavage of WMV Capsid and Cylindrical Inclusion Proteins


Capsid proteins of each virus were cleaved on the carboxyl side of

methionyl peptides with cyanogen bromide (CNBr) (Gross and Witkop, 1962;

Gross, 1967). The purified viruses were resuspended in either 0.02 M

Tris, pH 8.2 or 0.02 M Tris, 0.01 M Na2EDTA, pH 8.2, at concentrations

between 1-7 mg/ml, and then were cleaved by an excess of CNBr (10-20

mg) in 70% formic acid. The reaction was carried out at room temperature

for at least 18 hr. The cleaved peptides were concentrated by lyophili-

zation, resuspended in 2 ml of water, and stored at 4 C. Each uncleaved

virus preparation was examined on analytical Weber-Osborn gels just

prior to CNBr cleavage to insure that the preparation contained only

undegraded virus capsid proteins (Hiebert and McDonald, 1976; Hiebert

et al., 1979).

Partial proteolytic digestion and analysis on polyacrylamide gels

of purified inclusion proteins dissociated in SDS was performed using

Staphylococcus aureus V-8 protease (Miles Laboratories) as described by

Cleveland et al. (1977). The method involved a slight modification of

the procedure described by Cleveland et al. (1977). The acrylamide con-

centration of the stacking gel was increased from 3% to 5.6% for use

on 1.5 mm thick slab gels.

Conditions were optimized based on a kinetic study in which

purified inclusions at concentrations of 300 g/ml were treated by









incubation at 37 C with either no protease or 25 pg/ml of S. aureus

protease for times ranging up to 150 min. Proteolysis was stopped by

adding SDS and 2-ME to final concentrations of 2% and 10%, respectively.

The samples were heated at 100 C for 2 min. Fifteen, 30, or 60 pg of

partially digested inclusions were added to each well. Electrophoresis

was performed in a slab gel apparatus (Studier, 1973) at 100 volts, 21

mA for 4-6 hr. Gels were stained with Coomassie brilliant blue and photo-

graphed. Based on these kinetic studies, the standard conditions used

were 15 pg of inclusion protein (per well) which had been incubated for

30 min at 37 C with 25 pg/ml of S. aureus protease.


Polyacrylamide Gel Electrophoresis of
Viral and Inclusion Proteins

Polyacrylamide slab gel electrophoresis (PAGE) was performed

according to the method of Weber and Osborn (1969) as modified by

Hiebert and McDonald (1973).


Gel Electrophoresis of Cyanogen Bromide
Cleaved Viral Capsid Proteins

WMV capsid proteins which had been cleaved by treatment with

CNBr were electrophoresed on a low pH gel modified from Mauer's gel

system #7 (1971). System #7 required three stock solutions: stock

buffer #17 had a final phase of 4.3 and consisted of approximately 48

ml of IN KOH and 17.2 ml of glacial acetic acid plus sufficient water

to bring the total volume to 100 ml. Stock buffer #19 had a final

pH of 6.7 and consisted of 48 ml of IN KOH, 2.87 ml of glacial acetic

acid and was brought up to a total volume of 100 ml with water. The

electrode buffer stock had a pH of 4.5 and consisted of 31.2 g of










3-alanine, and 8 ml of glacial acetic acid brought up to one liter.

The electrode buffer was a 10% aqueous solution of the electrode

buffer stock.

Thirty milliliters of the running gel body consisted of 11.75 ml

of water, 3.75 ml of buffer #17, 12.5 ml of the acrylamide stock (30 g

monomer and 0.8 g Bis in 100 ml) and 0.2 ml of TEMED. The gel solution

was degassed for 5 min, then 1.8 ml of freshly prepared ammonium

persulfate solution (15 mg/ml) was added and the mixture was poured to

a height of about 72 mm in an Ortec casting stand. The gel was over-

laid with a solution consisting of 0.3 ml of stock #17 and 2.7 ml of

water. Polymerization occurred after transferring the casting stand

to a water bath set at 40 C. The casting stand with the polymerized

gel was removed from the hot water bath and allowed to cool to room

temperature. After cooling, the overlay was removed and a stacking gel

of about 4.5 mm in height was added. The stacking gel consisted of

0.3 ml of buffer #19, 0.4 ml of acrylamide stock, 0.03 ml of TEMED and

1.5 ml of water. To this was added 0.18 ml of ammonium persulfate and

the gel was mixed rapidly and poured on top of the running gel. The

stacking gel was immediately overlaid with a solution consisting of

0.3 ml of buffer #19 and 2.1 ml of water. The cap and well gel

solution was composed of 1 ml of undiluted electrode buffer stock,

2.7 ml of acrylamide stock, 6.3 ml water and 0.01 ml of TEMED. The

combined gel was divided into two 5 ml portions for the well and

capping gels, respectively. Polymerization of both well and capping

gels was achieved by adding 0.7 ml of ammonium persulfate just prior

to pouring. A twelve tined comb was used to form the wells.










The sample buffer was made fresh each time and consisted of 1.08 g

of ultrapure urea (Schwartz/Mann), 0.25 g of sucrose, 0.25 ml of #19

buffer and 0.025 ml of a 1% solution of methylene blue. The sample

buffer was brought up to a total volume of 2 ml with water. Approxi-

mately 50 pg of each CNBr cleaved and dissolved protein was dried on a

spotting plate and redissolved in about 30 pl of sample buffer. Samples

were immediately transferred to the appropriate wells. The capping gel

was pipetted into the wells and the gel former was transferred to the

Ortec vertical slab electrophoresis apparatus, Model 4010,4011. The

anode was attached to the upper tank and the cathode was attached to

the lower tank. Electrophoresis was performed for 3 to 4 hr at 200

volts, with a pulsed power of 300 pulses per second and a current of

approximately 70 mA. After electrophoresis, the gel was stained over-

night in a solution containing 50% methanol, 10% glacial acetic acid

and 0.1% Coomassie brilliant blue stain R 250. The gel was destined

in several changes of a solution consisting of 10% methanol and 7%

glacial acetic acid. The gels were photographed and then scanned using

an automatic gel scanner attached to a Beckman Model 25 recording

spectrophotometer.


