Formation of reassortant bunyaviruses in dually infected mosquitoes

VIROLOGY

111,662-665

(1981)

Formation B. J.

of Reassortant

BEATY,**’

Bunyaviruses

in Dually Infected Mosquitoes

E.J. ROZHON,-~' P. GENSEMER,t

AND

D. H.L.

BISHOP?

*Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale School of Medicine, 60 College St., P.O. Box 3833, New Haven, Connecticut 06510, and tDepartment of Microbiology, The Medical Center, University of Alabama in Birmingham, Birmingham, Alabama 85294 Accepted Febwaq

2.9, 1981

and temperature-sensitive (ts) bunyaviruses were recovered from Aedes &isafter l-3 weeks extrinsic incubation following intrathoracic inoculation with homologous virus mixtures of Group I and II ts mutants of showshoe hare (SSH), or La Crosse (LAC) viruses (e.g., SSH I and SSH II mutants, or LAC I and LAC II mutants). Wild-type and ts viruses were also recovered from mosquitoes dually infected with heterologous virus mixtures of SSH I and LAC II ts mutants. However, only ts viruses have been obtained from mosquitoes infected with LAC I and SSH II ts mutants. RNA fingerprint analyses of one of the wild-type viruses recovered from a mosquito infected with the SSH I and LAC II ts mutants showed that it had the reassortant virus large/medium/small RNA genotype of SSH/LAC/SSH. Wild-type and ts viruses were also recovered from suckling mice on which mosquitoes that had been infected with homologous virus Group I and II SSH, or LAC, ts mutants were allowed to feed. Likewise wild-type and ts viruses were recovered from mice on which mosquitoes inoculated with SSH I and LAC II ts mutants were allowed to feed. RNA fingerprint analyses of one of these wild-type viruses indicated that it had the reassortant virus genotype of SSH/ LAC/LAC. From mice on which mosquitoes infected with SSH II and LAC I ts mutants had fed, no wild-type viruses were recovered. The inability to detect reassortants having LAC/SSH/LAC and LAC/SSH/SSH genotypes from the in vivo infections expected to yield them (i.e., SSH II plus LAC I) was consistent with previous observations in which such reassortants were only recovered at very low frequencies from in vitro recombination analyses (E. J. Rozhon, P. Gensemer, R. E. Shope, and D. H. L. Bishop, Virology, 1981, in press). Wild-type

eriatus mosquitoes

determined, although a candidate is the 150-180,000-dalton virion polypeptide (2). High-frequency genetic reassortment has been demonstrated in vitro in homologous and certain heterologous virus infections using ts mutants of the California group bunyaviruses SSH, LAC, Tahyna, Lumbo, and trivittatus ((5-g), and unpublished results). The ts mutants have been categorized into groups based on their reassortment capabilities. As exemplified in Table 1, bunyavirus Group I ts mutants have M RNA defects; Group II mutants have L RNA defects (6). Homologous virus genetic reassortment has also been shown for Lumbo virus (9) and for the Bunyamwera serogroup members, Germiston virus (IO), and Guaroa virus (8). The studies with the California group viruses have indicated that closely

Viruses in the family Bunyaviridae have genomes comprised of three segments of RNA, designated large (L), medium (M), and small (S), (2). Analyses have indicated that the members of Bunyavirus genus have an M RNA species that codes for the viral glycoproteins (Gl, G2) and an S RNA species that codes for the viral nucleoprotein (N) (8, 4). The respective N polypeptides of SSH and LAC viruses can be distinguished from each other both by polyacrylamide gel electrophoresis and by tryptic peptide analyses (8). The information encoded in the L RNA has not been ’ To whom ’ Present Northwestern Chicago, Ill.

reprint requests should be addressed. address: Department of Neurology, University McGaw Medical Center 60611.

