J. gen. Virol. (1989), 70, 1011-1016.
Printed in Great Britain
I011
Key words: Wallal virus/reassortant/serotype
Detection of Reassortant Orbiviruses (Wallal Serogroup) in a Prototype
Strain Isolated from a Pool of Biting Midges (Culicoides dyceO
By P. J. W A L K E R , * t J. T A Y L O R AND B. M. G O R M A N
Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane,
Queensland 4006, Australia
(Accepted 18 November 1988)
SUMMARY
Genetic variation between clones selected from early passage pools of Wallal virus
(Reoviridae, Orbivirus) was investigated. The virus had been isolated in 1970 from a
pool of 100 insects (Culicoides dycei), caught in the wild, by laboratory passage in
suckling mice. Gel electrophoresis and oligonucleotide fingerprint analysis of clones
indicated that multiple reassortant genotypes were present in early passages of the
original isolate. The virus, previously described as the prototype strain of Wallal virus,
was one of the reassortant clones. A virus recovered during reisolation from the same
insect pool was genotypically and serologically distinct from the prototype strain and
represents a second Wallal serotype. We have concluded that the clonal variation was
due to the presence of two orbivirus serotypes in the original insect pool and that a
range of reassortant viruses were generated during early passages of the material in
mice.
Details of the isolation of the original strain of Wallal virus (Ch12048) and its reisolation
(Ch12048R) from a pool of biting midges collected in south-west Queensland, have been
provided by Doherty et al. (1973). The strain Ch12048 is registered in the International
Catalogue of Arthropod-borne Viruses (Karabatsos, 1985) as the prototype of the Wallal
serogroup of orbiviruses.
Like other orbiviruses, the genomes of Wallal serogroup viruses consist of 10 segments of
double-stranded RNA. Reassortment of genes between two serotypes of the Wallal serogroup
has been described previously (Gorman et al., 1978; Walker et al., 1980). During the course of
biochemical and genetic studies of Wallal virus, variations in the P A G E migration patterns
have been observed for genome segments of different plaque clones derived from the original
strain (P. J. Walker & B. M. Gorman, unpublished observations; Gonzalez & Knudson, 1988).
In this paper we show that these variations were due to genome segment reassortment between
two orbivirus serotypes that were present in the insect pool from which Wallal virus was
isolated.
The passage histories of the clones used are shown in Fig. 1. Two plaque clones derived from
the original strain were designated A and B. The uncloned virus recovered on reisolation of virus
from the pool of Culicoides dycei was designated Ch12048R. Comparison of the patterns of
migration in P A G E of the R N A of these viruses (Gorman et al., 1978) showed that the
migration of segment 6 differed between Ch12048 clones A and B. The reisolated virus,
Ch12048R, differed from clone A in segments 2, 3, 5 and 8, and from clone B in segments 2, 3, 6
and 8. To define more clearly the genetic relationships between the viruses, individual segments
of each virus were compared by RNase T1 mapping (Walker et al., 1980). The fingerprints of
segments 1 of clone A and Ch12048R differed by a single oligonucleotide. By contrast the
fingerprint of segment 1 of clone B was distinct. Segments 2 and 3 of clone A were almost
t Present address: CSIRO Division of Tropical Animal Production, Private Bag No. 3, Indooroopilly,
Queensland 4068, Australia.
0000-8596 © 1989 SGM
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1012
Insect pool
(100 C. dycei)"~---..~
Reisolation (Ch12048R)
Isolation (Ch12048)
Three passages suckling mice
Thr~.ff.assages suc~.~mice
One passage
suckling mice °
Ten passages
suckling mice
Eight plaques
selected
Plaquelclone B
Six passages
PS-EK cells
Two passages
suckling mice
One passage
PS-EK cells
t,
Plaque clone A
Four pasSages
PS-EK cells
Three passages
suckling mice
Seven passages
PS-EK cells
RNA analysed
RNA analysed
Eight plaques
selected
One passage
suckling mice
RNA analysed
RNA analysed
Mutagen treatment
and selection of
temperature-sensitive
mutants
Wallal tsl01
Three passages
PS-EK cells
RNA analysed
Fig. 1. Passage histories of Wallal viruses derived from the Ch12048 insect pool
Table 1. Relationships between ribonuclease T1 oligonucleotide fingerprints of corresponding
genome segments of Ch12048 clone A, Ch12048 clone B and Ch12048R
Fingerprint type
Segment
Clone A
Clone B
1
X
Y
Ch12048R
X*
2
3
X
X
X*
X*
Y
Y
4
X
X
X
5
6
7
X
X
X
X*
Y
X*
Y
X*
Y
8
X
X
Y
9
10
X
X
X
X
X
X
* Fingerprints that differed from type X in a small number of oligonucleotides only.
identical to those o f clone B yet distinct from Ch12048R (Fig. 2). The complete analysis is shown
in Table 1. F o r each segment no more than two fingerprint types, designated X and Y, were
present. Some segments were shared by all three viruses, while others were shared by only two.
The observed pattern of segment distributions was consistent with the viruses representing the
progeny of a mixed infection in which segment reassortment had occurred.
