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

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.

Figure 1

RFLP electrophoresis result to identify the amplified 829 bp segments of CDV vaccine strains and field strains. A) Lane 1-10: LDL05-M1, LDL05-F1, SDWD06-M1, LHLD06-M1, LHC06-F1, JSY06-R1, LLY06-M1, SN07-R1, HCL07-R1, LHS07-R1; Line 11,12: CDV3 and Spanish; M: 100 bp Ladder DNA Marker; B) Lanes 1-9: JDH07-F1, JDAN07-R1, LDL05-M2, LDL05-F2, SDWD06-M2, LHLD06-M2, JSY06-R2, HSN07-R2, HCL07-R2; Lanes 10-12: France, Holland and USA; M: 100 bp Ladder DNA Marker. Three bands were seen in Figure 1A(Lanes 1-10) and Figure 1B(Lanes 1-9): 455 bp, 220 bp and 153 bp, which belong to CDV field strains; two bands were showed in Figure 1A(Lanes 11,12) and Figure 1B(Lanes 10-12): 675 bp and 153 bp, which are CDV vaccines.

Figure 2

A single nucleotide difference leads to the existence of an addition BamHI site of CDV field strains. All CDV isolates have an A at 610 nt in N gene 829 bp fragments, but vaccine strains have a G. (BamHI site GGATCC showed in box). Underline indicates the 19 Chinese CDV field strains analyzed in this study. Underline with broken line means commercial CDV vaccines analyzed in this study.

Figure 3

Phylogenetic analysis of the N gene of field CDV strains. (1) vaccine strains, or America-1 genotype; (2) (3) (4) (5) four branches of the CDV field virus strains, (2) The branch belonging to Arctic genotype. Two strains (JSY06-R1 and JSY06-R2) of CDV isolated from Jinlin Songyuan raccoon dog, distinguished from other strains, are similar to HL, and were clustered into Arctic genotype; (3) America-2 CDVs genotype. JDH07-F1, isolated from Jilin Dehui slivery fox, which was most closely related to the standard field strains A75/17, which belong to America-2 CDVs; (4) Europe genotype. This cluster has not been identified in China. (5) Asia-1 genotype. The 16 field strains in present study are closely related to many field strains, which from China mainland or Taiwan China, and belong to Asia-1 genotype. Δ means CDV isolates in this study.