0042-6822/81/080662-04$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE VIRUSES

USED

FOR DUAL

Virus

cross

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INFECTION

1 OF

Aedes triseriatus

MOSQUITOES”

Expected

SSH/SSH*/SSH6 LAC/LAC*/LAC

I-l I-20

x X

SSH*/SSH/SSH LAC*/LAC/LAC

II-22 II-4

SSH/SSH/SSH LAC/LAC/LAC

SSH/SSH*/SSH SSH*/SSH/SSH

I-l II-21

X X

LAC*/LAC/LAC LAC/LAC*/LAC

II-5 I-20

SSH/LAC/SSH, (LAC/SSH/LAC,

wild-type

reassortant(s)

SSH/LAC/LAC LAC/SSH/SSH)

’ Virus titers were adjusted so that approximately lo’-lo5 PFU (0.6 ~1) of virus were inoculated into each mosquito by the intrathoracic route. b The RNA segment bearing the mutation responsible for the ts phenotype is indicated by an asterisk. The Roman numeral refers to the virus recombination group, the arabic number refers to virus ts isolate number. ‘These reassortants were not detected.

related bunyaviruses can reassort their genomes (5-8). Studies with more distantly related viruses (e.g., Guaroa and LAC) have, however, failed to detect genetic reassortment (8). In addition, particular crosses with closely related viruses have yielded unexpectedly low frequencies of the expected reassortants (e.g., LAC/SSH/SSH and LAC/SSH/LAC from LAC I X SSH II crosses), even though the reciprocal type of cross (i.e., LAC II X SSH I) yields high frequencies of the expected reassortants (SSH/ LAC/SSH and SSH/LAC/LAC) (1, 6-8). Analyses of the mosquito isolates Shark river and Pahayokee viruses (Patois serogroup, Bungavirus genus) have suggested that these two viruses represent naturally occurring reassortant viruses (11). Genotype analyses of LAC virus isolates recovered from the midwestern states of the United States have similarly provided evidence for the existence in nature of intertypic reassortant LAC viruses (R. A. Klimas and D. H. L. Bishop, unpublished data). In the study reported here we have investigated the question of whether reassortant bunyaviruses can be generated experimentally in the natural vector of LAC virus, Aedes triseriatus (12). Groups of 1520 colonized A. triseriatus mosquitoes, approximately 10 to 14 days old, were inoculated by the intrathoracic route with the virus doses and combinations given in Table 1. Mosquitoes were maintained at 23” and 80% relative humidity with 10% su-

crose solution provided ad libitum. At 7 days postinoculation, five mosquitoes were removed from each group, triturated, and frozen at -70”. The remaining mosquitoes in each group were permitted to engorge on a group of five suckling mice. The mice were then removed and observed for 14 days. Brains were recovered from moribund or dead mice on which the mosquitoes had fed. The brains were then triturated and frozen at -70”. Triturated individual mosquitoes, or mouse brain suspensions, were diluted in Eagle’s minimum essential medium and assayed for plaque-forming units (PFU) at 33” using BHK-21 cells (5). Plaques were picked (10 per assay) and the virus in each plaque directly assayed at both 33” (a permissive temperature for the SSH and LAC wild-type and ts mutant viruses) and 39.8” (a permissive temperature for the wild-type viruses, but not for their mutants) (5,s). The recovery of virus from the mosquitoes was usually of the order of l-5 X lo4 PFU, irrespective of whether the mosquitoes were processed 7,14, or 21 days postinoculation. From mouse brains usually 5 X 106-5 X 10’ PFU of virus were recovered. The results obtained for dually infected mosquitoes are expressed in terms of the percentage wild-type and percentage ts viruses recovered (Table 2). The values represent the average of several mosquitoes receiving dual infections. No differences were apparent in the distributions

