Arthropod-borne and Infectious Diseases Laboratory (AIDL), Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1682, USA1
Department of Entomology, North Carolina State University, Raleigh, NC 27965, USA2
Author for correspondence: Barry J. Beaty. Fax +1 970 491 8323. e-mail bbeaty{at}cvmbs.colostate.edu
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Abstract |
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Introduction |
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LAC virus is transovarially transmitted by A. triseriatus efficiently (Beaty & Bishop, 1988 ). Transovarially infected (TI+) mosquitoes may subsequently ingest a viraemic blood meal. If interference to superinfection also occurs in TI+ mosquitoes, virus evolution via gene segment reassortment would be restricted. Because virus titres in all tissues of TI+ mosquitoes are lower than in orally infected mosquitoes (Turell et al., 1982
), we hypothesized that TI+ mosquitoes could be superinfected with a second virus when exposed to a viraemic blood meal.
In this study, we demonstrate that TI+ mosquitoes do not exhibit complete interference to superinfection. Multiple approaches were utilized to demonstrate that a significant proportion of TI+ mosquitoes can become superinfected, and that segment reassortment can occur in dually infected mosquitoes.
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Methods |
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Mosquitoes.
A. triseriatus mosquitoes were from a colony that originated from mosquitoes collected in La Crosse, WI, USA. This colony has been maintained at AIDL continuously since 1982. Mosquitoes were reared at 23 °C, 80% relative humidity with a photocycle of 16 h light:8 h dark. Mosquitoes were provided sugar cubes, raisins and water ad libitum.
Establishment of TI+ A. triseriatus colonies.
The wt LAC and L-I-16 viruses were used to establish colonies of TI+ A. triseriatus mosquitoes. First generation mosquitoes were infected with a ts LAC virus by intrathoracic inoculation or with wt LAC virus orally. TI+ mosquitoes were used to found the next generation. Adult mosquitoes were screened for virus infection by fluorescent antibody (FA) testing. One leg was removed, squashed onto a microscope slide and assayed by FA (Beaty et al., 1985 ). TI+ mosquitoes were used in these superinfection studies.
Oral challenge of mosquitoes.
TI+ and control mosquitoes were orally challenged with virus within 2 weeks of eclosion. TI+ mosquitoes ingested either wt LAC or SSH virus via infected blood meal, and then were held for 2 weeks extrinsic incubation (EI). Control mosquitoes (TI-) ingested the same virus-containing blood meal. After 2 weeks EI, mosquitoes were assayed for superinfecting virus.
Quantification of virus in mosquito midguts.
Midguts of TI+ mosquitoes were individually tested by plaque assay in Vero cells to determine the virus titre. Midguts of orally infected mosquitoes were assayed at 1, 2, 3 and 7 days post blood feeding for infectious virus by plaque assay in Vero cells.
Assay for superinfection
Mosquitoes were assayed for the presence of superinfecting virus by three different techniques: (1) biological assay for infectious virus at a non-permissive temperature; (2) molecular hybridization; and (3) RTPCR using primers designed to distinguish LAC virus RNA segments from SSH virus RNA segments.
(1) Biological assay.
Mosquitoes that were TI+ with ts LAC virus and orally challenged with wt LAC virus were assayed for wt LAC virus by comparison of titres obtained by plaque assay in Vero cells at permissive (33 °C) and non-permissive (40 °C) temperatures (Beaty et al., 1985 ).
(2) Hybridization assay.
