Isolation and molecular characterization of canine distemper virus from India

Abstract

Ocular swabs from canine distemper virus (CDV) suspected live or brain tissue from dead dogs were tested for the presence of CDV nucleoprotein (N) gene using reverse transcriptase polymerase chain reaction (RT-PCR). Partial "N" gene sequencing of the RT-PCR-positive samples and the local vaccine virus revealed that the Ind/Andaman 01/07 virus was highly divergent from the rest of the CDV isolates and from the vaccine strain. Quantitative real-time PCR (qRT-PCR) using SYBR Green I chemistry for CDV haemagglutinin "H" gene quantification showed C(t) values ranging from 29.76-30.67 in the RT-PCR-positive samples. Two of the positive samples, designated Ind/TN 01/07 and Ind/Andaman 01/07 were used for virus isolation in B95a cell line. Characteristic cytopathic changes such as rounding of cells, syncytia formation, and ballooning were seen from the first passage onwards. Specific cytoplasmic fluorescence was seen in infected cells with a commercial reference serum against CDV. To the best of our knowledge, this is the first report of CDV isolation from clinical cases in India.

Isolation and phylogenetic characterization of Canine distemper virus from India

“…Based on sequences of the proteins (H, F, M and P) studied, Indian wild-type CDV showed a distinct clade in the phylogenetic tree. Similar finding has been reported earlier [18] based on partial N gene nucleotide sequences of two Indian CDV isolates.…”

“…Seventy percent seroprevalence of CDV infection has been reported from South India [13]. In addition, isolation of Indian CDV from B95a cells and sequence analysis of partial N gene has also been reported [18], but detailed molecular epidemiology of the Indian CDV isolates is lacking till date. Therefore, the present study focused on the cloning, sequencing of H, F, P, M genes and the phylogenetic analysis of an Indian CDV strain as compared to the GeneBank available reference CDVs and the commercial CDV vaccine strains.…”

Detection of Local Isolates of Canine Distemper Virus by Reverse-Transcription Polymerase Chain Reaction

“…7). The similar pattern has also been reported earlier based on partial N gene nucleotide sequences of two Indian CDV isolates (Pawar et al, 2011). Recently, another Indian CDV isolate was reported to make a separate branch compared to vaccine group of CDVs in phylogenetic tree not only based on H protein but also for F, M and P proteins (Swati et al, 2015).…”

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag

“…In addition, for mRNA analysis, a primer for the RT-step was designed that included an adapter sequence and a 5-nucleotide sequence at the 3 end that was specific for SIV gag and CDV N mRNAs, which significantly reduced background from cellular RNA and positive-sense vector genomic RNA and also helped to eliminate amplification of DNA caused by RNA self-priming (Craggs et al, 2001;Simon et al, 2010). More recently, an RT-qPCR assay was reported (Pawar et al, 2011) based on SYBR Green I chemistry for detection of the hemagglutinin gene in samples from infected dogs. This assay used oligo-dT primers to synthesize cDNA from mRNA in the RT step and a relative C T quantification method to determine fold changes in CDV gene expression.…”

“…These assays are very sensitive, but sensitivity varies with sample source, viral RNA extraction methods, and choice of primers. More recently, quantitative real-time PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) assays have been developed for the detection of CDV (Elia et al, 2006;Fischer et al, 2013;Meli et al, 2009;Pawar et al, 2011;Scagliarini et al, 2007;Wilkes et al, 2014) and many other viruses including rabies virus (Hughes et al, 2004), blue-tongue virus (Toussaint et al, 2007), SIV (Cline et al, 2005) and vesicular stomatitis virus (VSV) . These assays have been valuable in providing estimates of the viral load in plasma, and infected tissues and organs of animals.…”