Formation of recombinants between snowshoe hare and La Crosse bunyaviruses.

Wild-type recombinants were obtained at high frequency from coinfections of BHK cells involving temperature-sensitive, conditional-lethal mutants of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Analyses of two of the recombinants indicated that they have the genome compositions SSH/LAC/SSH and SSH/LAC/LAC for their respective L, M, and S virion RNA species. This evidence, together with that for the genetic stability of the recombinants, indicates that they were derived by segment reassortment of the competent genome pieces of the parental viruses. The SSH/LAC/SSH recombinant appears, from polypeptide analysis, to have the SSH type of nucleocapsid protein (N), whereas the SSH/LAC/LAC recombinant has the LAC nucleocapsid protein, suggesting that the viral S RNA codes for the N protein.     

Recombination between snowhoe hare and La Crosse bunyaviruses.

We have previously reported heterologous genetic recombination resulting from crosses involving temperature-sensitive (ts) mutants of La Crosse (LAC) group II and snowshoe hare (SSH) group I ts mutants (J. Gentsch, L. R. Wynne, J. P. Clewley, R. E. Shope, and D. H. L. Bishop, J. Virol. 24:893-902, 1977). From those crosses two reassortant viruses having the large/medium/small viral RNA segment genotypes of SSH/LAC/SSH and SSH/LAC/LAC were obtained. In this study it has been found that the reciprocal cross (SSH group II x LAC group I ts mutants) has not yielded the expected LAC/SSH/SSH or LAC/SSH/LAC reassortant viruses. The backcross of a SSH/LAC/SSH group II ts mutant with a LAC group I ts mutant has produced a new reassortant virus, LAC/LAC/SSH, whereas the backcross of SSH/LAC/LAC group I ts mutants with SSH group II ts mutants gave another reassortant, SSH/SSH/LAC. Backcross analyses of LAC/LAC/SSH group I ts mutants with Group II ts mutants of SSH have not yielded the expected LAC/SSH/SSH reassortant virus, nor have backcrosses of SSH/SSH/LAC group II ts mutants with group I ts mutants of LAC virus yielded the expected LAC/SSH/LAC reassortant. Possible reasons why certain reassortant viruses are not produced are discussed. A procedure to screen SSH-LAC reassortant viruses which differ in their virion N polypeptides is described.     

Comparison of the sequences and coding of La Crosse and snowshoe hare bunyavirus S RNA species.

The sequence of the S RNA of La Crosse bunyavirus was deduced from analyses of DNA copies cloned in the Escherichia coli plasmid pBR322. The S RNA is 984 nucleotides in length, has a base ratio of 31.8% U, 27.0% A, 23.2% C, and 18.0% G, and codes for two distinct gene products that are read from overlapping reading frames in the viral complementary strand. The larger gene product (N, 26.5 x 10(3) daltons) contains 235 amino acids, and the smaller gene product (NSS, 10.4 x 10(3) daltons) has 92 amino acids. Comparisons with the published sequences of the related snowshoe hare bunyavirus S RNA and its gene products (Bishop et al., Nucleic Acids Res. 10:3703-3713, 1982) indicate that there are a total of 114 nucleotide differences (6 additions or deletions and 108 substitutions). Also, there are 22 amino acid differences between the N proteins and 12 amino acid differences between the NSS proteins of the two S RNAs.     

The complete sequence and coding content of snowshoe hare bunyavirus small (S) viral RNA species.

The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322. The viral S RNA (negative sense strand) is 982 nucleotides long (3.3 x 10(5) daltons) with complementary 5' and 3' end sequences. It has a base composition of 30.5%U, 25.8%A, 24.9%C and 18.7%G. In the viral complementary (plus sense) strand there are two overlapping open reading frames initiated by methionine codons. One reading frame codes for a 26.8 x 10(3) dalton protein, the other for a 10.5 x 10(3) dalton protein. The larger gene product is presumably related to the viral nucleoprotein (N) that is coded by the S RNA (Gentsch and Bishop (1978) J. Virol. 28, 417-419). The smaller gene product is probably related to the recently identified S RNA coded nonstructural protein (NSS) induced in virus infected cells (Fuller and Bishop (1982) J. Virol. 41, 643-648).     

Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein.

Tryptic peptide analyses have been undertaken on the nucleocapsid (N) protein of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Similar analyses have been performed on the N proteins of two recombinant viruses which have the large/medium/small RNA genome configurations: SSH/LAC/LAC and SSH/LAC/SSH. The results provide conclusive evidence that the S RNA of bunyaviruses codes for the the viral N protein.     

Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.     

Nonviral heterogeneous sequences are present at the 5'' ends of one species of snowshoe hare bunyavirus S complementary RNA.

Analyses of the 5' ends of snowshoe hare bunyavirus plus sense S RNA species (including mRNA) recovered from infected cells have revealed two types of termini. These include ends that are essentially exact copies of the 3' end of the viral S RNA and others that are similar, but additionally have 13-14 nucleotide extensions that are heterogeneous in sequence. The former probably represent replicative plus sense RNA species, the latter mRNA species that have host cell derived primer sequences.     

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.     

Instability of the snowshoe hare and woody plant interaction

Summary We show that Alaskan woody plants respond to browsing in two ways that might destabilize a plantherbivore interaction and account for snowshoe hare population cycles. (1) Browse production of preferred, earliersuccessional woody plants increases in response to moderate levels of browsing. Such yield increases are potentially destabilizing. Later successional woody plants show decreases in yield after moderate browsing, which is consistent with the persistence of snowshoe hares in late successional refuge habitats (Keith 1966, Wolff 1980). (2) Many woody plants are destructively overbrowsed or girdled at the peak of the snowshoe hare cycle. The more palatable and plastic, early to mid successional plants respond by sprouting accompanied by juvenile reversion. Sprouts are markedly less palatable than mature shoots. We show here that sprout palatability and twig biomass are restored in 2–3 years for earlier successional plants, but palatability may not recover for 4–10 years in sprouts of some mid to late successional plants. The decrease in palatability helps to account for the snowshoe hare crash (assuming that damage to more palatable plants is widespread during the peak), and the 2–3 year time lag for recovery of more palatable species could account for (May 1974) the observed 8–11 year period of the hare cycles. Browse yield increases acting during the snowshoe hare population nadir and increase, and sprouting with juvenile reversion acting during the hare peak and decline can in principle account for the oscillatory nature and the observed 8–11 year periodicity of the snowshoe hare cycle.     

La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.     

Structural proteins of La Crosse virus.

Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm).     

Anti-mRNAs in La Crosse bunyavirus-infected cells.

Unlike some members of the family Bunyaviridae which contain ambisense genomes, all La Crosse virus reading frames are translated from antigenome sense mRNAs. Nevertheless, La Crosse virus genome sense mRNAs or anti-mRNAs are initiated from antigenome templates. These are characterized by the same range of capped, nontemplated sequences at their 5' ends as mRNAs, but their 3' ends are presumed to be heterogenous, as they were not seen on RNA blots. The anti-mRNAs are estimated to be 15 to 30 times less abundant than mRNAs, but remarkably, this ratio is similar to that of functional genome sense mRNAs made from other bona fide ambisense segments. A role for these anti-mRNAs during infection is unclear.     

