Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes

t%as Research, 20 0991) 181-191 0 1991 ElsevierScience Publishers B.V. 016%1702/91/$03.50

181

ADONIS 0168170291001011

VIRUS 00682

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes Laura J. Chandler

‘, Gary Hogge Neal Nathanson

‘, Michael Endres *, David R. Jacoby 2 and Barry J. Beaty i

2,

’ Department of Microbiology, Colorado State University, Fort Collins, Colorado and 2 Department of ~icrobioio~,

Universi@ of Penmylvania, Philadelphja, Pennsyfuania, U.SA (Accepted 17 April 1991)

Summary Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAI-I) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA, pLAC4.27: LAC M RNA, pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAB viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each btot was hybridized with 32P-IabeIled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LIT, 109 (15%); I-IT, 259 (36%); I-IL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%). Bunyauirus; Mosquito; Reassortment;

Corres~ndence

U.S.A.

La Crosse virus; Tahyna virus

to: B. Beaty, Dept. of Microbiology, Colorado State University, Fort Collins, CO 80523,

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Introduction The family Bunyaviridae is comprised of over 250 viruses, which are subdivided into 5 genera based on serological and biochemical studies (Elliott, 1990; Bishop, 1990; Gonzalez-Scarano and Nathanson, 1990). Most of the members of the family are arthropod-borne, and many are significant pathogens of veterinary and/or human importance (Beaty and Calisher, 1991). Bunyaviruses are enveloped RNA viruses, approximately 90 nm in size. The genome consists of three single-stranded segments of negative-sense RNA. The large (L) RNA codes for the polymerase protein (Endres et al., 1989), the middle (M) RNA codes for the two virion glycoproteins, Gl and G2, and the nonstructural protein, NS, (Bishop, 1990; Elliott, 1990; Schmaljohn and Patterson, 1990). The small (S) RNA codes for the nucleocapsid protein and a non-structural protein, NS,. The functions of the two nonstructural proteins are currently unknown (Elliott, 1990). Because of the segmented nature of the genome, bunyaviruses can reassort RNA segments during a mixed infection. Reassortment has been demonstrated in vitro for viruses in several serogroups (Bishop, 1990; Pringle et al., 19841, and reassortant bunyaviruses have also been recovered from nature (Klimas et al., 1981; Shope, 1988; Bishop, 1990). Reassortment could occur in either the vertebrate host or the vector. California serogroup viruses circulate in cycles consisting of vertebrate host and preferred mosquito vector, and many of these cycles are sympatric (Calisher 1983, LeDuc, 1979). Certain vectors will feed on more than one species of animal, thereby intersecting alternate arbovirus cycles. The vector could become infected with two or more viruses, which could reassort. Homologous and heterologous reassortment of two strains of La Crosse (LAC) and snowshoe hare (SSH) viruses, respectively, has been demonstrated in Aedes trzketiatus (Beaty et al., 1985; Beaty and Bishop, 1988; Chandler et al., 1990). Although studies to determine genotypic frequencies of reassortant viruses derived from dual infection of cell culture with bunyaviruses have been performed (Elliott et al., 1984; Pringle et al., 1984), determination of genotypic frequencies of viruses selected during in vivo virus vector interactions has not been attempted. Available technologies, such as oligonucleotide fingerprint analysis, are too expensive and laborious to use to genotype large numbers of virus isolates. We report here the use of a previously developed blot hybridization technique (Pringle, et a1.,1984; Endres et al., 1989; Bishop, 1990) to rapidly genotype LAC, Tahyna (TAH), and LAC-TAH reassortant viruses and the application of the technique to determine genotypic frequencies of LAC and TAH RNA segments in progeny viruses isolated from dually infected Aedes trisetiutus mosquitoes, the natural vector of LAC virus.

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Materials and Methods Mosquitoes Aedes trkeriatus mosquitoes were from a colony that originated from mosquitoes collected in La Crosse, Wisconsin in 1981. Mosquitoes were provided by Dr. Wayne Thompson of the University of Wisconsin. This colony has been maintained at Colorado State University for 7 years. Mosquitoes were reared at 25°C 80% relative humidity with a photoperiod of 16L8D. Mosquitoes were provided with sugar cubes, raisins and water ad libitum.