Mosquitoes that were TI+ with wt LAC virus and orally challenged with SSH virus were assayed for SSH virus by nucleic acid hybridization (Chandler et al., 1990 ). Triturated mosquitoes were subjected to plaque assay in Vero cells. Well-isolated plaques were identified and picked individually. Virus from each plaque was amplified in BHK-21 cells and total cytoplasmic RNA was extracted from the cells (White & Bancroft, 1982
). The RNA was blotted onto Nytran membranes and subjected to hybridization with a cDNA probe specific for the LAC virus L RNA segment (pLAC 4.16; Chandler et al., 1990
, 1991
). Blots were prehybridized for 6 h at 46 °C in a solution containing 5x SSC, 5x Denhardts solution, 20 mM NaPO4, 200 mg/ml herring sperm DNA, 50% formamide and 0·5% SDS, then hybridized in the same solution containing labelled probe (12x107 c.p.m./ml) for 20 h at 46 °C. Blots were washed at 55 °C for 10 min in each of the following: twice in 3x SSC, 0·1% SDS; twice in 1x SSC, 0·1% SDS; and twice in 0·1x SSC, 0·1% SDS. The blots were air-dried and then exposed to DuPont Cronex 4 film for 24 h.
(3) RTPCR assay.
Mosquitoes that were TI+ with wt LAC virus and orally infected with SSH virus were assayed for the presence of both wt LAC virus and SSH virus S segment RNA using RTPCR. Each mosquito was triturated in 500 µl MEM. Approximately 250 µl homogenate was removed and stored at -70 °C for use in plaque isolation of viruses. The remaining 250 µl was centrifuged, the supernatant was removed and 200 µl denaturation solution (containing guanidine thiocyanate and 2-mercaptoethanol) was added. RNA was extracted [using 20 µl 2 M sodium acetate (pH 4·0) and 200 µl phenol:chloroform:isoamyl alcohol solution), precipitated, washed and resuspended in 20 µl RNase-free water. For synthesis of LAC or SSH virus S segment cDNA, total cellular RNA [approximately 0·5 µg RNA in 5 µl nuclease-free H2O (Promega)] was mixed with 15 pmol virus-complementary (VC; Table 1) primer in 7·7 µl nuclease-free water. Samples were denatured at 70 °C for 10 min, then cooled to 20 °C for primer annealing. RNA was reverse transcribed in a 20 µl buffer solution containing 10 mM of each dNTP and 60 U SuperScript II reverse transcriptase (Gibco-BRL) at 42 °C for 1·5 h, then stopped at 95 °C for 10 min. For PCR, 2 µl cDNA was added to a 50 µl reaction mixture containing 1·5 mM MgCl2, 50 mM KCl, 10 mM TrisHCl (pH 9·0), 0·1% Triton X-100, 200 mM each dNTP, 1·5 U Taq DNA polymerase (Promega) and 50 pmol each VC and virus-sense (VS) primers (Table 1
). The sample was subjected to PCR under the following conditions: 92 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min for 30 cycles, followed by a final extension at 72 °C for 10 min. PCR products were analysed by electrophoresis in 2% agarose gels. Stored homogenates of mosquitoes shown by RTPCR to be dually infected were subjected to plaque isolation of viruses.
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Results |
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Quantification of virus in midguts
To determine if a lower virus titre was present in the midguts of TI+ mosquitoes than in orally infected mosquitoes, virus titre was quantified in the midguts of TI+ mosquitoes and in the midguts of TI- mosquitoes that had been orally infected with LAC virus. TI+ mosquitoes contained low virus titres in their midguts. Titres in nine mosquitoes ranged from 3·2x101 to 3·2x102 p.f.u. per midgut and averaged 1·4x102 p.f.u. per midgut. Midgut titres of mosquitoes that had been fed virus were dramatically higher, ranging from 3·4x103 p.f.u. per midgut at 24 h after feeding to 5·4x104 p.f.u. per midgut at 7 days after feeding (Sundin & Beaty, 1988 ).
Oral superinfection of TI+ mosquitoes
TI+ mosquitoes were susceptible to superinfection with homologous and heterologous viruses. When mosquitoes that were TI+ with ts LAC virus were orally challenged with wt LAC virus, 18·6% became superinfected (Table 2). Mosquitoes TI+ with ts LAC virus and challenged with SSH virus were also superinfected (19·6%; Tables 1
and 2
) as determined by blot hybridization (Fig. 1
). The proportion of plaque-purified viruses containing the SSH virus L RNA ranged from 4% to 29% per mosquito (Table 3
). When mosquitoes that were TI+ with wt LAC virus were orally challenged with SSH virus, 19·5% became superinfected as determined by RTPCR (Tables 2
and 4
).