Serology


Anitsera for virus and cytoplasmic inclusions were obtained by

injecting adult New Zealand white rabbits with either undegraded virus

capsid or cylindrical inclusion protein or with SDS dissociated capsid

or inclusion proteins obtained from preparative polyacrylamide gels.

The WMV-M capsid immunogens used in the production of antisera were

checked by analytical Weber-Osborn polyacrylamide gels to insure that










partial proteolytic cleavage (Hiebert and McDonald, 1976 had not occurred.

All rabbits were bled for normal serum prior to immunization. The

initial immunizations consisted of intramuscular injections of 1 to 2 mg

of protein in 1 ml 0.02 M Tris buffer, pH 8.2, emulsified with 1 ml

Freund's complete adjuvant (Difco). Rabbits were boosted with one or

two subsequent intramuscular injections two to four weeks apart using

similar quantities of protein emulsified in Freund's incomplete adjuvant

(Difco). All other immunizations involved essentially the same procedure

except that at least one toepad was injected with 0.15 to 0.2 mg of

protein. Antisera to formaldehyde fixed antigens were obtained with

only a single toepad and intramuscular injection. Rabbits were bled

according to the procedure of Purcifull and Batchelor (1977) at approxi-

mately weekly intervals and for three to nine months beginning 10 to

15 days after the first injection.

Antisera also were made against formaldehyde fixed viruses. Dur-

ing virus purifications, antigens were fixed in 1.8% formaldehyde for

10 min at room temperature prior to equilibrium density gradient

centrifugation in CsCI, and prepared for immunization as with unfixed

viruses.

Initially, antisera were freeze-dried and stored at room tempera-

ture. After one year a loss in titer was detected in these antisera

versus frozen antisera. Therefore, the freeze-dried sera were sub-

sequently stored at 4 C or -20 C, and all new serum collections were

frozen at -20 C.


Cross-Absorption of Antisera


Some antisera reacted with healthy plant antigens forming non-

specific precipitates in SDS-double immunodiffusion tests. These









antisera were cross-absorbed with concentrated healthy plant antigens

according to the method of Purcifull et al. (1973). Sixty grams of

frozen SSP were thawed and homogenized in 120 ml of 0.1 M potassium

phosphate buffer, pH 7.4, which contained 1% Na2SO3. The homogenate

was frozen for 3 hr, thawed, and centrifuged at 27,000 g for 10

min. The supernatant was centrifuged at 250,000 g (max) for 3 hr in

a Beckman Ti 60 rotor. The resulting pellet was resuspended in 4 ml

of 0.02 M Tris buffer, pH 7.4. Concentrated host antigens were

combined with the antisera to be cross-absorbed according to the

method of Purcifull and Zitter (1973). Host proteins were mixed with

antisera (1:4, v/v) and incubated overnight at 4 C. The mixture was

centrifuged at 81,000 g for 1 hr in a Beckman Type 40 rotor and the

supernatant containing antisera was used immediately or frozen at

-20 C.


Serological Tests

Double immunodiffusion tests in agar gels, microprecipitin tests,

and enzyme linked immunosorbent assays (ELISA) were preformed during

parts of this study.

Double immunodiffusion tests (Ouchterlony, 1962) were carried out

in agar gels consisting of 0.8% Noble agar (Difco), 0.5% SDS (Sigma),

and 1.0% sodium azide (NaN3) (Sigma) in deionized water (Purcifull and

Batchelor, 1977), or in a medium consisting of 0.8% Noble agar, 0.2%

SDS, 0.1% NaN3, and 0.7% NaC1 in deionized water (Tolin and Roane,

1975). Reactant wells (7 mm in diameter) were arranged in a hexagonal

array produced by an adjustable gel cutting device (Grafar Corp.,

Detroit, Mich.) with a spacing of 4.5 to 5 mm between wells. Plant









tissues were extractedby triturating with a mortar and pestle 1 g of

tissue in either 2 ml of deionized water or in 1 ml of deionized water

followed by the addition of 1 ml of 3% SDS. The extracts were expressed

through two layers of cheesecloth. For routine tests, sap from 5 to 10 g

of tissue were prepared in this manner and frozen in 3 to 4 ml aliquots.

When titering an antiserum, either normal serum or bovine albumin was

used as antiserum diluent (Purcifull and Batchelor, 1977).

Sometimes serological distinctions were demonstrated by the intra-

gel cross-absorption technique (Lima, 1978; Lima et at., 1979). Heterolo-

gous or homologous antigens were added to the center wells of a hexagonal

array. The peripheral wells were cut but the agar was not removed until

antigens in the center well had diffused into the agar. After 16 to

18 hr any remaining fluid in the center well was removed by aspiration

and the agar in the peripheral wells was removed. Appropriate anti-

serum and antigens were then added in the usual manner to the center and

peripheral wells, respectively.

Microprecipitin tests were sometimes used to titer antiserum

according to the procedures of Ball (1974), except that uncoated plastic

Petri dishes were used instead of Formvar-coated glass Petri dishes.


