Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

Fengxue Wang,Xijun Yan,Yongjun Wen,Jianjun Zhao,Hailing Zhang,Wei Wu,Xiuli Chai

doi:10.1186/1743-422x-8-85

Abstract

In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV), a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). We selected an 829 bp fragment of the nucleoprotein (N) gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

Highlights

Canine distemper virus (CDV) is an enveloped negativestrand RNA virus classified into the genus Morbillivirus within the family Paramyxoviridae
A specific 829 bp fragment of N gene was obtained from 19 CDV field strains and 5 CDV attenuated strains contained in the commercial vaccines
restriction fragment length polymorphism (RFLP) analysis RFLP analysis was carried out on the purified target segments and the results were used to identify the corresponding virus as vaccine or field strain

Summary

Introduction

Canine distemper virus (CDV) is an enveloped negativestrand RNA virus classified into the genus Morbillivirus within the family Paramyxoviridae. The accurate diagnosis of CDV infection can be made with the use of methods based on molecular biological techniques. The application of nucleic acid hybridization, PCR and other techniques [6,7,8,9] has improved the accuracy, sensitivity and specificity of CDV diagnosis. The detection of CDV based on N gene has been established by many researchers [10,11,12,13,14]. Real-time RT-PCR targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene was established and shown to be more sensitive and effective [15]. The use of RTPCR-RFLP to detect the hemagglutinin (H) gene of CDV was reported by Calderon [16]. The CDV N gene is more conserved than H gene, and provides a better target for CDV detection

Methods

Results

Conclusion