猪伪狂犬病病毒的分离鉴定及致病性试验OA

In this study,one virus strain was isolated from the brain and lung tissues of dead pigs from a swine farm in Wuhan.This virus was capable of inducing typical cytopathic effects(CPE)in swine testicle(ST)cells.After cultivation and harvesting,the virus titer was determined using ST cell plates.The mor-phology of viral particles was observed via electron microscopy.An equal volume of pseudorabies virus-spe-cific positive serum was mixed with the virus suspension for neutralization,followed by inoculation into ST cells to observe the neutralization efficacy.Purity testing was performed in accordance with the appendices of the Chinese Veterinary Pharmacopoeia.Molecular biological methods were employed to identify the gB and gE genes of the isolated virus.The full-length open reading frame(ORF)sequences of its gC,gD,gE,and TK genes were aligned with those of other strains retrieved from GenBank,and corresponding phyloge-netic trees were constructed.The virus suspension was diluted to a concentration of 106.0TCID50/mL and in-oculated into 5 piglets.Each piglet received 3.0 mL via intramuscular injection in the neck and 3.0 mL via intranasal administration.The incidence of disease and mortality of the piglets were observed and recorded.The results showed that the virus titer of the isolated strain in ST cells reached 107.75TCID50/mL.Under e-lectron microscopy,the viral particles appeared spherical with a diameter of approximately 150 nm.The iso-lated virus could be neutralized by pseudorabies virus-specific positive serum and was free from bacterial,mycoplasmal,and exogenous viral contamination.PCR amplification yielded specific bands corresponding to the gB and gE genes.Phylogenetic tree analysis indicated that multiple virulence genes of the isolated strain were distantly related to those of early-isolated strains,but closely related to the epidemic strains isolated after 2011.The virulence test in piglets demonstrated that all 5 inoculated piglets exhibited typical pseudor-abies symptoms,with 3 deaths recorded.These findings confirmed that the isolated virus is a pseudorabies virus strain with strong virulence,belonging to the epidemic clade.It was therefore designated as pseudora-bies virus strain WH-13.