Fractionation of Gamma Globulin for ELISA


Enzyme linked immunosorbent assays (ELISA) were carried out using

a modified procedure of Clark and Adams (1977). The gamma globulins

(yG) used to coat microtiter plates and to conjugate with alkaline

phosphatase were fractionated from antisera collected at least 2 months

after the initial immunization as recommended by Koenig (1978). One

milliliter of antiserum was diluted with 9 ml of deionized water and









stirred at room temperature while 10 ml of a saturated ammonium sulfate

solution was slowly added. The precipitated gamma globulin fraction

was allowed to incubate at room temperature for30 min and.was then collected

by centrifugation at 10,000 g for 10 min. The pellet was resuspended in

2 ml of half strength phosphate buffered saline (PBS), pH 7.4, having

a IX concentration per liter of 8.0 g NaC1, 0.2 g KH2P04, 2.51 g Na2HPO 4

7H20, 0.2 g KC1, and 0.2 g NaN3. A 10X PBS stock solution was maintained

at room temperature. The yG was dialyzed 3 times (approximately 4 hr

each) against 500 ml of half strength PBS, and then filtered through

a 5 ml DEAE Sephacel column previously equilibrated with half strength

PBS. The yG was washed through the column with half strength PBS, and

collected in 1 ml fractions in siliconized glass tubes. Each aliquot of

the first protein to elute was read on a Beckman Model 25 recording

spectrophotometer. Those tubes containing 0.8 O.D. or more were

combined and the yG concentration adjusted to 1 mg/ml (1.4 0.D.280

with half strength PBS (Clark and Adams, 1977). The yG was stored at

-10 C in silicone-treated glass tubes.


Conjugation of Alkaline Phosphatase
with Gamma Globulin

A crystalline suspension (usually 2.5 mg) of alkaline phosphatase

(Sigma No. P4502, 1000 units/mg), in (NH4)2SO4 was centrifuged at 10,000

g for 10 min. The pellets were dissolved in 1 ml (1 mg) of purified yG

and dialyzed 3 times against 500 ml of half strength PBS. A 25%

(v/v) solution of glutaraldehyde was added to the mixture to yield a

final concentration (v/v) of about 0.1%. The solution was incubated

at room temperature for 4 hr during which a slight yellow-brown color










developed. The conjugated yG was then dialyzed 3 times against half

strength PBS as described previously. Five milligrams of bovine serum

albumin was then added per milliliter of conjugate and the mixture stored at

4 C.


Preparation of ELISA Plates

Two hundred microliters of coating buffer containing purified gamma

globulin was added to all except the peripheral wells of round bottom

microtiter plates (Cook MicroELISA Substrate Plates #1223-24, Dynatech

Labs, Inc.). The coating buffer contained 1.5 g Na2SO3, 2.93 g NaHCO3,

and 0.2 g NaN3 per liter and had a pH of 9.6. The optimum concentration

of yG had to be determined for each conjugate, but it was usually in

the range of 1 to 2 yl/ml. The plates were usually incubated 2-6 hr at

room temperature though overnight incubation gave similar results.

Plates were washed three times with PBS-Tween (PBST) which consisted

of the PBS buffer to which had been added 0.5 ml of Tween 20 per liter.

The PBST was dispensed from a wash bottle with great care to prevent

contamination from one well to the next, particularly during all first

washes. Wells were filled with PBST and after at least 3 min the PBST

was removed by shaking the wash solution into a sink. Plates were

blotted on paper towels and washed two more times.

The test antigens were made by triturating leaf tissue in a

buffer consisting of PBST plus 2% (w/v) polyvinyl pyrrolidone (Sigma

PVP-40), using 9 ml buffer per g tissue. Two hundred microliters of each

test antigen usually were added to duplicate wells. Plates were

incubated at 4 C overnight or at 37 C or room temperature for 4-6 hr.










The optimum concentration of enzyme-labelled conjugate was determined

for each conjugate.

Plates were washed 3 times with PBST, and 250 pl of freshly

prepared substrate buffer containing 0.6 to 1.0 mg/ml of p-nitrophenyl

phosphate (Sigma #104-105 tablets) was added to each well. Each liter

of substrate buffer contained 97 ml diethanolamine, 0.2 g NaN3, and

sufficient HCI to give pH 9.3. The plates were incubated at room

temperature for 30 to 60 min, and the reactions were stopped by the

addition of 50 ul of 3 M NaOH to each well.

Reactions were assessed visually by rating on a 0, +, ++, +++

scale and by measurement of absorbance at 405 nm with a Beckman Model 25

spectrophotometer. Absorbance measurements required a dilution (1:2)

with water. Results were sometimes recorded photographically, using

backlighting and Kodak Verachrome 64 Film.

The determination of optimum concentrations of coating gamma

globulin, test and control samples and dilutions of enzyme-labelled

conjugates was similar to the general procedure except that the concen-

trations and setups were as shown in Figure 1.
































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RESULTS


Purification and Properties of Watermelon Mosaic
Viruses and Inclusions

Purification schemes for virus and virus-induced inclusions are

shown diagramatically in Figures 2, 3, and 4. Virus yields were

0.1%
determined using an extinction coefficient for TEV of E = 2.4
261
(Purcifull, 1966). The typical ultraviolet absorption curve (Figure 5)

obtained for all purified WMV isolates had a maximum absorbance between

260 and 262 nm and a minimum at about 245 nm. The 260/280 ratio was

approximately 1.2 after correction for light scattering. This value

is consistent with those found for other potyviruses (e.g., Lima, 1978).

WMV-2 infected pumpkin or N. benthamiana tissue yielded approximately

10 mg of virus per 100 g fresh weight when n-butanol (Figure 2) was

used in the clarification process, as opposed to only 3-4 mg per 100 g

fresh weight when a 1:1 (v/v) mixture of carbon tetrachloride-chloroform

(Figure 3) was used. Both the Florida and Jordan isolates were unstable

in n-butanol and pumpkin infected with these viruses routinely yielded

approximately 10 mg virus per 100 g fresh weight with the carbon

tetrachloride-chloroform clarification method. WMV-M, also unstable

in n-butanol, gave maximum yields of only 3-4 mg per 100 g fresh weight

with the carbon tetrachloride-chloroform method.