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given in Table 2, which were obtained for mosquitoes harvested at ‘7, 14, or 21 days postinoculation. On reassay, 20 out of 20 cloned wild-type viruses recovered from either the homologous virus crosses, or from the SSH I-l X LAC II-5 cross, were found to be phenotypically stable wildtype viruses, indicating that they were not mixtures of ts progeny. No wild-type reassortant viruses were recovered from mosquitoes inoculated with the SSH II-21 and LAC I-20 ts mutants. Likewise, out of 40 virus clones analyzed, no wild-type reassortants were detected in mosquito harvests when alternate ts mutants representing the same viruses and their recombination groups were used (SSH II22 and LAC I-13). The failure to detect reassortant wild-type viruses from these crosses is similar to the difficulty we have experienced in recovering LAC/SSH/SSH and LAC/SSH/LAC reassortants from in vitro recombination assays with SSH Group II and LAC Group I ts mutants (I, 6-8). The reason is not known. In order to prove that the wild-type viruses obtained from the dually infected mosquitoes were reassortant viruses, RNA oligonucleotide fingerprint analyses were TABLE VIRUSES

Virus

2

RECOVERED FROM DUALLY INFECTED triseriatus MOSQUITOES Percentage

cross

t.9

Aedes

Percentage wild type“

SSH LAC

I-l I-20

x SSH x LAC

II-22 II-4

95 45

5 55

SSH SSH

I-l II-21

X LAC X LAC

II-5 I-20

35 100

65b 0

cI Viruses recovered from dually infected mosquitoes were plated on BHK-21 cells at 33”, plaques picked and reassayed at both 39.S0 and 33’ to score for ts and wild-type viruses. The results for each cross represent the averages of 40 clonal analyses of viruses obtained from mosquitoes processed after 7,14, and/or 21 days post inoculation. b For different virus clones, both virus-induced intracellular polypeptides, and RNA oligonucleotide fingerprint analyses indicated that SSH/LAC/SSH reassortants were present.

TABLE VIRUSES Virus

3

RECOVERED

FROM MICE

Percentage ts”

cross

Percentage wild type”

SSH LAC

I-l I-20

X SSH X LAC

II-22 II-4

90 20

10 80

SSH SSH

I-l II-21

X LAC X LAC

II-5 I-20

55 100

45* 0

a Viruses recovered from the brains of moribund or dead mice were plated on BHK-21 cells at 33”, plaques picked and reassayed at both 39.8” and 33” to score for ts and wild-type viruses. The results for each cross represent the averages of 40-50 clonal analyses of viruses recovered from one or more mice processed 7,14, and/or 21 days after mosquito feedings. b For different virus clones, both virus-induced intracellular polypeptides and RNA oligonucleotide fingerprint analyses indicated that SSH/LAC/LAC reassortants were present.

used (12). Three of the cloned wild-type progeny from the SSH I-l- and LAC 11-5infected mosquitoes were grown into stocks and analyzed with respect to the intracellular polypeptides they induced (7). Each induced a SSH type of N polypeptide. One of these three viruses was fingerprinted and it was found to have the reassortant virus genotype of SSH/LAC/SSH. The phenotypes of the virus recovered from mouse brains are presented in Table 3. Wild-type and ts viruses were obtained from mice fed upon by mosquitoes that had been inoculated with the homologous combinations of Group I and II ts mutants of SSH, or LAC, or the heterologous combination of SSH I and LAC II ts mutants. Only ts viruses were detected in the brain extracts of mice on which mosquitoes inoculated with SSH II-21 and LAC I-20 viruses were allowed to feed. The virulence of the SSH or LAC ts viruses, and their recovery from mouse brains, was not unexpected since the temperature cutoffs for the SSH and LAC ts mutants that were used are between 3’7” and 39”. Stocks of five cloned wild-type viruses were prepared from the brain extracts of a mouse that had been fed on by mosquitoes

