Antigenic Variation Of New Epidemic Isolate Of Porcine Pseudorabies Virus And Humoral Immunity Of Killed Vaccine In Mice

Pseudorabies virus (PRV), also called Aujeszky disease virus or suid herpesvirus type 1, is a double-stranded linear DNA virus belonging to the Alphaherpesvirinae subfamily within the family Herpesviridae. PRV can infect a broad range of vertebrates, which including cattle, sheep, dogs, cats, goats, chickens, raccoons and wild animals. Among them, pigs have been confirmed to be the primary hosts and reservoir of this virus. PRV infection is characterized by nervous system disorders and death in newborn piglets and reproductive failure in sows. In recently years, PRV suddenly outbreaks in pigs in many farms in China, and it cause devastating disease in pigs and economic losses worldwide.At the end of 2011, a disease that is clinically similar to pseudorabies occurred in pigs in a large pig farm in a herd of Zhejiang province, which have been vaccinated with gE-deleted vaccines. We isolated a pseudorabies virus from the brain tissue of dead piglets. We perform this study to further understand the neutralization antibody characteristic of PRV. The killed vaccines of PRV were prepared and the immunogenicity was examined in mice. The contents of this paper are including:1. Neutralizing antigenic analysis of a new epidemic isolate of pseudorabies virus from swineIn order to know the antigenic characteristics of the new pandemic strain ZJ-01,30 sera of swine vaccinated with PRV vaccine (Bartha-K61、Bucharest and HB-98 strains) and 10 piglets serum against ZJ-01 isolate were collected for the cross-neutralization tests with ZJ-01 strain and traditional PRV LA strain. The results showed the serum from the 3 different vaccinated swine had significantly lower neutralizing effect on the PRV ZJ-01 strain, comparing with those on PRV LA strain. But the neutralizing effect of the anti-ZJ-01 serum on ZJ-01 was similar to that on LA strain. The antigenic relatedness R between ZJ-01 and LA strain was 0.255～0.378. Meanwhile, the anti-PRV vaccines sera were negative in gE antibody. It suggests that the new pandemic strain ZJ-01 changed greatly in antigen. It should be useful to control this disease efficiently and develop new vaccines in the future.2. Preparation of inactivated PRV vaccine and humoral immunity in miceIn this study, we used formaldehyde and hydrogen peroxide (H2O2) to inactive recombinant PRV ZJ-011G strain. The results showed that the viruses could be inactivated completely by a 0.1% concentration of formaldehyde for 12 h and 14h, or a 1% concentration of H2O2 for 4h and 5h. Then the killed viruses were mixed with water-based adjuvant CPH separately for preparing the four inactivated vaccines. ICR mice (n=35) aged 6～8 weeks were randomly assigned to 5 groups:Mice in groups 1-4 were injected subcutaneously with a 0.2-mL inoculums of the 4 inactivated vaccines, respectively. The fifth group was injected with 0.2 ml DMEM and used as a control. Booster vaccination was performed after an interval of 3 weeks with the same methods. The clinical symptoms were checked daily throughout the experiment. The PRV-specific antibodies in serum samples were detected by indirect ELISA at 3 and 6 weeks post inoculation, and neutralizing antibody were detected at 6 weeks post inoculation. The results showed that the ELISA antibody titers in the four vaccines groups were detectable three weeks post primary vaccination. After 6 weeks of immunization, the ELISA antibody peak up to 1:17000. The pigs in PRV vaccine group with 0.1% formaldehyde for 12h developed anti-PRV neutralizing antibody (1:8) after immunization, while no significant neutralizing antibody could be detected in the groups inactivated by H2O2. These data indicated that formaldehyde-based inactivation of a virus is better than other recently used approaches to virus inactivation such as H2O2. It should be useful for further development of PRV vaccines.