To prevent irreversible aggregation of WMV-2, it was necessary to

add 0.01 M Na2EDTA (final concentration) to each solution throughout

the procedure. Neither WMV-1 nor WMV-M required Na2EDTA though it was




































Figure 2. Flow diagram outlining the procedure of purification
of WMV-2 using n-butanol as the clarifying agent,
polyethylene glycol (PEG) for virus concentration,
and CsCI equilibrium density gradient centrifugation
for separation of virus from host components. See
description in Materials and Methods for further
details.








SYSTEMICALLY INFECTED TISSUE
0.5M KPO4 pH 7.5 + 0.01T! Na2EDTA + 0.5% Na2SO3
HOMOGENIZATION
FILTER
I
CENTRIFUGATION: 14,600 g-10min
PELLET
(discard)
(discard) UPERNATANT

8% n-BUTANOL
STIR 6hr
CENTRIFUGATION: 10,400 g-i0min
PELLET -
(discard) SEAAN
SUPERNATANT
I
FILTER
8% PEG
STIR lhr
CENTRIFUGATION: 16,300 g-15min
SUPERNATANT
(discard) PELLET

0.02M KPO4 + 0.01M Na2EDTA pH 8.2
I -
CsC1 GRADIENT CENTRIFUGATION
d=1.28 g/cc 120,000 g max-15-18hr
COLLECT VIRUS ZONE
DILUTE 3X WITH 0.02M KPO4 + 0.01M Na2EDTA pH 8.2
CENTRIFUGATION: 12,000 g-lOmin
PELLET
(discard)
SUPERNATANT
8% PEG
STIR lhr
CENTRIFUGATION
SUPERNATANT
(discard) PEL
PELLET

0.12M TRIS
0.01M NaEDTA pH 8.2
V I R U S

































Figure 3. Flow diagram outlining the procedure of purification
of WMV isolates and the initial separation of the
cytoplasmic cylindrical inclusions from the virus.
The procedure uses chloroform and carbon tetra-
chloride as the clarifying agents and a low speed
centrifugation to pellet the inclusions (see
Figure 4). Polyethylene glycol (PEG) is used to
concentrate the virus, Triton X-100 (TX-100) for
solubilization of the pigments, CsCI or Cs2SO4 for
separation of the virus from host components. For
detailed description, see Materials and Methods.










SYSTDMICALLY INFECTED TISSUE

0.5M KPO0 pH 7.5 0.35 4aSO, (- 0.01M Na2,OTA for 4Y-2)

CHC13 CC

HOMOGENIZATION

CENRIFUGATICN: 6 0xg-.min

DELL-ET SUPERNATANT

3.5HM .- 0.3!M Na,EDTA 'for PV-2)

4OMOGENIZATION CCGSIME SUPERNAT.NTS

CENTRIFUGATION: 50xg-5min :E'ITRIF7 GATION: ;6,200xg-i5min

i--SUPEP.NATANT--FILTER-- PELLET (Inciusians I
(See Next Fiourq)
13-L' .E

'dslcar3) SUPERNATANT 'Virus)


ST:5 1hr

CE:ITRIFUGAT:3N: 0,400oxq-;Cmin

SUPERNATANT----
.,iscard) E~LLET

Q.02M. (P, -- T.Z1M a.. Ar JMV-2)
p.n .L ;1 "'ITIN-4CCO

STIR Thr

:ENTRIFUATlSN: 12,.00xg-;0 nin

PELLET,
'inscara) SU'PERNATANT

%3 ?ES

STIR 30m2n

CEITI JGATION: i0,400xq- 10min

.'PERNATANT-
;discard) 'ELLET

3.32.1 KOPC (- 3.31M Ia,E)TA "cr .M-2E OH 3.2

CsC; GRAOIENT CENTRIFUGATION cssE0, sr MWV-A)
d-I.23g:cc 1C0,00Oxg -15-ihr-

;CLLECT V'IPUS ZONE

DiL'T: 3X :. U1

CE.NTRIFUGATION: 10,400xg-10min

PELLET
1'i sca-'a SUFERNATANT

8% PEG

STIR 3min

CZIEFRIFGATION: 10,Coox3-!0i

SUPE2NATAr.---N-
;discard) PELET

3.02B TRIS pH 13.
(3 0.Ol '4a.,ETA for 4AV-2)'
SY !




























Figure 4. Flow diagram outlining second stage of purification
of cytoplasmic cylindrical inclusions (for first
stage, see Figure 3). Remaining pigments were
solubilized with Triton X-100 (TX-100) and inclusions
separated by centrifugation. Large aggregates of
inclusions were broken up by homogenization at
9,000 rpm 3 min (max.) in a Sorvall omnimixer micro-
homogenizer.

Inclusionsto be purified as dissociated protein sub-
units were separated from a hard starchy-like
pellet by very low speed centrifugation. The
inclusions found in the resulting soft pellet and
supernatant were dissociated in 1% SDS and purified
by preparative PAGE. Inclusion subunits were
eluted from the gel and freeze dried.

Whole inclusions were further purified on a
sucrose step gradient, washed, concentrated
and stored at 4 C in 0.02 M Tris buffer, pH 8.2.









