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Query: EC:2.7.7.48 (transcriptase)
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In this article we describe two new complete genomic sequences of Old World Arenaviruses: the Mopeia (MOP) virus and the reassortant MOP/LAS virus, clone 29, or ML29. This reassortant has the large (L) RNA from MOP virus and the small (S) RNA from Lassa (LAS) virus, Josiah strain. Recent studies showed that the ML29 virus is not pathogenic for mice, guinea pigs, or macaques, can completely protect guinea pigs from Lassa virus, and elicit vigorous cell-mediated immunity in immunized monkeys (Lukashevich, I. S., Patterson, J., Carrion, R., Moshkoff, D., Ticer, A., Zapata, J., Brasky, K., Geiger, R., Hubbard, G. B., Bryant, J., and Salvato, M. S., J Virol 79, 13934-13942, 2005). This is a molecular characterization of a reassortant virus, which has been put forward as a live attenuated vaccine candidate against Lassa Fever. Sequence analysis of this reassortant virus revealed 5 non-conservative amino acid substitutions that distinguished it from the parental LAS and MOP viruses. Three substitutions were found outside the conserved RNA-dependent RNA polymerase (RdRp) motifs. A fourth substitution was located between the glycoprotein (GPC)-cleavage site and the putative fusion peptide of GP2. The nucleocapsid protein (NP) contained a fifth substitution in the carboxyl-terminal region of the protein. Two mutations were found within each non-coding terminus of the L segment and one mutation was located in the 3' non-coding region of the S segment of the MOP/LAS virus. ML29 mutations in its genomic termini may have implications for the genetic stability and replication efficiency of ML29 reassortant.
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PMID:Molecular characterization of a reassortant virus derived from Lassa and Mopeia viruses. 1714 22

Four RNAs from a new plant-pathogenic virus, which we have tentatively named European mountain ash ringspot-associated virus (EMARAV), were identified and sequenced completely. All four viral RNAs could be detected in previous double-stranded RNA preparations. RNA 1 (7040 nt) encodes a protein with similarity to the RNA-dependent RNA polymerase of different members of the Bunyaviridae, a family containing five genera with viruses infecting invertebrates, vertebrates and plants. RNA 2 (2335 nt) encodes a 75 kDa protein containing a conserved motif of the glycoprotein precursor of the genus Phlebovirus. Immunological detection indicated the presence of proteins with the expected size of the precursor and one of its processing products. The amino acid sequence of protein p3 (35 kDa) encoded by RNA 3 shows similarities to a putative nucleocapsid protein of two still unclassified plant viruses. The fourth viral RNA encodes a 27 kDa protein that has no significant homology to any known protein. As is typical for members of the family Bunyaviridae, the 5' and 3' ends of all viral RNAs are complementary, which allows the RNA to form a panhandle structure. Comparison of these sequences demonstrates a conserved terminal part of 13 nt, similar to that of the bunyaviral genus Orthobunyavirus. Despite the high agreement of the EMARAV genome with several characteristics of the family Bunyaviridae, there are a few features that make it difficult to allocate the virus to this group. It is therefore more likely that this plant pathogen belongs to a novel virus genus.
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PMID:A novel, multipartite, negative-strand RNA virus is associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.). 1737 80

Individual lyssavirus genes were evaluated for phylogenetic studies from available full genome sequences. The full genome of the ERA rabies virus was sequenced and its accuracy was confirmed through virus recovery by reverse genetics. The full length of the ERA is 11,931 nucleotides (nt), with a leader sequence of 58 nt, the nucleoprotein (N) gene of 1350 nt, phosphoprotein (P) gene of 891 nt, matrix protein (M) gene of 606 nt, glycoprotein (G) gene of 1572 nt, RNA-dependent RNA polymerase (L) gene of 6384 nt, Psi-region (or G-L intergenic region) of 400 nt, and a trailer region of 70 nt. The five mono-cistrons are separated by intergenic regions of 2, 5, 5 and 24 nt, respectively. One obvious difference between the ERA and SAD-B19 rabies virus strains was the putative stop/polyadenylation signal of the G gene, with a poly(A(8)) tract for ERA, and a poly(A(5)) for SAD-B19. The TGpoly(A(8)) sequence tract was identified to be a leaky termination signal in the ERA strain. Through analyses of nt diversity, protein co-variations, structural and functional constraints, and reconstruction of phylogenetic trees from comprehensive datasets, we propose lyssavirus genes probably are of similar value for phylogenetic analyses.
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PMID:Are all lyssavirus genes equal for phylogenetic analyses? 1768 31

In June 2006, 150 wild common carp were sampled from Hamilton Harbour, Lake Ontario, Canada. Tissue pools consisting of kidney, spleen and encephalon were screened for viruses as a condition facilitating the export of live carp to France. Cytopathic effect (CPE), indicative of a viral infection, became evident after 8 days of incubation at 15 degrees C. Eighteen of 30 tissue pools (five fish per pool) eventually demonstrated viral CPE. The viral pathogen was initially cultured and isolated on the epithelioma papulosum cyprini cell line and subsequently shown to produce CPE in the fathead minnow and bluegill fin cell lines. Electron microscopy demonstrated the virus to be a rhabdovirus. Reverse transcriptase-polymerase chain reaction assay and nucleotide sequence analysis identified the isolate as spring viraemia of carp virus (SVCV). Phylogenetic analysis of a 533 bp region of the glycoprotein gene grouped the Canadian isolate in SVCV genogroup Ia together with isolates from Asia and the USA. Sequence comparisons revealed the Hamilton Harbour, Lake Ontario isolate to be most similar to an isolate obtained from common carp in the Calumet Sag Channel in Illinois in 2003 (98.9% nucleotide identity). This is the first report of the detection of SVCV in Canada.
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PMID:First detection and confirmation of spring viraemia of carp virus in common carp, Cyprinus carpio L., from Hamilton Harbour, Lake Ontario, Canada. 1795 10