The earliest available stored pools of the original isolate (Ch12048) and that from the
reisolation (ChI2048R) were examined by P A G E for variations in genotype (Walker et al.,
1980). Eight well isolated limit dilution plaques were selected from each pool. The d s R N A
patterns of clones selected from the Ch12048R pool were homogeneous. By contrast, the
migrations of segments 2, 3, 5, 6 and 9 were variable among the plaque clones from the Ch12048
pool (Fig. 3). One migration pattern, which occurred twice, was identical to that of Ch12048R in
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Fig. 2. RNase T1 oligonucleotide fingerprints of dsRNA genome segments 1 (a, d and g), 2 (b, e and h)
and 3 (c,fand/) of Ch12048 clone A (a, b and c), Ch 12048 clone B (d, e and f) and Ch12048R (g, h and i)
viruses. Oligonucleotides a, b, b', c, c', d, d', e and e' represent minor differences between fingerprints of
corresponding genome segments.
mixed electrophoresis (clones ii and vii, lanes 2 and 7 respectively). The genomes of the other six
clones appeared to be constructed from permutations of the segments selected in the genomes of
ChI2048R and Ch12048 clone A. The specific patterns of segment migration observed for
Ch12048 clone A and Ch12048 clone B were not represented in this series of eight plaque clones
from the Ch12048 pool.
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1014
1
2
3
4
5
6
7
8
Fig. 3. Electrophoresis of dsRNA genome segments of eight plaque clones, i to viii (lanes 1 to 8
respectively), selected from third mouse brain passage of Ch12048 virus.
Table 2. Relationship between Ch12048 clone A ( Wallal), Ch12048R and Mudjinbarry viruses
by plaque reduction neutralization test
Virus
Antiserum
Ch12048 clone A
Ch12048R
Mudjinbarry
Ch12048
clone A
2.6*
0.8
0.1
Ch12048R
1.8
2.6
- 0.3
Mudjinbarry
1.3
1.0
1.8
* Neutralization index = loglo titre (virus + control serum)/titre (virus + antiserum).
Plaque reduction neutralization tests ( G o r m a n et al., 1975) were carried out on Ch12048 clone
A, Ch12048R and the second recognized serotype of the Wallal serogroup, Mudjinbarry virus
(Doherty et aL, 1978). The results in Table 2 indicate the presence of three distinct serotypes, but
the reactions of each virus with Ch12048 clone A antiserum and of Ch12048R antigen with each
antiserum suggests that Ch12048 clone A m a y be more closely related to Ch12048R than to
Mudjinbarry virus.
These data suggest that Wallal virus was generated by gene reassortment between two
serotypes of the Wallal group of orbiviruses. The two serotypes were present in a pool of 100
biting midges which were collected in south-west Queensland. The original isolate had been
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1015
designated the prototype strain (Karabatsos, 1985) but contained multiple genotypes. A
reisolate from the same pool had been confirmed as Wallal virus by a complement fixation test
but by a neutralization test was defined as a distinct serotype. Several other 'Wallal viruses' were
isolated from other pools of biting midges collected at the same time, but none was of a genotype
suitable for generating the set of reassortants detected in the Ch12048 pool (Gorman et al.,
1977). It has not been established whether the reassortants existed in a single insect or were
generated during virus isolation in mice.
The potential for collection of multiple virus types in large pools of wild-caught insects has
been recognized previously (Doherty et al., 1963). In Charleville (Queensland, Australia) in
February 1970, five Wallal strains were isolated from 2707 C. dycei processed in pools of 50 to
150 insects (Doherty et al., 1973; H. A. Standfast, personal communication). At this prevalence
of infection (0.15%), the probability that a pool of 100 insects would contain two or more
infected individuals is approx. 1%, which is significant in view of the large number of insect
pools that are often processed. Doherty et al. (1963) have suggested that during such periods of
intense arbovirus activity the isolation of two different viruses on the same or successive
inoculations had to be expected in a proportion of pools tested. However, this problem is
significantly heightened when viruses with a capacity for genetic interaction are involved, as the
isolation process could lead to the generation of new virus types. There is clearly a need for
extreme caution during the isolation and characterization of segmented genome viruses from
wild-caught insects. Efforts should be made to minimize pool size and, if possible, definitive
identification of isolates should be conducted on plaque clones selected from first cell culture
passage of the insect homogenate.
Gorman (1983) has discussed the concept of speciation in orbiviruses and emphasized the
limitations associated with the use of serotypes to define virus species. Serotype specificity in
orbiviruses appears to be determined by one or possibly two genome segments which encode
polypeptides located on the virion surface (Gorman et al., 1983; Kahlon et al., 1983; Mecham et
al., 1986; Huismans et al., 1987; Mertens et al., 1986). In virus populations that are actively
exchanging genome segments, the serotype relates only to the properties of the surface proteins
of viruses with a common gene pool (Gorman, 1983). The serotype may exist as a multitude of
segment constellations, sharing only type-specific genetic determinants. As such, the mixture of
reassorted viruses obtained during the isolation of Wallal virus may accurately illustrate the
interactive characteristics of orbiviruses in the natural environment.
We wish to thank Professor R. L. Doherty and M r H. A. Standfast for critical review of the manuscript.
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