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Seven viruses that were genotyped were diploid for one of the three segments (Table 4). All of the diploid viruses had LAC virus L, M and S segments and one additional segment from the SSH virus parent. The S segment was the most common diploid segment (71%) (Table 4
, Fig. 2
). Two other diploid viruses with incomplete genotypes were also identified; one was diploid for the S segment and the other was diploid for the L segment. These two diploid viruses were not included when calculating the percentage of diploid viruses due to their incomplete genotypes.
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SSH virus from infected mosquitoes tended to produce smaller plaques than the LAC virus-infected controls. Therefore, plaques from three mosquitoes (numbers 163, 135 and 129) were tested to determine if plaque size correlated with genotype. Each plaque was numbered and the size of the plaque was recorded as large (approximately 4 mm or greater), medium (23·5 mm), small (approximately 12 mm) or very small (1 mm or less). The plaque size did not appear to correlate with the genotype (data not shown). Viruses that were genotyped as having all LAC virus segments produced plaques of all sizes. Two reassortant viruses (163-20 and 163-22) that had the same genotype (SSH/LAC/LAC) had different sized plaques: medium and very small. No parental SSH virus genotypes were detected in the experiment to correlate plaque size and genotype.
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Discussion |
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The ability of TI+ mosquitoes to become superinfected with homologous or heterologous virus was demonstrated by assays using temperature sensitivity, blot hybridization and virus-specific RTPCR. All three techniques demonstrated that approximately 20% of the TI+ mosquitoes became superinfected when orally challenged. The biological ts assay was used to determine if superinfection had occurred. The RTPCR assay allowed us also to determine if reassortment had occurred, and allowed reassortant viruses to be completely genotyped. Four reassortant viruses were detected using RTPCR (Table 4). Interestingly, three of these four reassortant viruses had LAC virus M segments. LACSSH reassortant viruses that have the LAC virus M segment are much more efficient at dissemination in and transmission by A. triseriatus mosquitoes (Beaty et al., 1981
, 1982
; Schopen et al., 1991
). Other important phenotypic characteristics also segregate with the M segment, including neuroinvasiveness (Beaty et al., 1981
; Gonzalez-Scarano et al., 1992
).
LAC virus and LAC virus RNA segments were over-represented in the analyses. Two of the three segments of each reassortant virus were LAC virus segments and the LAC virus parental genotype was present in 92·8% of the plaques tested. This may be due to the fact that the mosquitoes were TI+ with LAC virus, which may have limited the replication sites for SSH virus. In addition, A. triseriatus mosquitoes are the natural host of LAC virus but not SSH virus, potentially providing a replicative advantage to the former virus.
Several studies have documented the presence of diploid viruses belonging to the family Bunyaviridae (Rozhon et al., 1981 ; Iroegbu & Pringle, 1981
; Hampson, 1987
; Urquidi & Bishop, 1992
; Rodriguez et al., 1998
). Bunyavirus virions are pleomorphic (Talmon et al., 1987
) and this may allow the packaging of additional nucleocapsid segments (Urquidi & Bishop, 1992
). The techniques used to genotype viruses (e.g. RTPCR, blot hybridization, ts mutants) would not detect viruses that are diploid or polyploid for segments of the same parent. Thus, polyploid viruses may be relatively common (Rodriguez et al., 1998
). The S segment was the segment that was most commonly heterodiploid (Table 4
). Urquidi & Bishop (1992)
also found that a majority of LACSSH reassortant diploid viruses contained two S segments, which are the most abundant intracellular RNA species (Gentsch et al., 1977
; Bishop & Shope, 1979
). Their abundance and small size may make it structurally more feasible for the virus to package two or more of them.
