Isolation, Differentiation, PCR Detection And B2L Gene Sequence Analysis Of CPDV

The paper has three parts. The first is isolation and identification of contagious pustulardermatitis virus(CPDV). The second is detection of CPDV by polymerase chain reaction(PCR). The last is clone and sequencing analysis of B2L gene of vaccine strain and threeisolation strains CPDV. 1.Isolation and identification of contagious pustular dermatitis virus(CPDV) fromclinical scabs using cell culture,electron microscopy detection and animal inoculation test.11of 17 clinical scabs specimens had regular cytopathic effect (CPE) over 8 passages on primarybovine testis cell culture. Electron microscopy demonstrated that typical brick-shaped virionswere found in the cell culture and the positive specimens. After the inoculation, all theanimals showed clinical signs of CPD, namely vesicle formation,which became pustularfollowed by scabbing. All the teats provide perfect information for CPDV diagnosis andepidemic survey. 2.One pair of primers was designed to detect contagious pustular dermatitis virus(CPDV)in cell culture by the PCR test, and the expected 594bp DNA fragment was obtained in thetest. The results of PCR were also confirmed by the cytopathic effect (CPE)on the cellculture,electron microscopy detection and animal infection. The negative results wereachieved from normal primary bovine testis cell,foot-and-mouth virus (FMDV),sheep poxvirus and bluetongue virus(BTV). Nucleotide sequences Comparison of the PCR productswith CPDV NZ-2 strain revealed that the homology of them was 97%. 11 Of 17 clinical scabssamples, were positive by PCR. The results were consistent with isolation and identificationof pathogeny. These findings suggested that the established the CPDV PCR technique providea specific and reliable diagnostic tool for CPDV rapid detection, especially for discriminationof CPDV from other pathogen that cause the similar clinical symptoms. 3.The B2L gene of vaccine strain (CPDVy), GanSu (CPDV1), ShanXi (CPDV2) andShanDong (CPDV3) strains were amplified by polymerase chain reaction (PCR) with anotherpair of primers. The amplified fragments were all about 1130bp in length by agarose gelelectrophoresis test. Then the fragments were cloned respectively into pMD-18T vector andsequenced. Comparison of the nucleotide sequences and deduced amino acid sequence of thefive CPDV strains showed that the nucleotide sequences homology of CPDVy strain withCPDV1, CPDV2, CPDV3 and NZ-2 strains were 99.7%, 96.9%, 97.4% and 96.1%Abstract IIIrespectively, the amino acid sequence homology were from 92.0% to 96.0% and the fourCPDV strains of China have a nucleotide deletion at 1111. These proved that the B2L gene ishighly conserved. The PCR detection method for CPDV was established which was the first in China, andthe B2L gene nucleotide sequences of four China strains were also reported firstly.