Two California group viruses, LAC and TAH, were used in these studies. LAC virus is restricted to the United States; TAH virus to Europe and perhaps Eurasia. LAC virus is maintained in a cycle involving A&es triseriatus mosquitoes and forest rodents (Grimstad, 1988). The source and passage history of the LAC virus stock has been described (Beaty et al., 1985). TAH virus is maintained in a cycle involving Aedes vexans and European hares and rabbits (Grimstad, 1988). The prototype TAH virus was obtained from the Yale Arbovirus Research Unit, New Haven, Connecticut. It had been passaged twice in BHK-21 cells. Stock viruses were prepared according to standard procedures. infection and processing of mosquitoes

Adult female Aedes trkeriatus mosquitoes were inoculated intrathoracically with a mixture of equal titers of LAC and TAH viruses. Each mosquito received approximately 30 TCID,, of virus. Control mosquitoes were inoculated with 30 TCID,, of LAC or TAH virus alone. Following inoculation, mosquitoes were held for two weeks extrinsic incubation (EI). Following EI, mosquitoes were triturated individually in 1 ml of tissue culture medium (Hanks MEM-10% fetal bovine serum), and held at -80°C until the time of assay. l&-us isolation and cytoplasmic RNA extraction

Plaque assays were performed as previously described (Chandler et al., 1990). After 6 days, cells were overlaid with neutral red (1: 1000) to allow visualization of plaques. Plaques were picked at limiting dilutions to preclude multiple genotypes and reassortment in the cell culture assay (Chandler et al., 1990) and care was taken to obtain plaques of various sizes. Twenty-four plaques were picked from each mosquito assay. Each plaque was inoculated into a well of a 24-well tissue culture plate containing a confluent monolayer of BHK-21 cells. After allowing for virus replication for 40-48 h at 37”C, cytoplasmic RNA was extracted from the cells (White and Bancroft, 1982). Cells were detached from the well with trypsin,

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placed in a microcentrifuge tube, and pelleted in an Eppendorf microcentrifuge for 15 s. The supernatant was removed and discarded and the cells resuspended in 50 ~1 of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Following resuspension, 10 ~1 of 5% NP40 was added to lyse the cells, the tubes were mixed gently and held in ice for 5 min. Following lysis, samples were centrifuged for 4 min in an Eppendorf microcentrifuge (10K rpm, 4°C) to pellet the nuclei. Supernatant (50 ~1) was removed and placed in a new tube containing 30 ~1 of 20 X SSC (1 X SSC = 0.15 M NaCI-15 mM Na-Citrate, pH 7.0) and 20 ~1 of 37% formaldehyde, then incubated in a water bath at 60°C for 30 min to denature the RNA. Following the RNA denaturation step, 1 ml of 20 x SSC was added to each tube, and samples were stored at - 80 ’ C until the time of RNA analysis. To ensure that a plaque isolate yielded virus, at the time of RNA extraction a 50 ~1 sample of tissue culture fluid was removed from each well and placed in a well of a 96-well tissue culture plate. BHK-21 cells (3500/well) were added, and after incubation for 4 days, cells were examined for cytopathic effects (CPE). Wells which did not yield infectious virus at the time of RNA extraction were not genotyped by the hybridization procedure. As positive and negative controls for hybridizations, BHK-21 cells in 24-well plates were inoculated with LAC or TAH stock viruses (MO1 = 0.011, allowed to incubate at 37°C for 40-48 hours, then cytoplasmic RNA was extracted, blotted and hybridized as described for virus isolated from mosquitoes. Blotting and hybridization

To genotype each virus isolate, the RNA was blotted onto Nytran and subjected to hybridization with each of 3 probes specific for the LAC virus S, M or L RNA segments. For blotting, RNA samples were thawed in a water bath and diluted 1: 2 in 20 x SSC. One hundred microliters of the diluted sample was applied in triplicate to Nytran (Schleicher and Schuell, Keene, New Hampshire) using a Slot-Blot apparatus. Blots were baked at 80°C for 1 h to fix the RNA, then stored dry at 4°C until the time of hybridization. Blots were prehybridized in a solution containing 5 X SSC-5 X Denhardt’s (1 X Denhardt’s = 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 50 mM NaPO,, 250 pg/ml herring sperm DNA, 50% formamide, 0.5% SDS. Prehybridization was for 6 h at hybridization temperature. Following prehybridization, blots were hybridized in a solution containing 5 X SSC-5 X Denhardt’s, 20 mM NaPO,, 200 pg/ml herring sperm DNA, 50% formamide, 0.5% SDS plus labelled probe (l-2 x 10’ cpm/ml). Hybridization was for 20 h at 46°C (pLAC4.161, 50°C (pLAC4.27) or 56°C (pLAC4C-26). Following the hybridization reaction, blots were washed extensively under stringent conditions. Each wash was in 500 ml of solution for 10 min. Washes were: twice in 3 X SSC, 0.1% SDS, twice in 1 X SSC, 0.1% SDS and twice in 0.1 X SSC, 0.1% SDS. Washes were at 55°C (pLAC4.161, 60°C (pLAC4.27) or 65°C (pLAC4C-26). Following washing, blots were air dried for 1 h, then exposed to DuPont Cronex 4 film for 24 h.