It is possible that the diploid viruses detected may have actually been due to the presence of two overlapping plaques. However, only plaques that were well separated were picked to be genotyped and, in each case, only one of the viral segments was found to be diploid. If the diploidy were due to overlapping plaques, one would expect to detect all three segments of both parental viruses.
The S segment primers identified superinfected mosquitoes more effectively than the L or M primer sets did. Sixty-nine of the mosquitoes initially screened for superinfection using the S segment primers were later screened with the L and M segment primers. When screened with the S segment primers, 21·7% (15/69) of these mosquitoes were identified as superinfected. When the M segment primers were used, 10·1% (7/69) mosquitoes were identified as superinfected. When the L segment primers were used, 18·8% (13/69) were superinfected. These data indicate that the M segment primers are not as sensitive as the other two primer sets in detecting dual infections. Therefore, it is possible that some M segment diploid viruses were not detected by the RTPCR technique. In this regard, passage of reassortant bunyaviruses may result in loss of the heterologous segment (Rodriguez et al., 1998 ). Therefore, passage of plaques to BHK cells for further amplification before genetic analysis may have resulted in the underestimation of the number of diploid viruses initially produced.
These studies have demonstrated that interference to superinfection is not complete in TI+ mosquitoes. TI+ mosquitoes have dramatically lower titres of virus present in their midguts than orally infected mosquitoes, which may permit them to become superinfected when challenged with a second virus via blood feeding. It is interesting to note that while SSH virus overall infects a smaller proportion of previously non-infected mosquitoes (82% vs 96% for LAC virus), the superinfection rate in TI+ mosquitoes was equivalent to that of LAC virus. This suggests that there may be less interference to a heterologous superinfecting virus than to a homologous virus in infected mosquitoes, thereby facilitating dual infection and RNA segment reassortment.
The frequency of reassortment following superinfection of TI+ mosquitoes was relatively low compared to rates obtained when vectors were infected simultaneously or via interrupted feeding (Beaty et al., 1985 ). It is noteworthy in this regard that virus replication and reassortment is enhanced in the ovaries of female A. triseriatus that are provided with additional blood meals. Active transport of molecules into and rapid cell division in the ovarian follicles may overcome interference (Chandler et al., 1990
). The mosquitoes in this study received only one blood meal and the ovaries were not metabolically active at the time of superinfection. An additional blood meal may have increased the generation of reassortant viruses. Nonetheless, since transovarial transmission is a major maintenance and amplification mechanism for LAC virus in nature (Beaty & Thompson, 1975
; Watts et al., 1974
), the ability of even a small percentage of TI+ mosquitoes to become superinfected and to generate reassortant viruses may be epidemiologically significant.
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Acknowledgments |
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Footnotes |
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c Present address: Center for Tropical Diseases, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA.
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References |
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Beaty, B. J. & Bishop, D. H. (1988). Bunyavirus-vector interactions. Virus Research 10, 289-301.[Medline]
Beaty, B. J., Holterman, M., Tabachnick, W., Shope, R. E., Rozhon, E. J. & Bishop, D. H. (1981). Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment. Science 211, 1433-1435.[Medline]
Beaty, B. J., Miller, B. R., Shope, R. E., Rozhon, E. J. & Bishop, D. H. (1982). Molecular basis of bunyavirus per os infection of mosquitoes: role of the middle-sized RNA segment. Proceedings of the National Academy of Sciences, USA 79, 1295-1297.[Abstract]
Beaty, B. J., Sundin, D. R., Chandler, L. J. & Bishop, D. H. (1985). Evolution of bunyaviruses by genome reassortment in dually infected mosquitoes (Aedes triseriatus). Science 230, 548-550.[Medline]
Bishop, D. H. L. & Shope, R. E. (1979). Bunyaviridae. In Comprehensive Virology, pp. 1-156. Edited by H. Frankel-Conrat & R. R. Wagner. New York: Plenum Press.
