Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs

Abstract

A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.

Fig. 1

Schematic illustration of CDV differential primers strategy by P1/P1 and P3/P4 for detecting CDV wild-type strains. The * display as the identity nucleotide. The bold letters represent 3′ terminus mismatches between CDV wild-type strain and vaccine by primers P3/P4.

Fig. 2

(A) Amplification of genomes of different easily infected canine viruses by primers P1/P2 of RT-PCR reaction. M, 100 bp Ladder DNA Marker; lane 1, canine distemper virus (CDV) vaccine strain CDV3; lane 2, CDV wild-type strain CDV-PS; lane 3, canine parvovirus virus; lane 4, canine adenovirus virus; lane 5, canine coronavirus virus; lane 6, uninfected cells control; lane 7, negative control of healthy animal tissue. (B) Amplification of genomes of different easily infected canine viruses by primers P3/P4 of RT-PCR reaction. M, 100 bp Ladder DNA Marker; lane 1, canine distemper virus (CDV) vaccine strain CDV3; lane 2, CDV wild-type strain CDV-PS; lane 3, canine parvovirus virus; lane 4, canine adenovirus virus; lane 5, canine coronavirus virus; lane 6, uninfected cells control; and lane 7, negative control of healthy animal tissue.

Fig. 3

Sensitivity of RT-PCRs to detect CDV vaccine by primers P1/P2 and CDV wild-type strain by primers P3/P4. Lanes 1–7: 10⁵ TCID₅₀ CDV cell culture; 10⁴ TCID₅₀ CDV cell culture; 10³ TCID₅₀ CDV cell culture; 10² TCID₅₀ CDV cell culture; 10¹ TCID₅₀ CDV cell culture; 10⁰ TCID₅₀ CDV cell culture; and 10⁻¹ TCID₅₀ CDV cell culture.

Fig. 4

Differential diagnosis of canine distemper virus between wide-type strains and vaccine by the combined RT-PCR test. M, 100 bp Ladder DNA Marker; lane 1 and 7: negative control without CDV template by primers P3/P4 and P1/P2, respectively; lanes 2–6: vaccine strain CDV3, Vacc-A, Vacc-B, wild-type strain CDV-PS and CDV-SD amplified from RT-PCR by primers P3/P4, respectively; lanes 8–12: vaccine strain CDV3, Vacc-A, Vacc-B, wild-type strain CDV-PS and CDV-SD amplified from RT-PCR by primers P1/P2, respectively.

Fig. 5

The phylogenetic relationships among 27 CDVs based on the nucleotide sequence of H gene. Distance values were calculated by the Clustal W program within the MEGA 4.0 software package. The neighbor-joining algorithm was used to generate the tree. Bootstrap values were calculated on 1000 replicates. The local isolates indicate the 7 Chinese wild-type CDV strains analyzed in this study. GenBank accession numbers of the reference strains are as follow: CDV3 (DQ778941), Onderstepoort (AF378705), Convac (Z35493), SnyderHill (AF259552), Yanaka (D85755), Ueno (D85753), Hamamatsu (D85754), Dog98-002 (AB025270), Dog5B (AY297453), DogHM-3 (AB040767), Dog5VD (AY297454), DogTaiwan (AY378091), DogisolateADen (AF478543), DogisolateCDen (AF478547), Dog Denmark (Z47761), US89 (Z47764), Africa 1bn (FJ461713), Africa 4sp (FJ461715), dog GR88 (Z47760), dog 179-94 (DQ226087), 01-2676 (AY498692), 01-2690 (AY465925), H06Ny12HU (DQ889189)and 207/00 IT (DQ228166).

Fig. 6

Sequence alignment of partial hemagglutinin gene. The partial H gene nucleotide sequences of field strains from China and commercial CDV3 vaccine were analyzed. The numbering starts at the position nucleotide 961 and 1441 of the H gene. Only amino acids that differ from the majority sequence are shown. identical residues are represented by dots. The substitution of the AG and A present in the field strains, which was used to design the differentiating primers for RT-PCR, is indicated by a square box.

Fig. 7

Sequence comparison at nucleotide position 990nt and 1506nt of H gene with various CDV lineages. GenBank accession numbers of the reference strains are as follow, Asia-1 strains: CDwuhan17, CDwuhan24, PS-05, BJ-09 (GenBank: EU192013, EU191985, EU191988, EU327874, EU327875, EF596903, EF596904, EU934234), Asia Japan isolates: Yanaka (D85755), Ueno (D85753),Asia-2 strains: Dog5B (AY297453), DogHM-3 (AB040767), Dog5VD (AY297454), Europe strains: DogisolateADen (AF478543), DogisolateCDen (AF478547), Dog Denmark (Z47761), Europe wildlife strains: H06Ny12HU (DQ889189) and 207/00 IT (DQ228166), America-2 strains:US89 (Z47764), 01-2676 (AY498692), 01-2690 (AY465925), Africa isolates: Africa 1bn (FJ461713), and America-1 strains (contain vaccines): CDV3 (DQ778941), Onderstepoort (AF378705), SnyderHill (AF259552). Omitted sequences are represented by dots.