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Plasmids

Recombinant plasmids containing complementary DNA (cDNA) copies of the LAC virus L (pLAC 4.161, M (pLAC 4.27) or S (pLAC 4C-26) RNA segments were utilized as hybridization probes in these experiments. The preparation of pLAC 4.16 and 4.27 has been described (Endres et al., 1989). PLAC 4C-26 (Cabradilla et al., 1983) was kindly provided by Dr. J. Patterson. Plasmid DNA was purified from E. cofi cells following standard procedures (Maniatis et al., 1982). For pLAC 4.16 and 4.27, the cDNA inserts were removed by restriction endonuclease digestion with BarnHI. The cDNA inserts were recovered by electrophoresis in a 1% agarose gel followed by electro-elution in a Schleicher and Schuell Elutrap. This material was then ethanol precipitated and reconstituted in TE buffer to a concentration of 1 pg per ml. This purified cDNA insert was used in the labelling reaction. For plasmid pLAC4C-26, whole recombinant plasmid DNA was linearized with Pst I, ethanol precipitated and reconstituted in TE to a concentration of 1 Kg/ml. Probe preparation

Plasmids were labelled on the day of hybridization by the random-priming method of Feinberg and Vogelstein (1983). Random-primed DNA labelling kits were obtained from Boehringer Mannheim (Biochemicals Division, Indianapolis, IN). DNA was labelled with [32P]-dATP and dCTP (Amersham, Chicago, IL). Following the reaction unincorporated nucleotides were separated from labelled DNA by chromatography on Quick-Spin Sephadex G-50 columns (Boehringer Mannheim Biochemicals). The specific activity of the labelled DNA was approximately 1 X lo9 cpm/pg, with > 50% incorporation rates routinely obtained. For hybridization, labelled probe was denatured by heating at 96°C for 5 min, then quenched in ice, and added to the hybridization mixture. Approximately l-2 X 10’ cpm per ml of hybridization solution was added to the blots.

Results Hybridization of UC

probes to LAC or TAH viral RNA

When the LAC probes were hybridized to cytoplasmic extracts of LAC virus infected cells, a strong hybridization signal resulted. No hybridization to uninfected or TAH virus infected cells occurred. This indicated that the probes were specific for LAC virus under the hybridization and wash conditions employed. Hybridization of LAC probes to plaque isolate RNAs from mosquitoes

Twenty-four plaques from each mosquito were selected and propagated individually in BHK-21 cells. Only plaque isolates containing infectious virus were

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analyzed by hybridization. Strong hybridization occurred to the RNA of viruses isolated from mosquitoes inoculated with LAC virus alone (Fig. 1A). No hybridization occurred to the RNA of viruses isolated from mosquitoes inoculated with TAH virus alone (Fig. 1B). Analysis of mosquitoes dually infected with LAC and TAH viruses

A total of 708 plaques from 39 dually infected mosquitoes were analyzed. The genotype of progeny viruses was determined from the presence (LAC) or absence (TAH) of a hybridization band on the film, and expressed as parental source of L/M/S RNA segments. An example of the virus progeny from two mosquitoes, Nos, 32 and 34 is seen in Fig. 2. Of 708 isolates yielding infectious virus, 259 (36%) were TTT, while 150 (21%) were LLL (Fig. 3). The remaining 299 (42%) were reassortant viruses, indicating that high frequency reassortment occurred in mosquitoes dually infected with LAC and TAH viruses. Every genotype expected from a cross of these two viruses was obtained. The distribution of genotypes of progeny reassortant viruses was (as a percent of total progeny): LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTL,

B LAC Control Mosquito #5 Plaque Number L

M

TAH Control Mosquito #11 S

Plaque Number

1

.I

2

2

3

3

4

4

5

5

6

6

7

7

a

8

9

9

10

10

11

11

12

12

Fig. 1. Specificity of pLAC4.16, pLAC4.27 and pLAC4C-26 probes for LAC virus RNAs. Qtoplasmic extracts of BHK-21 cells inoculated with plaque isolates of virus derived from (A) LAC mosquito 3; (B) TAH mosquito 3.