PELLET (INCLUSIONS)
I
0.05M KPO4 pH 8.2 + 0.1% 2-ME

HOMOGENIZATION

5% TRITON- X 100

STIR 90min

CENTRIFUGATION: 27,000 g-l0min

SUPERNATANT
(discard)
PELLET

FOR WHOLE INCLUSIONS FOR INCLUSION SUBUNITS
I (PAGE purification)
WASH 3X 0.02M KPO4 pH 8.2 + 0.1% 2-ME
WASH 3X 0.02M KPO4 pH 8.2 +
HOMOGENIZATION SORVALL 0.1% 2-ME
OMNIMIXER MICROHOMOGENIZER
9000RPM 3min (MAX) CENTRIFUATION: 27,000 g-l0min

SUCROSE STEP GRADIENT -- SUPERNATANT
CENTRIFUGATION: 45,000 g-lhr (discard)

COLLECT INCLUSION ZONE PELLET

DILUTE 0.02M KPO4 pH 8.2 + 0.1% 2-ME 0.02M Tris pH 8.2

CENTRIFUGATION: 27,000 g-15min HOMOGENIZATION SORVALL OMNIMIXER
MICROHOMOGENIZER 9000 RPM
SUPERNATANT 3min (max)
(discard)
CENTRIFUGATION: 250 g-5min
PELLET
I -- HARD PELLET
0.02M TRIS pH 8.2 (discard
INCLUSIONS
DECANT SOFT PELLET AND SUPER-
NATANT

DISSOCIATE IN SDS
I
PREPARATIVE PAGE

ELLTE IN 0.02M TRIS

DIALYZE 0.02M TRIS

FREEZE-DRY

INCLUSION SUBUNITS




































Figure 5. Absorption spectra of purified preparations of
WMV-M in 0.02 M Tris-HC1 buffer, pH 8.2, and
WMV-M dissociated cylindrical inclusions in
0.02 M Tris, pH 8.2, containing 1% SDS.











0.8










I


0.6 L















i3











0.2


WAVELENGTH (nm)
0 34


PURIFIED WM V-M

VIRUS

INCLUSIONS --------


26u


300


320


340 360











usually included. All virus isolates at about 1 mg/ml in Tris

or phosphate buffer showed strong stream birefringence.

Most proteains were not detected by PAGE (Figure 6).

Cylindrical inclusions were unstable in n-butanol and were

always purified using chloroform-carbon tetrachloride for clarifi-

cation of homogenized plant saps (Figure 3). Yields of up to 40

A280 units (1 A280 = 1 mg protein) per 100 g fresh tissue weight

were obtained. A typical ultraviolet absorption is given in Figure

5. Highly purified inclusion proteins were obtained by elution

from preparative polyacrylamide gels. Yields from such gels

averaged about 1 mg purified eluted inclusion subunits for every 3-4

mg of crude inclusion preparation. These highly purified inclusion

proteins which reacted with antisera to untreated inclusions were

used as immunogens and for partial digestion by S. aureus

protease.


Infectivity of Purified Viruses


WMV-1 (Jordan) and WMV-2 (Florida) purified by these methods and

freeze-dried with 0.02 M Tris and 0.01 M NaEDTA at a final concen-

tration of 1 mg/ml were infective to a dilution of about 10-3 mg/ml

after lyophilization and storage for up to one month at room tempera-

ture. Infectivity of WMV-2 was determined by inoculation to a local

lesion host, C. amaranticolor, whereas WMV-1 (Jordan) infectivity was

determined by mechanical inoculation of pumpkin at various dilutions

and observing systemic symptom development.

















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Particle Length Determination of WMV-M


Measurement of 350 purified WMV-M virus particles gave a normal

length of 713 nm (Figure 7). Approximately 50% of the measured particles

were between 675 and 750 nm. Approximately 5% of the examined rods were

clustered between 1400 and 1440 nm as an apparent result of dimer

formation due to end-to-end aggregation.


Molecular Weight Determination


Molecular weights of capsid and inclusion proteins dissociated in

SDS were determined from 8% polyacrylamide analytical gels (Weber-Osborn,

1969) employing proteins of known molecular weights as standards

(Figures 8 and 9). The undegraded capsid proteins of WMV-1 (Florida

and Jordan isolates), WMV-2 (Florida), and WMV-M, each tested at

least four times, gave an average molecular weight for undegraded

capsid protein of 34,000 daltons with a range from 32,500 to 36,000

daltons. Molecular weights of the cylindrical inclusion subunits of

all four WMV isolates ranged from 68,000 to 71,000 daltons based

on 18 determinations, with an average of 69,000 daltons. WMV-1 and

IWMV-2 virus preparations which were stored at 4 C showed typical

proteolytic cleavage with time (Hiebert es al., 1979; Hiebert and

McDonald, 1976) to lower molecular weight forms of approximately 26,000

to 30,000 daltons (Figure 8). Purified preparations of WMV-M did

not have lower molecular weight forms resulting from proteolytic cleavage

on any of the preparations run on polyacrylamide gels. Other than the

apparent resistance of WMV-M to proteolytic cleavage, significant

differences in the molecular weights of the four isolates were not

observed.




































Figure 7. Histogram of lengths of WMV-M particles from
purified preparation negatively stained in
phosphotungstate. Normal length was 713 nm
based on measurement of 350 particles.
Class interval is 25 nm.





























































600 700 800
PARTICLE LENGTH (nm)

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Capsid Protein Digests by Cyanogen Bromide

Digests of capsidproteins of the four WMV isolates by CNBr (Gross and

Witkop, 1962) revealed at least three distinctive patterns when analyzed by

discontinuous PAGE. WMV-M and the two WMV-1 isolates (Florida and Jordan)

formed similar patterns with Mauer's gel system /7 (1971)(Figure 10) while

the position of bands from WMV-2 digests gave consistently different

pattern. All four isolates had two major bands and about seven to eight

minor bands. The digest patterns were similar from gel to gel and with

cleavages from different purifications of the same isolate. The two

major bands, 6 and 7, of WMV-1 Florida showed a slightly slower electro-

phoretic mobility than the two corresponding major bands of WMV-M and

WMV-1 Jordan. Band 9, which was present in WMV-M and WUV-1 Florida, was

missing in WMV-1 Jordan. Band 8, which was detected as a shoulder in WMV-M

and WMV-1 Florida, was not resolved clearly in WMV-1 Jordan Analysis of

peptide patterns on gels made with buffers having a lower pH than the system

described here suggest that WMV-M is distinct from WMV-1 Florida and

WMV-1 Jordan (data not shown).

