Organization of the simian hemorrhagic fever virus genome and identification of the sgRNA junction sequences.

SHFV is a member of the Arteriviridae family. Viruses within this family encode eight open reading frames (ORFs), two of which are translated from the full-length genome RNA. The remaining six ORFs are translated from a nested set of six or seven 3' co-terminal, subgenomic RNAs (sgRNAs). We have cloned and sequenced approximately 6000 nucleotides (nt) from the 3' end of the SHFV genome. Eleven ORFs, numbered ORFs 1a, 1b, 2a, 2b, 3, 4, 5, 6, 7, 8, and 9, were identified, three more than the other arteriviruses. The characteristics of the peptides encoded by ORFs 2a through 9 were determined from their computer-generated amino acid sequences. We also amplified the junction sequences from each of the SHFV subgenomic RNAs (sgRNAs) using RT-PCR analysis. Eight separate junction sequences were found which suggests that SHFV produces eight sgRNAs during replication. ORFs 2a and 2b appear to be encoded on the same sgRNA implying that RNA 2 is polycistronic. Sequence analysis identified the conserved SHFV junction sequence as 5'-(U/C)(C/U)N(U/C)(U/C)(A/C/G)AC(C/U)-3'. Since SHFV encodes additional ORFs and produces additional sgRNAs during replication, these data suggest that SHFV may be more complex than the other arteriviruses.

Identification of the leader-body junctions for the viral subgenomic mRNAs and organization of the simian hemorrhagic fever virus genome: evidence for gene duplication during arterivirus evolution.

Simian hemorrhagic fever virus (SHFV) was recently reclassified and assigned to the new virus family Arteriviridae. During replication, arteriviruses produce a 3' coterminal, nested set of subgenomic mRNAs (sgRNAs). These sgRNAs arise by discontinuous transcription, and each contains a 5' leader sequence which is joined to the body of the mRNA through a conserved junction sequence. Only the 5'-most open reading frame (ORF) is believed to be transcribed from each sgRNA. The SHFV genome encodes nine ORFs that are presumed to be expressed from sgRNAs. However, reverse transcription-PCR analysis with leader- and ORF-specific primers identified only eight sgRNA species. The consensus sequence 5'-UCNUUAACC-3' was identified as the junction motif. Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and ORF 2b. SHFV encodes three more ORFs on its genome than the other arteriviruses. Comparative sequence analysis suggested that SHFV ORFs 2a, 2b, and 3 are related to ORFs 2 through 4 of the other arteriviruses. Evidence which suggests that SHFV ORFs 4 through 6 are related to ORFs 2a through 3 and may have resulted from a recombination event during virus evolution is presented.

Sequence of the 3' end of the simian hemorrhagic fever virus genome.

SHFV is a member of a new virus family which includes the genus arterivirus. We have cloned and sequenced 6,314 nt from the 3' end of the SHFV genome. This sequence encompasses nine complete ORFs which is three additional ORFs as compared to the other arteriviruses. We have numbered these ORFs 2a, 2b, 3, 4, 5, 6, 7, 8 and 9. At the 5' end of this sequence is a partial ORF (ORF 1b) of 1590 nt and at the 3' end is a poly(A) tract preceded by a 76 nt noncoding region. The coding capacity for each of the SHFV ORFs as well as the potential mass, pI and number of N-linked glycosylation sites for each of the encoded peptides was determined.

Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome.

The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.

Analysis of simian hemorrhagic fever virus (SHFV) subgenomic RNAs, junction sequences, and 5' leader.

Full-length simian hemorrhagic fever virus (SHFV) genome RNA (about 15 kb in length) and six subgenomic RNAs, ranging in size from 0.65 to 4.7 kb, were detected by Northern blot hybridization in MA104 cytoplasmic extracts with a 3' genomic antisense probe. The 5' regions of the two smallest subgenomic RNAs (RNAs 6 and 7) were cloned and sequenced. Sequence analysis indicated that these two RNAs contained a common 5' leader sequence joined to the subgenomic RNA bodies via a highly conserved junction sequence; the junction sequence of RNA 7 was 5'-TTAACC-3', while that of RNA 6 was 5'-TCAACC-3'. The complete 5' leader sequence (208 nt) was obtained from genomic RNA. The genomic 5' junction sequence is identical to that of RNA 7. Northern blot hybridization with an antisense 5' leader probe confirmed the presence of the complete leader sequence in all six species of subgenomic RNA. In its virion morphology, genome size, gene order, and replication strategy, SHFV is most similar to viruses such as equine arteritis virus, lactate dehydrogenase-elevating virus, and Lelystad virus/porcine respiratory and reproductive syndrome virus.

Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (LDV).

