Construction And Efficacy Of A Recombinant Pseudorabies Virus Expressing GP5 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious diseases in pigs, causing late-term reproductive failure in sows and severe pneumonia in neonatal pigs. The disease is caused by PRRS virus (PRRSV), a member of the Arteriviridae family. Current vaccines against PRRSV have some drawbacks. Killed vaccines have been proved to be less effective in prevention from both infection and disease. Modified live vaccines typically provide protection against clinical disease but not infection in some cases. Additionally, live PRRSV vaccines are likely to revert to virulence. Development of safe and efficacious vaccines against PRRS remains a big challenge. GP5 is the major envelope glycoprotein of the virus. Circulating antibodies in PRRSV-infected pigs responsible for virus neutralization (VN) are mainly directed against GP5. GP5 possesses a small putative ectodomain comprising approximately the first 40 residues of the mature protein. The ectodomain contains a variable number of N-glycosylation sites, and it has been proposed that linear neutralizing epitopes is located in this region. In addition, young pigs immunized with a DNA vaccine encoding GP5 gene developed PRRSV-specific neutralizing antibodies and protective immunity. The establishment of an effective mucosal immunity against glycosylated and conformation dependent epitopes is necessary to control virus persistence and shedding. In this study, the GP5 gene of PRRSV under the control of human cytomegalovirus (CMV) immediate early promoter and a LacZ reporter gene expression cassette regulated by the SV40 early promoter were inserted into the universal pseudorabies virus(PRV) transfer vector, pBdTK-Uni, giving rise to a new PRV transfer vector, named as pGP5-LacZ. Purified genomic PRV Bartha-K61 strain DNA and transfer vector pGP5-LacZ were co-transfected into Vero cells using Lipofect Reagent. A recombinant PRV harboring PRRSV GP5 gene, designated as rPRV-GP5, was obtained after ten cycles of blue plague purification and PCR identification. The expression kinetics and immunogenicity of the GP5 expressed by rPRV-GP5 were analyzed by single-step growth analysis, Western blot, and indirect immunofluorescence test. GP5 expression was first detected 15 hours post-infection in the cellular extracts of rPRV-GP5-infected Vero cells. The results showed that PRRSV GP5 could be expressed authentically and efficiently, GP5 is expressed as intracellular forms and not incorporated into rPRV-GP5 virions. Compared to its parental virus, rPRV-GP5 showed no obvious difference with respect to virus replication and cytopathogenic effects in several cell cultures, including Marc-145, IBRS2, Vero cells and chicken embryo fibroblasts. Sequence analysis of 30th passage of rPRV-GP5 indicated that GP5 gene remain stable in rPRV-GP5. To test the safety and efficacy of rPRV-GP5, ten 5-to 6-month-old, PRV free sheep were randomly divided into three groups. Sheep in Groups I (n= 4) and II (n=3) for safety test were immunized intramuscularly with 106.0 and 108.0 PFU of rPRV-GP5, respectively, and Group III (n=3) served as mock vaccinated control. Fourteen days post-inoculation, animals in Groups I and III were challenged intramuscularly with 103 LD50 of highly virulent PRV S strain of porcine origin. The gE gene of PRV S strain couldn't be detected from vaccinated sheep 7 and 15 days post challenge by PCR. The results showed that vaccinated sheep were completely protected from lethal PRV challenge and insertion of GP5 gene into PRV genome doesn't alter the replication and virulence of PRV Bartha-K61 strain. Thirty PRV-and PRRSV-negative healthy piglets were assigned to one of 6 groups (1 through 6, 5 pigs per group). Animals in Group 1 for safety test were each inoculated intranasally(i.n) 108.0 PFU of rPRV-GP5; Group 2 were each inoculated i.n with 107.0 PFU of rPRV-GP5;Group 3 were each inoculated i.n and intramuscularly interval 28 days with 107.0 PFU of rPRV-GP5; Group 4 were each inoculated i.n with 107.0 PFU of its parent Bartha-K61; Group 5 were each vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 6 was served as non-vaccinated control. At 63 days post-inoculation, all animals were all challenged with 2×105.0 TCID50 of virulent PRRSV CH1a. The piglets inoculated with rPRV-GP5 or its parental virus developed PRV-specific neutralizing antibodies, and there was no significant difference in the antibody titers between Groups 1 and 3. While Group 5 produced antibodies to PRRSV 35 days post-inoculation as indicated by indirect immunofluorescence test(IIF), enzyme-linked immunosorbent assay test(ELISA), and 7 days post-challenge neutralization antibodies were developed. Group 2 and 3, developed antibodies against PRRSV 3, 5, 14 days post-challenge, as revealed by indirect IIF, ELISA and virus neutralization test. The results suggest that rPRV-GP5 is capable of inducing anamnestic immune response to PRRS in inoculated animals. All animals in Groups 2,3,5 remained clinically healthy before and after challenge, with only a short period of fever (less than 39.5℃and 7 days), mild and gradually improving lung and kidney lesions, more pronounced reduction numbers of virus distribution in organs and short-term viremia (3 weeks). On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 40℃) after challenge, and severe lung, kidney and spleen lesions, the numbers of virus distribution in organs and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathological lesions induced by PRRSV challenge in vaccinated piglets. In summary, the rPRV-GP5 is a good candidate recombinant virus vaccine to control PRRS and pseudorabies.