Cylindrical Inclusion Digests

Purified cylindrical inclusion proteins eluted from preparative

gels and cleaved by Staphylococcus aureus V-8 protease (Cleveland et al.,

1977) gave different peptide fragment patterns for WMV-1 Jordan, WNV-2,

and WMV-M when analyzed on Laemmli discontinuous gels (Laemmli, 1970)

(Figure 11). While the digest patterns for the inclusions of each isolate

were distinctive, there was no apparent variation in the inclusion

patterns of the same isolate purified on different dats.

Serology

Antisera specific for both virus and cylindrical inclusions of

WMV-1, WMV-2, and WMV-M were obtained (Tables 3 and 4). Unless































Figure 10. Electrophoretic analysis of cyanogen bromide cleaved
WMV capsid protein. Undegraded capsid proteins were
cleaved by cyanogen bromide and electrophoresed on a
12.5% polyacrylamide gel using a discontinuous buffer
system (pH 4.5) of acetic acid and 6-alanine. Gels
stained with Coomassie brilliant blue were scanned
at 565 nm. Lanes from left to right contain the
following digests:


WMV-l Florida

M V-1 Jordan
WMV-2

WMV-M


Lane "lA" and the next lane to
its right
Lane "J"
Lane "2" and the next three
lanes to its right
Lane "M" and the next lane
to its right.









CN Br Cleavage


C -iaIiT,;1 B SIf StW F




WMV-2 Florida (2)
(-)


WMV-M Morocco (M)



WMV-1 Jordan (1)





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Table 4. Serological reactions with WMV inclusion antisera.



WMV-1 WMV-2
Antisera Jordan Florida WMV-M

Rabbit No. 903 904a 942 943 928 935b
Immunogen Subunitsc Subunitsc
Treatment None None None (PAGE) None (PAGE)

Inclusion Reaction with Antigensd
Antigens

WMV-M + +

WMV-1
Jordan + + 0

WMV-1
Florida + + 0

WMV-2 + + + +e 0

DMV 0 0 + + 0

BCMV 0 0 + 0

SoyMV 0 0 + 0

LMV 0 0 + + 0

PRSV 0 0 0 + 0

Notes:

bAntisera from all bleedings gave strong healthy reactions
Weak homologous reactions to all bleeding
cSubunits purified by SDS polyacrylamide gel electrophoresis (PAGE)
Reaction with antigens in SDS double immunodiffusion tests:
+ = positive reaction
- = negative reaction
0 = not tested
eReacted with purified inclusions, but not with sap.










specifically noted, antisera did not give a positive reaction with

healthy antigens in gel immunodiffusion tests. All the antisera gave

positive homologous reactions. In reciprocal SDS double immunodiffusion

tests, reactions were negative between WMV-1, WMV-2, and WMV-M with

virus antisera obtained during the first four months following

immunization (Figure 12). The formaldehyde-fixed WMV-M antisera

reacted with WMV-1 (Florida) and WMV-1 (Jordan) after four months

(Figure 13). This antiserum was made from a virus which had a capsid

protein molecular weight of approximately 34,000 daltons, indicating

that it had not undergone proteolytic cleavage (Hiebert and McDonald,

1976). In SDS double immunodiffusion tests, the fixed antigen reacted

with WMV-M antisera but not with WMV-1 antisera. WMV-M antisera

bleedingss taken for three months following immunization) made against

nondegraded capsid which was not formaldehyde fixed did not react

with either isolate of WMV-1 (Figure 13). Antisera collected after

four months formed only faint precipitin bands with WMV-M, precluding

further studies of cross-reactivity with WMV-1 isolates. None of the

other antisera to WMV-1, WMV-2, or WMV-M, representing bleedings taken

up to one year after immunization, gave heterologous reactions.

Florida and Jordan isolates of WMV-1 gave reactions of identity

in reciprocal double immunodiffusion tests. Intragel absorption tests

failed to detect any serological differences between these two isolates

(Figure 14).

In reciprocal SDS double immunodiffusion tests, WMV-2 reacted

with bean common mosaic virus, blackeye cowpea mosaic virus, soybean

mosaic virus (Figure 15). These viruses did not react with WMV-M or

WMV-1 in reciprocal tests.




























Figure 12. Reciprocal SDS-double immunodiffusion tests between
WMV-1, WMV-2, and WMV-M, with antisera obtained
during the first four months after initial injection
of immunogen. Media contains 0.8% Noble agar, 0.5%
SDS and 1% sodium azide.

Center wells were charged with:

Ivs = WMV-1 Florida antiserum
2vs = WMV-2 Florida antiserum
Mvs = WMV-M antiserum.

Antigens in peripheral wells were as follows:

1 = WMV-1 Florida in sap
J = WMV-1 Jordan in sap
2 = WMV-2 in san
M = WMV-M in sap
V = corresponding purified virus preparation,
50 pg/ml
H = healthy pumpkin sap.





