Chandler, L. J., Beaty, B. J., Baldridge, G. D., Bishop, D. H. L. & Hewlett, M. J. (1990). Heterologous reassortment of bunyaviruses in Aedes triseriatus mosquitoes and transovarial and oral transmission of newly evolved genotypes. Journal of General Virology 71, 1045-1050.[Abstract]
Chandler, L. J., Hogge, G., Endres, M., Jacoby, D. R., Nathanson, N. & Beaty, B. J. (1991). Reassortment of LaCrosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes. Virus Research 20, 181-191.[Medline]
Gentsch, J., Wynne, L. R., Clewley, J. P., Shope, R. E. & Bishop, D. H. (1977). Formation of recombinants between snowshoe hare and LaCrosse bunyaviruses. Journal of Virology 24, 893-902.[Medline]
Gonzalez-Scarano, F., Jacoby, D., Griot, C. & Nathanson, N. (1992). Genetics, infectivity and virulence of California serogroup viruses. Virus Research 24, 123-135.[Medline]
Hampson, J. (1987). A study of the genetics and growth of a tripartite RNA virus using ts mutants. PhD thesis, University of Warwick.
Iroegbu, C. U. & Pringle, C. R. (1981). Genetic interactions among viruses of the Bunyamwera complex. Journal of Virology 37, 383-394.[Medline]
Rodriguez, L. L., Owens, J. H., Peters, C. J. & Nichol, S. T. (1998). Genetic reassortment among viruses causing hantavirus pulmonary syndrome. Virology 242, 99-106.[Medline]
Rozhon, E. J., Gensemer, P., Shope, R. E. & Bishop, D. H. (1981). Attenuation of virulence of a bunyavirus involving an L RNA defect and isolation of LAC/SSH/LAC and LAC/SSH/SSH reassortants. Virology 111, 125-138.[Medline]
Schopen, S., Labuda, M. & Beaty, B. (1991). Vertical and venereal transmission of California group viruses by Aedes triseriatus and Culiseta inornata mosquitoes. Acta Virologica 35, 373-382.[Medline]
Sundin, D. R. & Beaty, B. J. (1988). Interference to oral superinfection of Aedes triseriatus infected with LaCrosse virus. American Journal of Tropical Medicine and Hygiene 38, 428-432.[Medline]
Sundin, D. R., Beaty, B. J., Nathanson, N. & Gonzalez-Scarano, F. (1987). A G1 glycoprotein epitope of La Crosse virus: a determinant of infection of Aedes triseriatus. Science 235, 591-593.[Medline]
Talmon, Y., Prasad, B. V., Clerx, J. P., Wang, G. J., Chiu, W. & Hewlett, M. J. (1987). Electron microscopy of vitrified-hydrated La Crosse virus. Journal of Virology 61, 2319-2321.[Medline]
Turell, M. J., Reeves, W. C. & Hardy, J. L. (1982). Transovarial and trans-stadial transmission of California encephalitis virus in Aedes dorsalis and Aedes melanimon. American Journal of Tropical Medicine and Hygiene 31, 1021-1029.[Medline]
Turell, M. J., Saluzzo, J. F., Tammariello, R. F. & Smith, J. F. (1990). Generation and transmission of Rift Valley fever viral reassortants by the mosquito Culex pipiens. Journal of General Virology 71, 2307-2312.[Abstract]
Urquidi, V. & Bishop, D. H. (1992). Non-random reassortment between the tripartite RNA genomes of LaCrosse and snowshoe hare viruses. Journal of General Virology 73, 2255-2265.[Abstract]
Watts, D. M., Thompson, W. H., Yuill, T. M., DeFoliart, G. R. & Hanson, R. P. (1974). Overwintering of LaCrosse virus in Aedes triseriatus. American Journal of Tropical Medicine and Hygiene 23, 694-700.[Medline]
White, B. A. & Bancroft, F. C. (1982). Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. Journal of Biological Chemistry 257, 8569-8572.
Received 17 December 1998;
accepted 24 February 1999.