Isolation And Identification Of Porcine Pseudorabies Virus And Establishment Of Detection Methods

Porcine pseudorabies virus was identified from a field isolate collected in Hebei province. The specimen was the brain of a postabortive sow. To identify the virus microbiologically, chicken embryos were infected by field isolate. The typical pathology and pockmark formation on allantoic sac were observed. Infected PK-15 cell line shrunk, aggregated, and detached from the culture system in 20h approximately. Tissue culture infectious dose(TCID₅₀) in PK-15 cell line transfected with isolated virus was 10^-6.11/20μl. It was shown that the virus was sensitive to heat, chloroform, and acid. It was proved that the virus could be neutralized by PRV positive serum through neutralization test, and neutralization index was 479. Circular and enveloped virus could be seen under the transmission electron microscope. Mock-infected rabbits were itching severely. We get the specific bands of 217bp through PCR with DNA of isolated virus, which testified that it was PRV. These data indicate that the isolated virus could be PRV and referred to as JiA strain in this report.It is very difficult to differentially diagnose PRV from PPV because both of them are etiologic agents of infectious abortion with similar symptoms. For clinically differentiate these diseases at the level of nucleic acid, two pairs of specific primers were designed in accordance to the conservative sequences of PRV(gp50) and PPV(VP2). The zone of DNA amplification of single PCR appeared in 217bp and 158bp, respectively. To improve PCR assay for the detection of PRV and PPV, double PCR was performed on pooled specimens. It was shown that two specimens which contained PRV and PPV DNA could be amplified by the double PCR using these two pairs of primers, yielding two specific bands of 217bp and 158bp. This established PCR assay was capable of detecting target viruses at minimum of 10^-6.11/20μl TCID₅₀ of PRV and 0.008HA unit of PPV DNA. This detection assay could be performed in no more than 24h.The method of SPA co-agglutination is conceived for diagnosing PRV from other viruses popularly, maneuverablely and intuitionisticly.We establish the SPA plate co-agglutination test through making SPA combine hyperimmune serum.It is showed by the sensitive test .specific test, contrastive test and animal test that this method is more sensitive, more specific and more reduplicate, furthermore it is handy, fast, cost material and not requiring special apparatus,which is fit for fast identification of PRV in common field and epidemiological investigation.