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Antisera to inclusions induced by WMV-1 (Jordan), WMV-2 (Florida)

and WMV-M each gave strong homologous reactions and none reacted with

their respective purified viruses (at antigen concentrations of 50-100

Ug/ml) in SDS double immunodiffusion tests (Figure 16). The WMV-1 and

WMV-2 inclusion antisera reacted homologously only, whereas the WMV-M

inclusion antiserum reacted heterologously with purified inclusion

preparations (400 ug/ml) or crude extract preparations of either WMV-1

or WMV-2 (Figure 17, Table 4) and with sap containing DMV or PRSV

(Figure 17). Intragel absorption of WMV-M inclusion antisera with

purified inclusion preparations at 1 mg/ml of WMV-1, WMV-2, or WMV-M

was performed. Intragel absorption wtih WMV-1 or WMV-2 inclusions

resulted in the formation of a precipitin band between WMV-M inclusions

and its homologous antiserum but not between WMV-M inclusion antiserum

and the cross absorbing heterologous antigens. Intragel absorption by

WMV-M inclusions prevented the formation of any precipitin bands by

WMV-1, WMV-2, or WMV-M inclusions and WMV-M inclusion antisera (Figure

18). Intragel absorption of WMV-1 Jordan inclusion antisera with

either WMV-1 Florida and WMV-1 Jordan purified inclusion preparations

(1 mg/ml) was complete (Figure 19).


Enzyme-Linked ImmunosorbentAssay (ELISA)

Reciprocal tests were conducted with enzyme labeled gamma globulins

specific for WMV-M, WMV-1 (Florida), and WMV-2 (Florida) and their

corresponding antigens (Table 5). The serum conjugates also were

tested against samples containing the following viruses: WMV-1

Jordan, zucchini yellow fleck virus (kindly supplied by C. Volvas),

turnip mosaic virus, potato virus Y, blackeye cowpea mosaic virus,


















Figure 16. Reciprocal SDS double immunodiffusion serology of
WMV cylindrical inclusions.
Center wells were charged with the following anitsera:
Jis = WMV-1 Jordan
2is = WMV-2
Mis = WMV-4
Peripheral wells contained the following antigens:
1 = WMV-1 Florida in sap
Ji = WMV-1 Jordan purified inclusions (400 Hg/ml)
Jv = WMV-1 Jordan purified virus (50 Ug/ml)
2 = WMV-2 in sap
2i = WMV-2 purified inclusions (400 ug/ml)
2v = WMV-2 purified virus (50 ug/ml)
M = WMV-M in sap
Mi = WMV-M purified inclusions (400 Hg/ml)
Mv = WMV-M purified virus (50 Hg/ml)
H = Healthy pumpkin sap
Arrow denotes spur formation. Medium consisted of 0.8%
Noble agar, 0.5% SDS, and 1% sodium azide.







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ONE















Table 5. ELISA serology of WMV isolates.


ELISA Test Antigens in Sap WMV-Mbd WMV-


1. Healthy N. benthamiana 0.00

2. WMV-1 Jordan 0.16

3. WMV-1 Florida 0.10

4. WMV-2 Florida 0.00

5. Zucchini Yellow Fleck Virus 0.00

6. WMV-M 1.10

7. Healthy Cowpea 0.00

8. Turnip Mosaic Virus 0.00

9. Potato Virus Y 0.00

10. Healthy Bean 0.00

11. Healthy Pumpkin 0.00

12. PBS-Tween 0.00

13. Soybean mosaic Virus 0.00

14. Bean Common Mosaic Virus 0.00

15. Healthy Nicotiana eduardsonii 0.00

abused at dilutions (w/v) of 10% and 1% in each.
Coating antiserum conc.: 1 pg/ml; antiserum-enzyme
dilution.
CCoating antiserum cone.: 1 pg/ml; antiserum-enzyme
dilution.
At dilution of test antigens, value represents the
four replications of each test antigen.


-1(Fl)bd


0.00

2.40

0.88

0.01

0.04

3.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00


WMV -2(Fl)cd


0.00

0.00

0.00

5.20

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

3.40

0.20

0.00


conjugate: 1/800


conjugate: 1/300


average OD405 for






85



bean common mosaic virus (Siratro isolate; Lima, 1978), and healthy

saps of pumpkins, bountiful bean, cowpea, Nicotiana benthamiana and

N. ecardsonii Christie and Hall. Only the homologous reactions with

the Moroccan conjugate was clearly positive, though a weak reaction at

the threshold level (Rochow and Carmichael, 1979) occurred with the

Florida and Jordan isolates of WMV-1. The WMV-1 (Florida) conjugate

reacted only with the Florida and Jordan isolates of WMV-1. The Florida

WMV-2 conjugate gave a strong homologous reaction, and heterologous

reactions with soybean mosaic virus and bean common mosaic virus.















DISCUSSION


This study provides new evidence that the relationships among

watermelon mosaic viruses are considerably more complex than was realized

previously (Milne and Grogan, 1969; van Regenmortel, 1977). The evidence

provided herein, however, supports the observations of Webb and Scott

(1965) and Purcifull and Hiebert (1979) that WMV-1 and WMV-2 are

serologically distinct.

Evidence also is presented in this dissertation that WMV-M is indeed

serologically distinct from WMV-1 and WMV-2, as suggested previously by

Purcifull and Hiebert (1979). Gel immunodiffusion tests with antisera

specific for the capsid proteins of WMV-1, WMV-2, or WMV-M, showed that

the three isolates are serologically distinct. These distinctions

applied to antisera collected up to 4 months after the initial injections;

one of the WMV-M antisera collected later than 4 months after immunization

reacted with WMV-1. These results were generally upheld by the ELISA

tests. The WHV-M antiserum showed a weak heterologous reaction with

WMV-1, but failed to react either with WMV-2 or the zucchini yellow

fleck virus from Italy in ELISA tests. Neither the WMV-1 nor WMV-2

antisera reacted with WMV-M in ELISA.

It is of limited value to define WMV-1 as isolates of WMV which

have a host range confined to the Cucurbitaceae (Webb and Scott, 1965).

Purcifull and Hiebert (1979) obtained serological reactions of identity

using Florida WMV-1 antiserum and Mediterranean isolates of WMV whose

host ranges included members of the Chenopodiaceae as well as the










Cucurbitaceae. In this study, antisera were made to the virus and the

cylindrical inclusions of one of the Mediterranean isolates, WMV-1

Jordan. Intragel absorption in reciprocal double immunodiffusion tests

failed to detect differences between the capsids of the two viruses.