187 LAC

X TAB

MOSQUITO Plaque Number L

M

LAC X TAH MOSQUITO #34

#32

S

Plaque Number

L

M

S

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Fig. 2. Hybridization of pLAC4.16, pLAC4.27 and pLAC4C-26 to cytoplasmic extracts for genotyping reassortant viruses derived from LAC x TAH dually infected mosquitoes (A, mosquita 32; B, mosquito 34). Cytoplasmic extracts of BHK-21 cells infected with plaque isolates were extracted, blotted and hybridized as described in the text. Genotypes are determined by the presence or absence of a hybridization band. L, pLAC4.16; M, pLAC4.27; S, pLAC4C-26.

188

45’ 2

36

4o 35.’

g

30’

z

25’

2 0 2

20’

k

10’

15’

5’ 0-

_ TTT

LLL

LTT

LLT

TLT

LTL

TTL

TLL

GENOTYPE Fig. 3. Distribution

of progeny

virus genotypes (as a percent of total) resulting from dual infection of mosquitoes (n = 708).

16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.3%) (Fig. 3). The distribution of these genotypes is not random (x2 = 550, P < 0.01). The distribution of reassortant viruses was also not random: x2 = 138, P < 0.001. Analysis of reassortant viruses by parental origin of RNA segments

The L RNA segments of reassortant progeny viruses were derived primarily from the LAC parent virus: 73% were from LAC and 27% from TAH (Fig. 4). The

100 90 80 b

5

60 IO

n

LAC

z

50

t

TAH

E

:;

viruses.

(n = 299).

20 IO LARGE

MIDDLE

RNA Fig. 4. Parental

source of RNA segments

SMALL

SEGMENT (as a percent

of total) in reassortant

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frequency

of distribution

of large RNA segments

was not random

(x2 = 64,

P < 0.01). The M RNA segment appeared to segregate freely: 55% of M RNAs were derived from TAH virus, while 45% were derived from LAC virus. This distribution is random (x2 = 2.8, P = > 0.05). In contrast, the S RNA was derived primarily from the TAH parent: 79% from TAH and 21% from LAC (Fig. 5). The frequency of the S RNA segments was not random (x2 = 98, P < 0.01). If all of the reassortant viruses are compared by contingency analysis, the distribution is not random (x2 = 162, P < 0.001).

Discussion

These studies describe the use of the blot hybridization technique (Elliott et al., 1984; Pringle et al., 1983) to detect and genotype reassortant viruses resulting from dual infection of mosquitoes with two parental wild-type viruses. These studies confirm that Aedes triseriatus can serve as a site for the evolution of California group viruses by gene segment reassortment (Beaty and Bishop, 1988). In previous studies, temperature-sensitive mutant viruses from different complementation groups were used to ascertain the potential for Bunyavirus reassortment in dually infected mosquitoes (Beaty and Bishop, 1988). Detection of virus at non-permissive temperatures indicated the presence of reassortant viruses. Although such a biologic assay permitted ready detection of reassortant viruses, results from such studies were of limited value for two reasons. First, only two of the three Bunyavirus reassortment/complementation groups were represented; thus, reassortant events involving the third RNA segment (the S segment), which are not phenotypically detectable, were not detected. Oligonucleotide fingerprint (ONF) analyses could have been used to determine the parental origin of the S RNA segment, but ONF is a difficult, expensive and time-consuming procedure. Thus, genotyping of large numbers of virus isolates with this procedure is not realistic. In addition, parental viruses were mutagenized to obtain the ts phenotypes; thus, there are potential problems with silent mutations determining reassortment frequencies. The molecular hybridization technique described herein permits: (1) relatively easy and inexpensive (as compared to ONF) analysis of reassortment events, (2) genotypic identification of all three RNA segments in reassortant viruses, and (3) utilization of non-mutagenized parental viruses in the studies, thereby permitting a more biologically relevant analysis of reassortment potential of bunyaviruses. These studies suggest that reassortment of RNA segments and/or selection for certain preferred genotypes in mosquitoes is not random. The LAC virus L RNA and TAH S RNA were detected at higher than expected frequencies, whereas the M RNA segment was derived from the LAC and TAH parent in equivalent frequencies. These results differ from in vitro studies with Bunyamwera serogroup viruses in which cosegregation of the L and S segments was observed in vitro. However, the association between L and S segments was not observed when heterologous reassortant viruses were used as the parental viruses (Elliott et al.,

