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The purpose of this study was to generate complete genome sequences of Aravan (ARAV), Khujand (KHUV), Irkut (IRKV) and West Caucasian bat (WCBV) viruses, and to compare them with genomes of other lyssaviruses. We focused on RNA-dependent RNA-polymerase (L) and non-coding regions, because other genes of these viruses have been described previously. The L protein is organized into six conserved blocks (I-VI), previously detected in all Mononegavirales. Furthermore, lyssaviruses have two additional conserved regions, L1 and L2, located in the COOH part of the L. L1 may be responsible for methylation of viral mRNA cap structures, whereas the significance of L2 is unclear. Phylogenetic patterns based on the L are similar to those described for the nucleoprotein. The WCBV is the most divergent member of the genus. Besides phylogeny, it has a short trailer region (57 nucleotides versus 69-70 nucleotides in other lyssaviruses) and different intergenic region lengths, including an exceptionally long non-coding region of the glycoprotein (697 nucleotides) containing a potential open reading frame of 180 nucleotides. The absence of a flanking transcription initiation signal, as well as Northern and Western blot data, suggests that this region is not independently transcribed but is a part of G mRNA.

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers. [ABSTRACT FROM AUTHOR]

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms. [ABSTRACT FROM AUTHOR]

Abstract: 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(A8) tract for ERA, and a poly(A5) for SAD-B19. The TGpoly(A8) 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. [Copyright &y& Elsevier]

Abstract: Rabies in Asia is emerging as a serious public health issue. To explore the possible origin, phylogenetic relationships, and evolutionary dynamics of Asian Rabies viruses (RABV), we examined 200 complete nucleoprotein (N) gene sequences from RABV isolates in the region. Phylogeny supported the classification of Asian RABVs into five distinct clusters in lyssavirus genotype 1. Our geospatial and temporal analyses demonstrated that China appears to be the prime source of Asian RABVs. Understanding of rabies transmission and associated human activities, such as dog translocation, can help rabies control and elimination in Asia through collaborative efforts or programs. [Copyright &y& Elsevier]