Genomic and biologic analyses of snowshoe hare virus field and laboratory strains.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.

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).

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.

Radioimmune assays and molecular studies that place Anopheles B and Turlock serogroup viruses in the Bunyavirus genus (Bunyaviridae).

Molecular analyses indicate that Turlock virus (TUR, Turlock serogroup) and Boraceia virus (BOR, Anopheles B serogroup) have virion RNA species and polypeptides comparable in size to those of members of the Bunyavirus genus and unlike those of members of the newly defined Phlebovirus, Nairovirus, or Uukuvirus genera (Bunyaviridae). The 11 terminal 3' end nucleotides of the three virion RNA species of both BOR and TUR viruses (HOUCAUCACAUG...) are identical in sequence to the 3' end sequences of the viral RNA species of snowshoe hare (SSH) and La Crosse bunyaviruses (LAC, California serogroup, Bunyavirus genus). Competition radioimmune assays (RIA), using iodinated LAC nucleocapsid polypeptide (N), or LAC glycoproteins (G1, G2), and LAC rabbit hyperimmune antisera, or iodinated Oriboca (ORI, Group C, Bunyavirus genus) N, or G1 and G2 polypeptides and LAC antisera, or iodinated Bunyamwera (BUN, Bunyamwera serogroup, Bunyavirus genus) N, or G1 and G2 polypeptides and BUN or LAC antisera, have indicated that the virion polypeptides of BOR virus share antigenic determinants with these other bunyaviruses. Competition RIA analyses also have shown that TUR virus shares antigenic determinants with LAC virus. The competition RIA analyses have confirmed the antigenic relationships of LAC, SSH, trivittatus, Bwamba, Aino, Simbu, Mermet, Guaroa, Lumbo, Tahyna, ORI, Anopheles A, BUN, Capim, Guama and Shark river viruses (Bunyavirus genus members), and lack of antigenic relationships between Karimabad, or Chagres, or sandfly fever, Sicilian, Viruses (Phlebovirus genus members), and the bunyaviruses, LAC, ORI, or BUN.

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, Virology102, 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' (HO)OUCAUCAAAUGA ... 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 x 10(3)) and two glycoproteins (PAH: G1 118 x 10(3), G2, 35 x 10(3); SR: G1 113 x 10(3), G2, 35 x 10(3)). In SR-infected cells several immune precipitable polypeptides have been detected. These include 11-, 54-, 64-, 93-, and 104 x 10(3)-dalton polypeptides. In addition, both SR and PAH viruses induce a 74 x 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.

Oligonucleotide sequence analyses indicate that vesicular stomatitis virus large defective interfering virus particle RNA is made by internal deletion: evidence for similar transcription polyadenylation signals for the synthesis of all vesicular stomatitis virus mRNA species.

RNase T(1) oligonucleotide fingerprint analyses of three vesicular stomatitis virus Indiana serotype small defective interfering (DI) particle RNA species indicate that they only have oligonucleotides derived from the 5' region of the viral genome. These studies also indicate that these three DI RNAs have partial L gene sequences as well as two 5' viral oligonucleotides (59 and 70) that are not transcribed into L (or other) mRNA species (J. P. Clewley and D. H. L. Bishop, J. Virol. 30:116-123, 1979). Analyses of the large DI RNA (LT DI) reveal a different origin. The LT DI RNA has oligonucleotides derived from both the 3' end of the genome (including all the large oligonucleotides identified for N, NS, M, and G genes), in addition to at least one of the 5'-proximal L gene oligonucleotides (47), as well as all seven oligonucleotides (3, 38, 42, 43, 44B, 59, and 70) that are not protected from nuclease digestion after the formation of mRNA-viral RNA duplexes (Clewley and Bishop). It appears therefore that the genesis of LT RNA involves a deletion of internal L gene sequences from the viral RNA. Oligonucleotide sequence analyses have been undertaken on several of the vesicular stomatitis viral RNA oligonucleotides, including all seven (3, 38, 42, 43, 44B, 59, and 70) that are not transcribed into mRNA. The analyses confirm that oligonucleotides 59 [3'...GAACACCAAAAAUAAAAAAUA(G)...5'] and 70 [3'...GACCAAAACACCA(G)...5'] are at the 5'-end region of the viral genome. Oligonucleotide 38 [3'...GAAAUUCAUACUUUUUU(U)(G)...5'] may represent the termination signal for L mRNA synthesis (R. A. Lazzarini, personal communication). Oligonucleotide 43 [3'...GUAUACUUUUUUU(G)...5'] corresponds to the sequence shown to be the N gene mRNA polyadenylation signal (D. J. McGeoch, Cell 17:673-681, 1979). The other three oligonucleotides share a common feature with oligonucleotides 43 and 38, viz., a stretch of 6 or 7 U residues preceded by an AUAC sequence. Thus the sequence of oligonucleotide 3 is 3'...GAAUUAAUAUAAAAUUAAAAAUUAAAAAUACUUUUUU(U)(G)...5', whereas that of oligonucleotide 42 is 3'...GAUACUUUUUUUCAU(U)(G)...5', and that of oligonucleotide 44B is 3'...G(U)AUACUUUUUU(G)...5'. These sequence analyses suggest a common polyadenylation signal for the synthesis of all vesicular stomatitis virus mRNA species, i.e., the sequence (3')...AUACUUUUUU(U)...(5').

Host and virus specific RNA polymerases in alfalfa mosaic virus infected tobacco.

The particulate fraction of a homogenate of alfalfa mosaic virus infected tobacco was found to contain the viral replicase, and one or more host-specific RNA-polymerases. Using an endogeneous template, the membrane-bound replicase incorporates 3H-CTP in virion-type RNA. The synthesized RNA is part of a replicative intermediate; after RN-ase-treatment the product comigrates with viral double-stranded RNA in polyacrylamide gels. A preliminary characterization of this double-stranded RNA was made by RNA-RNA hybridization. Treatment of the particulate fraction with lubrol releases a host-specific RNA-polymerase. The activity of this enzyme is completely dependent on exogeneous template-RNA. Probably only a very small region of the template is transcribed. After washing with lubrol, the particulate fraction still contains the viral replicase. When this fraction is resuspended in a Mg++-deficient buffer, about 60% of the enzyme activity is released into the supernatant. No such activity is found in a comparable extract of healthy leaves.