Snowshoe Hare Virus Research Articles

Nucleotide sequence of the M segment of Germiston virus: Comparison of the M gene product of several bunyaviruses

The complete nucleotide sequence of the M RNA segment of Germiston bunyavirus was determined from plasmids containing overlapping M cDNA inserts. The M segment is 4534 nucleotides long and contains a 50-base-long inverted terminal repeat which can form a stable hydrogen-bonded secondary structure with a ΔG of −45.8 kcal/mol. The RNA molecule complementary to viral RNA contains a single large open reading frame that encodes a 1437 amino acid-long protein with hydrophobic amino and carboxy terminal regions, which could represent signal and anchor sequences, respectively. It is presumed that this gene product is the polyprotein precursor to glycoproteins G1 and G2 and to the nonstructural polypeptide NS M. The nucleotide and amino acid sequences of the M RNA of Bunyamwera virus (prototype of the serogroup) and snowshoe hare and La Crosse viruses (California serogroup) (Lees et al., 1986; Eshita and Bishop, 1984; Grady et al., 1987) were compared to those of Germiston virus. An overall amino acid sequence homology of 44% was found between Germiston and snowshoe hare viruses and of 61 % between Germiston and Bunyamwera viruses. Most of the cysteines, three out of seven of the potential glycosylation sites, as well as the N and C terminal hydrophobic domains, are conserved between the four viruses.

The sequence of the M RNA of an isolate of La Crosse virus.

The middle-size (M) genomic RNA of a New York State, U.S.A. isolate of La Crosse (LAC) virus has been cloned by a random priming procedure and its nucleotide sequence determined by the dideoxy method. The RNA was found to be 4526 nucleotides in length and to have a base composition of 34.2% U, 27.8% A, 20.6% C and 17.4% G. There is a single, long open reading frame in the viral complementary RNA that contains sufficient information to code for a protein of 1441 amino acids. In these respects, as in many others, the LAC virus M RNA and its encoded protein were very similar, if not identical, to those previously reported by other investigators for the closely related snowshoe hare virus. The M RNAs of the two viruses show 79% nucleotide sequence homology and 89% homology in the amino acid sequence of their encoded proteins. Several algorithms for predicting surface residues, as well as the Chou-Fasman rules for predicting secondary structure, were used to compare the LAC virus and snowshoe hare virus M gene proteins. These analyses identified 39 sites on the proteins as those most likely to be linear antigenic determinants that might contribute to the differences between the two viruses.

California serogroup and Powassan virus infection of cats

One hundred and seventy five sera from cats in Ontario, Canada, were tested for hemagglutination inhibition (HI) antibodies to three arboviruses; namely, Powassan (POW) of the Flavivirus serogroup, and Snowshoe hare (SSH) and Jamestown Canyon (JC) viruses of the California (CAL) serogroup. All sera were negative for antibodies to POW virus. Twelve cats possessed CAL serogroup antibodies including 3 with antibodies to SSH alone, 6 with antibodies to JC alone, and 3 with antibodies to both SSH and JC antigens. POW virus was inoculated into seven cats, one intracerebrally and six intravenously. Neurologic signs were not detected in any of the cats. Histologic lesions of a nonsuppurative encephalitis and encephalomyelitis were observed in the intracerebrally inoculated cat and in one of the intravenously inoculated cats, respectively. POW virus was not isolated from the brain or spinal cord of either of these two cats. HI antibodies were detected in the sera of all inoculated animals. HI antibodies were not detected in the CSF of any animal.

The S segment of the Germiston virus RNA genome can code for three proteins

The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids ( M r 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids ( M r. 11,800) which corresponds to the NSg protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NS s proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.

Host-adaptive Antigenic Variation in Bunyaviruses

Bunyamwera virus and snowshoe have virus (family Bunyaviridae) were passaged up to six times through mosquito cells in culture and the resultant viruses were compared to the input, mammalian cell-passed virus using monoclonal antibodies raised against the input virus. The mosquito cell-adapted virus population consisted of mutants which were better adapted to replication in the new host than was the input mammalian cell-passed virus and were differentially susceptible to neutralization by antibody.

