MOLECULAR DETECTION, N GENE SEQUENCING AND PHYLOGENETIC ANALYSIS OF RABIES VIRUS
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Date
2016
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AAU, Anand
Abstract
Rabies is an ancient global fatal disease of central nervous system (CNS) and most significant zoonotic and neglected viral disease that affects almost all kinds of mammals, including humans. The causative agent for rabies is Rabies virus, the prototype member of the genus Lyssavirus of the family Rhabdoviridae under the order Mononegavirales. India stands highest for the incidences of rabies with an estimated 20,000 human death and 17.4 million exposures to animal bites every year. The study of rabies dynamics in the field required to understand the genetic variants in circulation and their evolutionary relationships thereby to help devise effective control measures. The genetic characterization of the virus provides an opportunity to elucidate the epidemiologic and evolutionary relationships between rabies virus (RV) and rabies related viruses (RRVs). The present study was aimed to study the ante mortem detection of rabies virus from saliva samples as well as post mortem detection of rabies virus from brain samples by Immunochromatographic test kit and molecular technique (RT-PCR), and virus characterization by sequencing of partial N gene of rabies virus and phylogenetic analysis to know the genotype and lineage of rabies virus present in Gujarat state. A total of 32 samples (18 brain samples and 14 saliva samples) were aseptically collected from live and dead animals viz. dog, buffalo, cow, goat, donkey and hyena for rabies virus detection. Brain and saliva samples were examined by Immunochromatographic test (ICT) and RT-PCR, and the results of both these tests were compared. Out of 18 brain samples, 17 brain samples were found positive by ICT, whereas 16 brain samples were found positive by RT-PCR. In 14 saliva samples, seven samples were positive by ICT, whereas eight samples were positive by RT-PCR. On comparison of both these tests, one brain sample was found false positive and one saliva sample was found false negative by ICT when compared with RT-PCR. Both the tests, thus, yielded comparable results with an additional advantage of using them in live animals. Sequencing of 20 samples having good concentration of RT-PCR product was performed by Sanger sequencing method using ABI 3500 Genetic analyzer. Obtained sequences had nucleotide length ranging from 584 to 606 bp and deduced amino acid length of 193-196 amino acids. Sequences were submitted to NCBI Genebank using Bankit online sequence submission tool. ClustalW alignment of nucleotide sequences and amino acid sequences of field rabies isolates revealed 95.20-100% and 97.95-100% similarity among themselves, respectively. Multiple nucleotide sequence alignment of field rabies isolates with Pasteur and CVS reference strains revealed 89.10 - 90.75% and 88.07 - 90.84% similarity, respectively. The multiple sequence alignment revealed single nucleotide variations at total 91 different positions. Multiple amino acid sequence alignment showed that all the field rabies isolates were 94.38 - 95.91% and 94.89 - 96.42%) similar with Pasteur and CVS reference rabies strains and also revealed single amino acid variations at total 17 different positions. Phylogenetic analysis was done using MEGA version 6 with Neighbor Joining algorithm using a consensus of 1000 bootstrap replicates with a total of 21 isolates/ sequences of Indian and foreign origin along with Pasteur and CVS reference strains. Phylogenetic analysis of nucleotide sequences revealed three distinct clusters with Indian and foreign isolates. Our 20 field rabies isolates were highly similar with earlier reported five Indian sequences and were grouped together in cluster I. The cluster I was at 0.03 genetic distance with cluster II and 0.10 with cluster III. The reference vaccine strains Pasteur and CVS were present in cluster II. The genetic distance of Pasteur strain with our field rabies isolates and whole cluster I was 0.10 and 0.08, respectively. CVS strain was at 0.11 genetic distance with our field isolates and at 0.09 genetic distance with whole cluster I. The present study revealed that the Immunochromatographic test and RT-PCR yielded comparable results for detection of rabies virus from brain and saliva samples with an additional advantage of using them in live animals. Multiple sequence alignment of field rabies isolates and reference vaccine strains (Pasteur and CVS strain) indicated single nucleotide variations-(SNVs) at total 91 positions and amino acid variations at total 17 different positions. Phylogenetic analysis of N gene sequences using our 20 field rabies isolates and 21 other reported isolates in Genbank resulted in 3 phylogenetic clusters. All the field rabies isolates showed same genetic lineage among themselves and with other earlier reported Indian rabies isolates placing them in Arctic like lineage of Genotype 1 Rabies virus. However, they were at genetic distance with reference Pasteur and CVS strains, which grouped in different phylogenetic cluster.
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VERTINARY MICROBIOLOGY, AN ANALYSIS