In addition, these two WMV-1 isolates produced similar capsid digest

patterns following CNBr cleavage. Intragel cross-absorption of WMV-1

Jordan inclusion antisera with WMV-1 Florida purified inclusions was

complete. These tests confirmed the close relationship between WMV-1

Florida, which has no known hosts outside the Cucurbitaceae (Purcifull

and Hiebert, 1979), and the Jordan isolate, which has a broader host

range (Martelli and Russo, 1976). The concept (Webb and Scott, 1965)

that North American isolates of WMV-1 are limited to the Cucurbitaceae

may also need revision, because an isolate of WMV has recently been

found in South Carolina which caused local lesions on C. canaranticolor

but which was closely related serologically to WMV-1 Florida (personal

communication by 0. W. Barnett).

The serological tests indicated that WMV-1, WMV-2, and WMV-M

inclusions were distinct, although the Moroccan isolate was related

to both WMV-1 and WMV-2. Antisera to inclusions gave stronger reactions

in SDS double immunodiffusion tests with purified inclusions than with

sap extracts. The medium of Tolin and Roane (1975) gave stronger

reactions, but was more likely to result in nonspecific reactions, than

the medium consisting of 0.8% agar, 0.5% SDS, and 1.0% sodium azide

(Purcifull and Batchelor, 1977). These results suggest that it could

be useful to investigate the efficacy of these media, in order to

optimize results in serological detection of inclusion body proteins.









The WMV-1, WMV-2, and WMV-M isolates were compared in other ways.

Unlike WMV-1 and WMV-2, the WMV-M was unstable in cesium chloride. Both

WMV-1 and 1MV-M were unstable in n-butanol, although this solvent was

useful for WMV-2 purification. The CNBr derived peptide pattern of WMV-M

capsid proteins had a degree of similarity with the peptide protein of

WMV-1 isolates when electrophoresed at pH 4.5. However, the peptide

pattern of WMV-M was distinct from those of the WMV-1 isolates when the

pH of the electrode and separating gel buffers were lowered approximately

one unit. The WMV-2 peptide pattern was always distinct from WMV-M and

both WMV-1 isolates. Molecular weights of the cylindrical inclusion

proteins of WMV-1 (both the Florida and Jordan isolates), WMV-2, and WMV-M

all averaged 69,000 daltons. The peptide fragment patterns following

digestion of inclusions with S. aureus protease indicated that WMV-M

patterns were distinct from those of WMV-1 or WMV-2.

This dissertation points to the need for augmenting the standard

techniques presently used to determine strain relationships among poty-

viruses. Peptide mapping of virus-specified proteins is one such approach.

The recently evolved techniques for immunochemical analysis of CNBr cleaved

fragments (Doyen and Lapresle, 1979; Vita et al., 1979) may in the future

be used to map antigenic sites and to expose new sites which may aid

further in determining serological relationships.

In conclusion, this study supports the proposition that there are

at least three serologically distinct viruses involved in the WMV complex.

The three types are represented by the WMV-1, W1MV-2, and WMV-M isolates.

It would be of particular interest to determine the serological relation-

ship of WMV-M to South African isolates (van Regenmortel et al., 1962),

which reportedly also may differ from WfV-1 and WMV-2 (debb and Scott, 1965).















LITERATURE CITED


Adlerz, W. C. 1969. Distribution of watermelon mosaic virus 1 and 2 in
Florida. Proc. Fla. State Horticultural Soc. 81: 161-165.

Anderson, C. W. 1951a. Further observations on some Cucurbit viruses
from central Florida. Plant Dis. Rep. 35: 396-398.

Anderson, C. W. 1951b. Viruses of Cucurbits in central Florida.
Proc. Fla. State Horticultural Soc. 64: 109-112.

Anderson, C. W. 1954. Two watermelon mosaic virus strains from central
Florida. Phytopathology 44: 198-202.

Arceaga, M. P. L., J. B. Quiot, and J. P. Leroux. 1976. Mise en
evidence d'une souche de watermelon mosaic virus (WMV-II) dans
sud-est de la France. Ann. Phytopathol. 8: 347-353.

Auger, J. G., 0. Escaffi, and F. S. Nome. 1974. Occurrence of water-
melon mosaic virus 2 on Cucurbits in Chile. Plant Dis. Rep. 58:
599-602.

Bakker, W. 1971. Notes on East African plant virus diseases: II.
Courgette leaf distortion incited by watermelon mosaic virus. East
African Agric. and Forestry J. 82: 78-85.

Ball, E. M. 1974. Serological tests for the identification of plant
viruses. Am. Phytopathol. Soc., St. Paul, Minn.: 31 pp.

Batchelor, D. L. 1974. Immunogenicity of sodium dodecyl sulfate
denatured plant viral inclusions. Ph.D. dissertation, University
of Florida, Gainesville. 81 pp.

Baum, R. H., D. E. Purcifull, and E. Hiebert. 1979. Purification and
serology of a Moroccan isolate of watermelon mosaic virus (WMV).
Phytopathology 69: 1021-1022.

Bhargava, B. 1977. Some properties of two strains of watermelon mosaic
virus. Phytopathol. Z. 88: 199-208.

Brandes, J. 1964. Identifizierung von gestreckten pflanzenpathogenen
Viren auf morphologischer Grundlage. Mitt. Biol. Bundesanst.
Land Forstwirtsch. (Berlin-Dahlem). 110: 1-130.

Campbell, R. N. 1971. Squash mosaic virus. C.M.I./A.A.B. Descriptions
of Plant Viruses No. 43. 4 pp.




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