Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive disorder in breeding pigs and respiratory symptoms in pigs of all ages. The virus continues to bring significant economic losses to swine industry, and is considered to be one of the most important swine pathogens. Vaccination has been utilized to aid or facilitate PRRS control. Among various types of vaccines used, attenuated live virus vaccines appear to be more efficacious than killed virus vaccines. However, the effect of vaccination with attenuated live virus vaccines is crippled by several safety and efficacy issues. First, the vaccine development process is time-consuming. Second, the vaccine has the potential to revert to virulent. Third, the vaccine tends to provide full protection only to homologous strains. Three independent studies were conducted to address these issues and provide a concept for future vaccine development.
The first study was based on a previous in vitro work in our laboratory which demonstrated that chimeric viruses containing mixed structural genes from two genetically and antigenically distinct strains (VR2332 and JA142) of PRRSV in an organized manner were susceptible to antisera generated against both of the donor strains. In the present study, three chimeric viruses (JAP5, JAP56 and JAP2-6) were constructed by replacing ORF5, ORFs 5 and 6, and ORFs 2-6 of VR2332 with the corresponding gene(s) of JA142 respectively and were evaluated in vivo for their ability to confer pigs the cross protection against the VR2332 and JA142 strains. A total of 114 pigs were divided into 6 groups and each group was intramuscularly inoculated with one of the three chimeric viruses (n=16 per group), VR2332 (n=24), JA142 (n=24), or sham inoculums (n=18). At 44 days post inoculation (dpi), these pigs were divided further into 15 groups (n= 6 or 8 pigs per group) and challenged intranasally with VR2332, JA142, or sham inoculum. Although no pigs demonstrated severe clinical signs or lesions after the 2nd inoculation, all pigs inoculated with chimeric viruses prior to challenge had significantly (p
The second study utilized the same concept of the first study but was expanded to assess if use of chimeric viruses can be universally applicable to obtaining broader cross protection among PRRS viruses. To evaluate the strategy of chimeric virus, this study selected four distinct wild-type 1648-02, 17198-6, MN184 and SDSU73 strains of PRRSV whose nucleotide identities range from 86.7% to 92.2% and generated 12 chimeric viruses by mixing their ORFs 3-4 or ORFs 5-6. Their susceptibilities to the neutralizing activity of hyperimmune serum generated against each one of the four wild-type strains were assessed in vitro by fluorescent foci neutralization (FFN) assay. No or minimum level of cross neutralization existed among the four wild-type strains, and not all chimeric viruses obtained broader cross neutralization. Broader cross neutralization could be obtained when chimeric viruses were constructed in an organized manner depending on the immunobiological importance of ORFs 3-4 and ORFs 5-6 products of each strain. The neutralization of MN184 and SDSU73 were mainly mediated by ORFs 5-6 products, while ORFs 3-4 products played very limited roles in virus neutralization. Products from ORFs 5-6 and ORFs 3-4 of 1648-02 were all implicated in virus neutralization, but ORFs 5-6 products were more important (p
The third study focused on assessing whether non-structural proteins play a role in the cross neutralization between viruses. In this study, eight different patterns of structural gene mixing between JA142 and VR2332 were constructed into three cDNA infectious clone backbones. Chimeric viruses were then tested for their susceptibilities to JA142 and VR2332 antisera. No matter which backbone was used to construct the chimeric viruses, chimeric viruses carrying the same structural genes showed almost the same degree of susceptibility to the JA142 and VR2332 antisera, suggesting non-structural gene of the backbone has limited impact on the specificity of viral immunity. On the other hand, chimeric viruses with different gene mixing patterns displayed significant diversity in their susceptibilities to the JA142 and VR2332 antisera. Cross neutralization testing demonstrated that determinants for VR2332 neutralization were located in ORF3 or ORF4 whereas determinants for JA142 neutralization were located in ORF6. ORFs 2 and 7 products of JA142 displayed conflicting roles in virus neutralization. Their impacts remain to be further studied.
In conclusion, chimeric virus strategy may be a way to induce broader cross-neutralizing antibody leading to better cross-protective humoral immunity against heterologous PRRSVs in pigs. As the nonstructural genes of backbone did not affect the specificity and sensitivity of virus neutralization, chimeric viruses containing avirulent non-structural genes of the backbone (i.e., infectious clone) and well-organized structural genes of PRRSV may be used to obtain the improved efficacy and safety of vaccination when a live virus based vaccine is used.
Sun, Dong, "Utilities of reverse genetics system as a platform for the development of Porcine Reproductive and Respiratory Syndrome (PRRS) virus vaccine" (2013). Graduate Theses and Dissertations. 13619.