Prevalence of helminths in a cyclic snowshoe hare population

Five species of helminths were monitored in a population of snowshoe hares (Lepus americanus) near Rochester, Alberta, during 1961-1977. Prevalence of both Obeliscoides cuniculi and Protostrongylus boughtoni among young hares averaged about 50% by age 2 mo, then tended to level off. Prevalence of Taenia pisiformis (cysticerci) and Dirofilaria scapiceps rose more slowly, but continued to increase steadily beyond their mean levels of 8% and 1% at age 2 mo. There were well defined seasonal (within-year) cycles in prevalence of O. cuniculi and P. boughtoni that were generated evidently to a major degree by arrested development of larvae in fall and renewed development in late winter. It was hypothesized that renewed larval development was triggered (in February) in O. cuniculi by the seasonal rise of circulating pituitary gonadotrophins, and (in April) in P. boughtoni by the seasonal rise of gonadal androgens and estrogens. Indices to gonadal hormone levels in hares indicated that these increased most rapidly among males, and may have accounted for the higher prevalences of P. boughtoni in males during April-May. Neither T. pisiformis nor D. scapiceps exhibited conspicuous seasonal changes in prevalence. Maximum prevalence of T. pisiformis was attained at about 1 yr of age, whereas D. scapiceps increased among adult snowshoes through age 2 yr before stabilizing. Long-term (between-year) changes in prevalence of O. cuniculi, T. pisiformis, and D. scapiceps were correlated significantly with the cyclic hare population which declined from a peak in fall 1961 to a low in 1965-1966, rose to another peak by fall 1970, and declined again to a low in 1975. There was no detectable time lage between this "10-yr" cycle in hare density and the cycles of parasite prevalence among juveniles (less than 1 yr of age). Among adult hares, the cycle of O. cuniculi prevalence was likewise synchronous with that of the hare population, but the cycles of D. scapiceps and T. pisiformis lagged by approximately 1 and 2 yr, respectively. This lag in T. pisiformis prevalence was largely inexplicable to us. Our data on P. boughtoni were not suitable for analyses of between-year trends; nor were those for the fifth helminth, Taenia serialis (coenuri), because mean prevalence was less than 1% among both juveniles and adults. An apparent decline in T. serialis after the early 1950's, and its continued scarcity thereafter, paralleled a major change in numbers of one important definitive host--the red fox (Vulpes vulpes).(ABSTRACT TRUNCATED AT 400 WORDS)     

M viral RNA segment of bunyaviruses codes for two glycoproteins, G1 and G2.

Tryptic peptide digests of the two viral glycoproteins (G1 and G2) of snowshow hare (SSH) virus, La Crosse, La Crosse (LAC) virus, and an SSH/LAC recombinant virus which has a large (L)/medium (M)/small (S) RNA segment genome composition of SSH/LAC/SSH were analyzed by ion-exchange column chromatography. The analyses prove that the M RNA species of bunyaviruses codes for the two viral glycoproteins.     

Segmented genome and nucleocapsid of La Crosse virus.

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Mortality by moonlight: predation risk and the snowshoe hare

Optimal behavior theory suggests that prey animals will reduceactivity during intermittent periods when elevated predationrisk outweighs the fitness benefits of activity. Specifically,the predation risk allocation hypothesis predicts that preyactivity should decrease dramatically at times of high predationrisk if there is high temporal variation in predation risk butshould remain relatively uniform when temporal variation inpredation risk is low. To test these predictions we examinedthe seasonably variable response of snowshoe hares to moonlightand predation risk. Unlike studies finding uniform avoidanceof moonlight in small mammals, we find that moonlight avoidanceis seasonal and corresponds to seasonal variation in moonlightintensity. We radio-collared 177 wild snowshoe hares to estimatepredation rates as a measure of risk and used movement distancesfrom a sample of those animals as a measure of activity. Inthe snowy season, 5-day periods around full moons had 2.5 timesmore predation than around new moons, but that ratio of theincreased predation rate was only 1.8 in the snow-free season.There was no significant increase in use of habitats with morehiding cover during full moons. Snowshoe hares' nightly movementdistances decreased during high-risk full-moon periods in thesnowy season but did not change according to moon phase in thesnow-free season. These results are consistent with the predationrisk allocation hypothesis.     

Electron microscopy of vitrified-hydrated La Crosse virus.

La Crosse (LAC) virions were cryopreserved by rapid freezing in a thin layer of vitreous ice. The vitrified-hydrated LAC virions were subsequently imaged at -170 degrees C in a transmission electron microscope equipped with a low-temperature specimen holder. This cryoelectron microscopic technique eliminates the artifacts frequently associated with negative staining. Images of vitrified-hydrated LAC virions clearly revealed surface spikes as well as bilayer structure. Size measurements of the vitrified-hydrated LAC virions showed heterogeneity, with diameters ranging from 75 to 115 nm. Regardless of the particle size, the spike was about 10 nm long, and the bilayer was about 4 nm thick. The spikes are interpreted to be one or both of the glycoproteins, and the bilayer is interpreted to be the membrane envelope of the virus. In contrast to the pleomorphic appearance of the negatively stained LAC virions, the vitrified-hydrated LAC virions showed uniform spherical shapes regardless of their sizes.