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inoculated ‘7 days prior to feeding with a mixture of SSH I-l and LAC II-5 viruses. All five virus stocks were shown to induce a LAC type of N polypeptide in infected cells (7). When one of these viruses was analyzed by oligonucleotide fingerprinting (12), it was found to have the reassortant virus genotype SSH/LAC/LAC. The results obtained indicate that reassortant viruses have been produced in A. triseriatus mosquitoes inoculated intrathoracically with ts viruses of LAC, or SSH, or LAC Group II and SSH Group I ts mutants. The recovery of ts viruses from mice on which the infected mosquitoes were allowed to feed indicates that the inoculated viruses were transmitted by the mosquitoes. Whether the reassortant viruses recovered from mice were formed in the mice from ts parents, or were produced in the mosquito prior to transmission, is not known. The biological significance of recombination of bunyaviruses in dually infected vector mosquitoes is yet to be ascertained. New strains of influenza viruses are thought to arise by recombination (reassortment) of animal and human viruses in dually infected hosts (14, 15). Recombination of bunyaviruses in vectors would seem to be a realistic mechanism for the generation of new virus genotypes in nature. Unlike vertebrates which have a brief viremic stage, viruses can replicate in the mosquito for the life of the insect (X-18). Since mosquitoes may feed several times, there is ample opportunity for vectors to become infected with more than one virus. The possibility is further enhanced in this system by the fact that LAC virus can be transovarially (19) and venereally (20) transmitted by the vector mosquito, A. trisdutus. Although LAC and SSH viruses are maintained in nature in distinct arbovirus cycles involving different vectors and different vertebrate hosts, they are nonetheless sympatric throughout much of their respective ranges. Thus there is ample opportunity for dual infection of vector species to occur. In these studies mosquitoes were infected by intrathoracic inoculation. Further studies are underway to determine if

recombination can occur under normal circumstances of mosquito infection. ACKNOWLEDGMENTS This study was of Health Grants zhon was supported doctoral fellowship

supported by National Institutes AI-15400 and AI-15426. E. J. Roby a Public Health Service post(Training Grant l-T32-AI-07041). REFERENCES

1. ROZHON, E. J., GENSEMER, P., SHOPE, R. E., and BISHOP, D. H. L., Virology, in press (1981). 2. BISHOP, D. H. L., and SHOPE, R. E., In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 14, pp. 1-156. Plenum Press, New York, 1979. 3. GENTSCH, J., and BISHOP, D. H. L., J. Viral. 28, 417-419 (1978). 4. GENTSCH, J. R., and BISHOP, D. H. L., J. Viral. 30, 767-776 (1979). 5. GENTSCH, J., and BISHOP, D. H. L., J. Virol. 20, 351-354 (1976). 6. GENTSCH, J., WYNNE, L., R., CLEWLEY, J. P., SHOPE, R. E., and BISHOP, D. H. L., J. Virol. 24,893~902 (1977). 7. GENTSCH, J. R., ROBESON, G., and BISHOP, D. H. L., J. Viral. 31,707-717 (1979). J. R., ROZHON, E. J., KLIMAS, R. A., 8. GENTSCH, EL SAID, L. H., SHOPE, R. E., and BISHOP, D. H. L., Virology 102,190-204 (1980). 9. OZDEN, S., and HANNOUN, C., Virology 84, 210212 (1978). 10. OZDEN, S., and HANNOUN, C., Virology 103, 232234 (1980). 11. USHIJIMA, H., CLERX-VAN HAASTER, C. M., and BISHOP, D. H. L., Virology 110.318-332 (1981). 12. LEDUC, J., J. Med. Entomol. 16,1-17 (1979). 13. CLEWLEY, J., GENTSCH, J., and BISHOP, D. H. L., J. Viral. 22,459-468 (1979). U., NAKAJIMA, K., ALFINO, P., 14. DESSELBERGER, PEDERSEN, F., HASELTINE, W., HANNOUN, C., and PALESE, P., Proc. Nat. Acad. Sci. USA 75. 3341-3345 (1978). 15. GARDNER, I., and SHORTRIDGE, K., Rev. Iqfect. Dis. 1,885-890 (1979). 16. CHAMBERLAIN, R., and SUDIA, W., Ann. Rev. EntomoL 6,371-390. (1961). 17. BEATY, B., and THOMPSON, W., Amer. J. TTOP. Med I&g. 25.505-512 (1975). 18. BEATY, B., and THOMPSON, W., J. Med Entomol. 14.499-503 (1978). 19. WATTS, D., PANTUWATANA, S., DEFOLIART, G., YUILL, T., and THOMPSON, W., Science 182. 1140-1141 (1973). 20. THOMPSON, W., and BEATY, B., Science 196,530531 (1977).