Nucleotide sequence of the Bunyamwera virus M RNA segment: Conservation of structural features in the Bunyavirus glycoprotein gene product

The complete nucleotide sequence of the Bunyamwera virus M RNA segment was determined from four overlapping cDNA clones and by primer extension. The RNA segment is 4458 bases in length, and encodes a single gene product in the viral complementary RNA. The predicted protein is 1433 amino acids long (mol wt 162,065), contains four potential glycosylation sites, and is relatively cysteine rich. It is presumed that the three proteins G1, G2, and NS M which have been mapped to the M RNA segment are synthesized as a precursor polyprotein which is subsequently proteolytically cleaved. A putative hydrophobic signal sequence at the amino terminus and a hydrophobic anchor sequence at the carboxy terminus of the predicted protein have been identified, in addition to internal regions of hydrophobicity of unknown function. The nucleotide and amino acid sequences of the Bunyamwera virus M segment have been compared with those of the snowshoe hare virus M segment ( Y. Eshita and D. H. L. Bishop, Virology 137,227–240, 1984 ). Common features include the overall architecture of the RNAs, single cysteine-rich primary gene products, and conservation of hydrophobic domains in the gene products. When aligned the amino acid sequences are 43% homologous, and 66 of 70 cysteine residues can be matched. The evolutionary significance of these findings is discussed.

The complete sequence of the M RNA of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein

The complete sequence of the viral M RNA of snowshoe hare (SSH) bunyavirus has been determined. The RNA is 4527 nucleotides long (mol wt: 1.5 × 10 6, base composition: 27.5% A, 33.5% U, 17.7% G, 21.3% C), and has 3′ and 5′ terminal sequences that, depending on how they are arranged, are complementary for some 44 residues. The viral RNA codes in its viral-complementary sequence, for a single primary gene product (the viral glycoprotein) that is comprised of 1441 amino acids (162,391 Da), and is rich in cysteine residues but poor in potential asparagine-linked glycosylation sites. Like the SSH S RNA, the M viral-complementary 5′ noncoding region is shorter than the 3′ noncoding sequence (61 as opposed to 142 nucleotides). The different functions of the M RNA are discussed in relation to those of the S RNA of SSH virus. No other large open reading frames have been identified in either the viral, or viral-complementary, M RNA sequences. Examination of the sequence of the M gene product reveals the presence of an 18 residue amino terminal hydrophobic sequence (putative signal) and a much longer 32 amino acid carboxy proximal hydrophobic region that is followed by a terminal sequence rich in charged amino acids (12 out of 20 residues). The size and constitution of the carboxy end regions are consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. In addition to these terminal hydrophobic sequences, a localized internal region of the gene product contains several hydrophobic sequences, 15 to 29 amino acids in length. Their possible role in the morphogenesis of bunyaviruses that occurs in the Golgi cisternae of infected cells is discussed.

Arthropod vectors of human disease in the United States

Arthropod vectors of human disease in the United States

Serological evidence of infection with California serogroup viruses (family Bunyaviridae) in residents of Long Hua, suburb of Shanghai, People's Republic of China

Sera from 126 residents of Long Hua, a suburb of Shanghai, in the People's Republic of China, were studied. Sera were tested for haemagglutination inhibiting antibodies to alphavirus (eastern equine encephalitis, western equine encephalitis), flavivirus (St. Louis encephalitis, Powassan, dengue) and California serogroup (snowshoe hare) antigens. Flavivirus antibodies were found in 14 (11·1%) and California serogroup antibodies in 5 (3·9%) individuals. Neutralizing antibodies with highest titres to snowshoe hare virus were demonstrated in 3 of the 5 California serogroup reactors. We believe this to be the first report of California serogroup virus antibodies in Chinese residents and the first evidence to suggest that California serogroup viruses may be circulating in the Orient.

Serologic evidence of arbovirus infections in humans and wild animals in Alaska.

Blood samples were collected from humans and several species of free-ranging wild animals in Alaska. Sera were tested for antibody to Jamestown Canyon (JC), snowshoe hare (SSH), Northway (NOR), Klamath (KLA), Sakhalin (SAK), Great Island (GI), and Silverwater (SIL) virus. JC antibody was found in 54% of 121 human, 89% of 97 bison (Bison bison), 51% of 84 Dall sheep (Ovis dalli), 43% of 68 snowshoe hare (Lepus americanus), and 3% of 33 arctic fox (Alopex lagopus) sera. SSH antibody was found in 42% of 121 human, 89% of 97 bison, 41% of 84 Dall sheep, and 65% of 68 snowshoe hare sera. NOR antibody was found in 14% of 121 human, 94% of 97 bison, 84% of 84 Dall sheep, 43% of 69 caribou (Rangifer tarandus), 3% of 68 snowshoe hare, 48% of 64 grizzly bear (Ursus arctos), 3% of 33 arctic fox, and 78% of 27 moose (Alces alces) sera. KLA antibody was found in 5% of 121 human and 40% of 97 bison sera. SAK antibody was found in 2% of 97 bison and 3% of 33 arctic fox sera. GI antibody was found in 1% of 97 bison sera. No SIL antibody was found in any sera tested. Thus the natural host ranges of JC, SSH, NOR, and KLA viruses have been extended by inference from the occurrence of antibody.