The apparently complete sequence of the RNA genome of the neurovirulent isolate of lactate dehydrogenase-elevating virus (LDV-C) has been determined. The LDV-C genome is at least 14,222 nucleotides in length and contains eight open reading frames (ORFs). ORF 1a, which encodes a protein of 242.8 kDa and is located at the 5' end of the genome, contains at least two putative papain-like cysteine protease domains, and one putative chymotrypsin-like serine protease domain. This ORF terminates with a UAG stop codon that can be bypassed if a -1 frameshift occurs. The frameshift region consists of a heptanucleotide "slippery" sequence, 5'-UUUAAAC-3', followed by a putative pseudoknot. ORF 1b encodes a protein of 155.4 kDa containing, in its N-terminal portion, an RNA-dependent RNA polymerase and an RNA helicase domain separated by a Zn finger domain. Another domain of unknown function that is also conserved in coronaviruses and toroviruses is located at the C-terminus of the ORF 1b product. Three cleavage sites in the ORF 1a polyprotein and three in the ORF 1b polyprotein were predicted for the chymotrypsin-like protease and tentatively delimit the mature nonstructural proteins of LDV. Six small, overlapping 3' ORFs (ORFs 2 through 7) encode proteins with calculated sizes of 25.8, 21.6, 19.8, 23.9, 18.9, and 12.3 kDa. ORF 7 encodes the virion nucleocapsid protein Vp-1, while ORF 6 encodes the nonglycosylated envelope protein Vp2. ORFs 5, 4, 3, and 2 each encode glycoproteins which may be virion envelope proteins. LDV is closely related to equine arteritis virus, Lelystad virus (LV), and simian hemorrhagic fever virus. These four viruses belong to a new group of positive-strand RNA viruses and are related to coronaviruses and toroviruses.

Map location of lactate dehydrogenase-elevating virus (LDV) capsid protein (Vp1) gene.

Lactate dehydrogenase-elevating virus (LDV) is currently classified within the Togaviridae family. In an effort to obtain further information on the characteristics of this virus, we have begun to sequence the viral RNA genome and to map the virion structural protein genes. A sequence of 1064 nucleotides, which represents the 3' terminal end of the genome, was obtained from LDV cDNA clones. A 3' noncoding region of 80 nucleotides followed by two complete open reading frames (ORFs) were found within this sequence. The two ORFs were in different reading frames and overlapped each other by 11 nucleotides. One ORF encoded a protein of 170 amino acids and the other ORF, located adjacent to the 3' noncoding region of the viral genome, encoded a 114 amino acid protein. Thirty-three N-terminal residues were sequenced directly from purified LDV capsid protein, Vp1, and this amino acid sequence mapped to the ORF adjacent to the 3' noncoding region. The presence of overlapping ORFs and the 3' terminal map position of Vp1 indicate that LDV differs significantly from the prototype alpha togaviruses.

The 3'terminus of lactate dehydrogenase-elevating virus genome RNA does not contain togavirus or flavivirus conserved sequences.

Lactate dehydrogenase-elevating virus (LDV) is currently considered to be an unclassified togavirus. The 3' terminus of the genome RNA of the C-strain of LDV was cloned and sequenced. A synthetic DNA oligomer complementary to the 3' portion of this cloned sequence was then used to prime dideoxy sequencing from the LDV-C genome RNA as well as from the genome RNAs of three additional LDV isolates. A high degree of sequence conservation was observed in the 3' terminal region among the four LDV isolates analyzed. Comparison of the LDV 3' sequence with those of the alpha togaviruses, rubella virus, and the flaviviruses showed that the LDV genome does not contain conserved 3' sequences characteristic of these viruses.

In situ immune autoradiographic identification of cells in heart tissues of mice with coxsackievirus B3-induced myocarditis.

In adolescent CD-1 male mice inoculated with a myocarditic coxsackievirus B3 (CVB3m) acute focal lesions containing necrotic myocytes, infiltrating mononuclear cells, and fibroblasts develop. With the use of an in situ immune autoradiographic method with rat monoclonal antibodies (MAb) and an 35S-labeled antibody, viral antigens were detected outside of lesions. Macrophages, T lymphocytes, and natural killer (NK) cells were identified within myocarditic lesions during the acute phase of the disease. Macrophages detected by anti-Mac-1 MAb were in focal areas within myocarditic lesions on Days 4-7 after inoculation. T lymphocytes were detected in myocarditic lesions on Days 4-10, with MAb to Thy-1 and Lyt-1 antigens showing diffuse reaction patterns, suggesting random distribution of these cells in lesions. Focal areas of reactivity were detected with MAbs to L3T4 and Lyt-2 antigens, suggesting clusters of helper and cytotoxic/suppressor T lymphocytes, respectively. NK cells were presumptively detected by asialo GM1 surface marker in lesions at all times. The presence of activated NK cells in lesions was confirmed by assay of mechanically dissociated heart tissues on Day 8. These data describe the temporal sequence and identity of leukocytes entering into CVB3-induced focal myocarditic lesions during the acute phase of disease in CD-1 mice.

Murine natural killer cells limit coxsackievirus B3 replication.

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.

Coxsackievirus group B replication in cultured fetal baboon aortic smooth muscle cells.

All six coxsackievirus B (CVB) serotypes replicated to various extents in fetal baboon aortic smooth muscle cells (SMC) in culture. CVB3 and CVB4 replicated to the highest titers and induced no cytopathology at the level of light microscopy. Maximum yields of CVB3 were produced between 12 and 24 hr postinoculation. Up to 15% of SMC cells became infected, as determined by immunofluorescence assays with anti-CVB3 antiserum, yet overall cell division in infected cultures did not differ from infected SMC cultures. Electron microscopy of CVB3-inoculated SMC cultures revealed changes in some cells: viruslike particles, secondary lysosomes containing dense bodies, and peripheral nuclear chromatin condensation. CVB3 replicated well in SMC passages up to the eighth, but did not replicate in eleventh-passage cells. Because of the cardiotropic and myotropic potential of this virus and its ability to replicate in aortic SMC with associated ultrastructural alterations, CVB3 (and other CVB) should be further examined as an etiologic agent(s) that could induce atherosclerosis.

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis.

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.