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1984; Pringle et al., 1984). Perhaps there are differences in the reassortment potential for viruses in different serogroups of the Bunyavirus genus or in mosquito vectors. It is necessary to emphasize that these mosquitoes were infected parenterally. Thus, normal determinants of vector-virus interactions, such as midgut infection and dissemination barriers (Beaty and Bishop, 1988; Paulson et al., 19891, were bypassed. Studies are in progress to determine genotypic frequencies resulting from dual oral (natural) infection of Aedes triseriatus mosquitoes with LAC and TAH viruses. Such studies may permit elucidation of the evolutionary potential of bunyaviruses in vectors and of determinants of arbovirus cycle integrity in nature. The molecular hybridization techniques, which have been used to differentiate Bunyavirus reassortants (Elliott et al., 1984; Pringle et al., 1984; Endres et al., 1989) may also be of value for identification of California group viruses isolated from nature. Certain California group viruses are closely related antigenically and difficult to differentiate using conventional serologic assays (Beaty et al., 1988). The probes and techniques reported here readily differentiated LAC and TAH virus RNA segments. More specific probes and techniques are available, which can be used to differentiate more closely related viruses, such as LAC, SSH and their reassortant viruses (Nolan et al., 1989).

Acknowledgements

The authors thank Virginia Wynne and Mandy Story for their excellent technical assistance. This work was supported by Public Health Service Grants AI19688, AI24888 and NS20904 from the National Institutes of Health.

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191 Chandler, L.J., Beaty, B.J., Baldridge, G.D., Bishop, D.H.L. and Hewlett, M.J. (1990) Heterologous reassortment of bunyaviruses in Aedes triseriatus mosquitoes and transovarial and oral transmission of newly evolved genotypes. J. Gen. Virol. 71, 1045-1050. Elliott, R.M. (1990) Molecular biology of the Bunyaviridae. J. Gen. Virol. 71, 501-522. Elliott, R.M., Lees, J.F., Watret, G.E., Clark, W. and Pringle, CR. (1984). Genetic diversity of bunyaviruses and mechanisms of genetic variation. In: A. Kohn and P. Fuchs (Eds), Mechanisms of viral pathogenesis from gene to pathogen, pp. 61-76. Martinus Nijhoff, Boston. Endres, M.J., Jacoby, D.R., Janssen, R.S., Gonzalez-Scarano, F. and Nathanson, N. (1989) The large viral RNA segment of California serogroup bunyaviruses encodes the large viral protein. J. Gen. Virol. 70, 223-228. Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. Gonzalez-Scarano, F. and Nathanson, N. (1990) Bunyaviruses. In: B.N. Fields (Ed.), Virology, 2nd edit., pp. 1195-1228. Raven Press, New York. Grimstad, P.R. (1988). California group virus disease. In: T.P. Monath (Ed.), The Arboviruses: epidemiology and ecology, Vol. II, pp. 99-136. CRC Press, Boca Raton, FL. Klimas, R., Thompson, W.H., Calisher, C.H., Clark, G.G., Grimstad, P.R. and Bishop, D.H.L. (1981) Genotypic varieties of La Crosse virus isolated from different geographic regions of the continental United States and evidence for a naturally occurring intertypic recombinant La Crosse virus. Am. J. Epidemiol. 114, 112-131. LeDuc, J. (1979) The ecology of California group viruses. J. Med. Entomol. 16, 1-17. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. Nolan, K.F., Urquidi, V., Emery, V.C. and Bishop, D.H.L. (1989). The design and use of specific genetic probes to identify closely related bunyaviruses and to determine the genotype of their recombinants. J. Gen. Virol. 70, 2201-2207. Paulson, S.L., Grimstad, P.R. and Craig, G.B. Jr. (1989) Midgut and salivary gland barriers to La Crosse virus dissemination in mosquitoes of the Aedes triseriutm group. Med. Vet. Entomol. 3, 113-123. Pringle, CR., Lees, J.F., Clark, W. and Elliott, R.M. (1984) Genome subunit reassortment among bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes. Virology 135, 244-256. Schmaljohn, C.S. and Patterson, J.L. (1990) Bunyaviridae and their replication. Part II. Replication of Bunyaviridae. In: B.N. Fields (Ed.), Virology, 2nd edit., pp. 1175-1194. Raven Press, New York. Shope, R.E. (1988) Group C viruses. In: T.P. Monath (Ed.), The Arboviruses: epidemiology and ecology, Vol. III, pp. 37-52. CRC Press, Boca Raton, FL. White, B.A. and Bancroft, F.C. (1982) Cytoplasmic dot hybridization. J. Biol. Chem. 257, 8569-8572. (Received 24 January 1991; revision received 16 April 1991)