Activity of California encephalitis group viruses in Entrelacs (province of Quebec, Canada)

During the summer of 1979, indicator rabbits were placed in three sites in Entrelacs (Laurentian area, province of Quebec) and mosquitoes were collected in order to monitor arbovirus activity in the area. Eight seroconversions to California encephalitis (CE) group viruses were detected in rabbits during June, July, and August. Twenty-five strains identified as members of the CE group were isolated: 3 were obtained from viremic rabbit sera, 1 from adult Aedes communis reared in the laboratory from field-collected larvae, and 21 from mosquito pools. Twenty-two of these were typed as snowshoe hare (SSH) virus. No evidence of La Crosse (LAC) virus was detected but three strains belonging to the CE group showed antigenic properties different from reference SSH, LAC, or Jamestown Canyon (JC) viruses. One isolate identified as Flanders virus was obtained from Culex pipiens. Three mosquito species (A. communis, A. punctor, and A. excrucians) were involved in the transmission cycle of SSH virus in Entrelacs. This is the first report, in the province of Quebec, of SSH isolation from animal sera and the first demonstration of its transovarial transmission.

Molecular basis of bunyavirus per os infection of mosquitoes: role of the middle-sized RNA segment.

The molecular basis of bunyavirus per os infection of mosquitoes was determined; La Crosse (LaC), snowshoe hare (Ssh), and LaC-Ssh reassortment viruses were compared for their ability to infect Aedes triseriatus, the natural vector of the LaC virus. The viruses were comparable in their ability to infect midgut cells; 115 of 117 (98%) mosquitoes ingesting viruses containing the LaC middle-sized RNa segment and 92/100 (92%) of mosquitoes ingesting viruses containing the Ssh middle-sized RNA segment became infected. However, those viruses containing the LaC middle-sized RNA segments disseminated efficiently (113/115, 98%) from the midgut to infect secondary target organs. Those viruses containing the Ssh middle-sized RNA segment efficiently infected the midgut and large amounts of viral antigen were detected in the midgut cells but antigen was detected in the secondary target organs only in 26% (24/92) of the mosquitoes with midgut infection. Thus, the middle-sized RNA segment seems to be the major determinant for successful dissemination of LaC virus from infected A. triseriatus midgut cells.

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.

Nucleotide sequence analyses and predicted coding of bunyavirus genome RNA species.

We performed 3' RNA sequence analyses of [(32)P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3'-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3'-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3'-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3'-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.

Antibodies to the California group of arboviruses in the moose (Alces alces americana Clinton) population of Nova Scotia.

Moose (Alces alces americana Clinton) blood collected by hunters during the 1977 and 1978 hunting seasons was tested for California group antibodies. Hemagglutination inhibition (HI) tests using snowshoe hare virus (SSH) as antigen yielded 37.02% HI-positive sera. Neutralization tests showed reactors reactors (67.5% positive) to SSH and classified six reactors as Jamestown Canyon virus (JC). This study reports the first finding of JC in a moose population; it also provides the first evidence of JC in Atlantic Canada and supports previous findings of SSH in Nova Scotia.

Antibodies to snowshoe hare virus of the California group in the snowshoe hare (Lepus americanus) and domestic animal populations of Prince Edward Island.

This study, the first of arbovirus activity in Prince Edward Island, has shown antibodies to the snowshoe hare strain of the California encephalitis group virus to be present in sera of wild and domestic animals; 15.35% of snowshoe hare, 20% of equine. and 2.5% of bovine sera were positive for hemagglutination inhibition antibody, confirmed by virus neutralization procedures.

Role of the middle-sized bunyavirus RNA segment in mouse virulence

The middle-sized (M) RNA segment of California group viruses (Bunyaviridae) is a major determinant of virulence for mice. This has been demonstrated by intraperitoneal (ip), or intracerebral (ic), inoculation of 4-week-old mice with prototype or reassortant bunyaviruses, the latter obtained from mixed infection of BHK-21 cells with temperature-sensitive ( ts) mutants of La Crosse (LAC), snowshoe hare (SSH), and Tahyna (TAH) viruses. Reassortant viruses were not more virulent than wild-type progenitor viruses. All reassortants, and wild-type cloned viruses having the LAC, or SSH, M RNA segment, killed mice following ip inoculation; reassortant and wild-type viruses having the TAH M RNA did not kill 4-week-old mice after ip inoculation. In addition, reassortant viruses with the TAH M RNA killed mice after is inoculation more rapidly than did viruses having the LAC M RNA segment, irrespective of the parental origin of the small (S), or large (L), viral RNA species. The correlation of the M RNA segment with virulence implies that G1 and/or G2 determine major murine virulence properties, perhaps through attachment which may influence tropism. These experiments are an initial step in understanding the mechanism of virulence for mice, and eventually for man. They could also lead to the rational design of live attenuated bunyaviruses as vaccines for man and domestic animals.