The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus.
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PMID:Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF. 1806 57

An isolate of a new tospovirus species, causing concentric zoned ringspots on fruits and necrotic lesions on leaves of infected plants, was characterised based on particle morphology, host range and serological properties. The complete nucleotide sequences of large (L), medium (M), and small (S) RNAs of this virus were found to contain 8919, 4945, and 3279 nts respectively. The L RNA encoded the RNA-dependent RNA polymerase (RdRp) (2885 aa, 332.7 kDa). The M RNA encoded a non-structural (NSm) protein (309 aa, 34.4 kDa) and a viral glycoprotein precursor (Gn/Gc) (1122 aa, 127.4 kDa). The S RNA encoded a non-structural protein (NSs) (459 aa, 51.9 kDa) and the nucleocapsid (N) protein (278 aa, 30.6 kDa). This N protein shared amino acid identities of 80.9% with those of calla lily chlorotic spot virus. Our results suggest that the virus studied here belongs to a new tospovirus species, for which the name tomato zonate spot virus is proposed.
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PMID:Characterization of tomato zonate spot virus, a new tospovirus in China. 1832 Jan 36

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Western blotting analysis revealed that three mAbs (1H5, 1E10, and 1H7) recognized specifically to a single protein of 47kDa (N), the mAb 3G4 reacted with two SVCV0504 proteins of 69kDa (G) and 47kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69kDa (G), 50kDa (P), and 47kDa (N). By indirect ELISA, two mAbs (1H5 and 1H7) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID(50) (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28h after inoculation with the virus (0.01PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus.
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PMID:Development and characterization of monoclonal antibodies to spring viraemia of carp virus. 1837 3

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. The ability of BHV-1 to transport anterogradely from neuronal cell bodies in trigeminal ganglia (TG) to nerve ending in the noses and corneas of infected cattle following reactivation from latency plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. We have constructed a BHV-1 bacterial artificial chromosome (BAC) clone by inserting an excisable BAC plasmid sequence in the long intergenic region between the glycoprotein B (gB) and UL26 genes. A BAC-excised, reconstituted BHV-1 containing only the 34-bp loxP sequence within the gB-UL26 intergenic region was highly infectious in calves, retained wild-type virulence properties, and reactivated from latency following treatment with dexamethasone. Using a two-step Red-mediated mutagenesis system in Escherichia coli, we constructed a gE cytoplasmic tail-truncated BHV-1 and a gE-rescued BHV-1. Following primary infection, the gE cytoplasmic tail-truncated virus was efficiently transported retrogradely from the nerve endings in the nose and eye to cell bodies in the TG of calves and rabbits. However, following dexamethasone-induced reactivation from latency, the gE mutant virus was not isolated from nasal and ocular sheddings. Reverse transcriptase PCR assays detected VP5 transcription in the TG of rabbits infected with gE-rescued and gE cytoplasmic tail-truncated viruses during primary infection and after dexamethasone treatment but not during latency. Therefore, the BHV-1gE cytoplasmic tail-truncated virus reactivated in the TG; however, it had defective anterograde transport from TG to nose and eye in calves and rabbits.
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PMID:A bovine herpesvirus type 1 mutant virus specifying a carboxyl-terminal truncation of glycoprotein E is defective in anterograde neuronal transport in rabbits and calves. 1848 Apr 34

Human rabies virus vaccine strain CTN181 from China was sequenced. The overall length of the genome was 11,923 nucleotides (nt), comprising a leader sequence of 58 nt, nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609 nt, glycoprotein (G) gene of 1575 nt, RNA-dependent RNA polymerase (RdRp, L) gene of 6387 nt, and a trailer region of 70 nt. The five monocistrons are separated by intergenic regions (IGRs) of 2, 5, 5 and 24 nucleotides (nt), respectively. Two obvious differences between CTN181 and the other rabies virus vaccine strains were (1) the putative stop/polyadenylation signal of the G gene has only one poly (A) tract for CTN181, and (2) the start of the open reading frame for L has two repeats of ATG for CTN181. Both were similar to the SHBRV-18 (silver-haired bat-associated RV strain 18) strain. In addition, some mutations and new functional regions were discovered that are presumed crucial to the function of leader region and L protein. There is an equal role for all five genes in the phylogenetics of rabies virus.
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PMID:Characterization of human rabies virus vaccine strain in China. 1850 87

The results of analyses of Z, RNA-dependent RNA polymerase, glycoprotein precursor, and nucleocapsid protein gene sequence data suggested that Guanarito virus was the most common cause of Venezuelan hemorrhagic fever in a 7-year period in the 1990s and that the evolution of Pirital virus in association with Sigmodon alstoni (Alston's cotton rat) has occurred at a significantly higher rate than the evolution of Guanarito virus in association with Zygodontomys brevicauda (short-tailed cane mouse) on the plains of western Venezuela. The results of analyses of the primary structures of the glycoproteins of the 8 strains of Guanarito virus isolated from humans suggested that these strains would be highly cross-reactive in neutralization assays. Thus, passive antibody therapy may prove beneficial in the treatment of human disease caused by strains of Guanarito virus that are enzootic in the region in which Venezuelan hemorrhagic fever is endemic.
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PMID:Genetic diversity between and within the arenavirus species indigenous to western Venezuela. 1858 98


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