Attenuation of virulence of a bunyavirus involving an L RNA defect and isolation of LAC/SSH/LAC and LAC/SSH/SSH reassortants

Unlike wild-type La Crosse (LAC), or snowshoe hare (SSH) viruses, or the reassortants SSH/SSH/LAC, LAC/SSH/SSH, LAC/SSH/LAC, SSH/LAC/LAC, and SSH/LAC/SSH (large/medium/small, L/M/S, RNA genotypes), LAC/LAC/LAC and LAC/LAC/SSH reassortant bunyaviruses recovered from mixed virus infections involving Group II temperature-sensitive ( ts) mutants of SSH/LAC/SSH and a LAC virus Group I ts 16 mutant were found to be significantly less virulent after intracerebral (ic) inoculation into 4-week-old mice. Mixed virus infections involving the same SSH/LAC/SSH ts mutants and an alternate LAC Group I ts mutant (LAC I-20) gave reassortants that were almost as virulent as wild-type LAC virus. It has previously been shown that bunyavirus Group II ts mutants have defects in their L RNA gene products, while Group I ts mutants have defects in their M RNA gene products (Gentsch et al., 1977b, J. Virol. 24, 893–902). In the analyses reported in this paper it has been determined that, in addition to a ts lesion in its M RNA species, the L RNA segment of LAC I ts 16 has a defect that is responsible for attenuation of the derived LAC/LAC/LAC and LAC/LAC/SSH reassortant progeny. The attenuated viruses were found to have lower ic LD 50 to PFU ratios than nonattenuated viruses. Two new SSH-LAC reassortant viruses used in this study (namely LAC/SSH/LAC and LAC/SSH/SSH), which had previously eluded detection by standard recombination analyses with unconcentrated viruses, have been obtained from SSH II-22 × LAC I-20 ts mutant virus crosses using polyethylene glycol-6000 (PEG) concentrated virus preparations. The difficulties encountered in obtaining these reassortants using unconcentrated viruses is in contrast to the ease of recovery of other SSH-LAC reassortants from their parental ts mutant virus crosses in standard recombination assays.

Analyses of patois group bunyaviruses: Evidence for naturally occurring recombinant bunyaviruses and existence of immune precipitable and nonprecipitable nonvirion proteins induced in bunyavirus-infected cells

Shark River (SR) and Pahayokee (PAH) bunyaviruses (Patois serogroup, Bunyavirus genus, family Bunyaviridae) have almost identical L and S RNA oligonucleotide fingerprints, but M RNA fingerprints that are different, suggesting that the two viruses may represent naturally occurring reassortant viruses. These observations are in agreement with serological studies ( B. N. Fields, B. E. Henderson, P. H. Coleman, and T. H. Work, 1969, Amer. J. Epidemiol., 89, 222–226 ) which have distinguished these two viruses by neutralization of infectivity tests (presumably reflecting differences in M RNA gene products, J. R. Gentsch, E. J. Rozhon, R. A. Klimas, L. H. El Said, R. E. Shope, and D. H. L. Bishop, 1980, Virology 102, 190–204 ), but not by complement fixation tests (which probably relate to the viral N polypeptide coded by the S RNA, J. Gentsch, L. R. Wynne, J. P. Clewley, R. E. Shope, and D. H. L. Bishop, 1977b, J. Virol. 24,893–902 ). The 3′ terminal 11 nucleotides of PAH S RNA (3′ HOOUCAUCAAAUGA … 5′) are identical in sequence to those of the S RNA species of snowshoe hare (SSH) and La Crosse (LAC) viruses, except for a position 7A residue which is a C residue in the SSH and LAC sequences. The major virion polypeptides of SR and PAH viruses include a nucleocapsid polypeptide (N, 22 × 10 3) and two glycoproteins (PAH: G1 118 × 10 3, G2, 35 × 10 3; SR: G1 113 × 10 3, G2, 35 × 10 3). In SR-infected cells several immune precipitable polypeptides have been detected. These include 11-, 54-, 64-, 93-, and 104 × 10 3-dalton polypeptides. In addition, both SR and PAH viruses induce a 74 × 10 3-dalton polypeptide (p74) that has not been detected in actinomycin D-treated infected cells, and is not immune precipitated from infected cell extracts.

Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.