| Porcine reproductive and respiratory syndrome virus(PRRSV) is the etiological agent of PRRS, characterized by reproductive failure in sows and respiratory disease in pigs of all ages. In 2006, a highly pathogenic PRRS(HP-PRRS) emerged in China, which caused enormous economic losses to the pig industry and spread in the field till now. In 2015, a disease with characteristics of PRRS emerged on one pig farm in Gansu province, and the lung tissues were collected from sick pigs to isolate and identify the virus. The virus was isolated by serial passage on Marc-145 cells and then detected by RT-PCR and N protein IFA, which confirmed that the causative agent was PRRSV. The complete genome sequence of GSWW strain was aligned with PRRSV reference strains after sequencing. The phylogenetic analysis showed that GSWW strain belonged to subgroup Ⅲ with a discontinuous deletion of 30 aa in nsp2 coding sequences. Amino acid mutation resulted in a new N-linked glycosylation site N34 and one amino acid mutation appeared in the primary neutralizing epitope of GP5. So, GSWW strain was a currently prevalent HP-PRRSV strain in China.In order to investigate the pathogenicity and immunologic characteristics of GSWW strain, 24 PRRSV-free piglets were obtained, among which 17 piglets were challenged with 108 copies of GSWW virus and 7 piglets were contact-infected by raising in the same room. Piglets started to show clinical signs on day five, like fever, lethargy and emaciation. Some piglets showed blue ears, anhelation and finally died. 10 piglets died during two months after infection, which showed high level of viremia and developed enlarged tonsil, swelling and diffuse hemorrhage in lung and lymph nodes. Fluorescence quantitive PCR was applied to detect virus titer in the blood, and piglets showed viremia about 4 days after infection. The viremia could last over a month and virus titer in the blood reached 108copies/mL. N protein antibody was detected 7 days after infection by an indirect ELISA. But γ-interferon was not detected in serum by ELISA. All these results showed that GSWW strain was a virulent PRRSV strain with high morbidity and mortality.Due to the rapid variation of PRRSV and low levels of protective immune responses, there are no ideal vaccines available for PRRSV with high safety and efficiency. It has been the highlight to improve the protective immune responses of vaccines. There were non-essential regions for replication in the genome of PRRSV, which could tolerate foreign gene insertion. So PRRSV could serve as a vaccine vector to express foreign genes. Using an infectious clone of PRRSV Fl12 strain, a separate transcriptional unit was inserted between ORF1 b and ORF2 a to express EGFP. The recombinant plasmid was linearized by AclⅠdownstream of Poly(A) tail, and then transcripted to viral RNA by in vitro transcription. The viral RNA was electroporated into BHK-21 cells to produce infectious virus, which then was passaged on Marc-145 cells. Marc-145 cells showed obvious CPE and green fluorescence, indicating that rescued virus vFl12-EGFP expressed EGFP correctly, but the recombinant virus lost EGFP gene by the third passage. However, the EGFP gene was stably expressed for at least 7 passages on PAM cells. Recombinant PRRSV expressing FMDV B7 multi-epitope gene was constructed and rescued by the same strategy. Marc-145 cells infected with vFl12-B7 showed typical CPE of PRRSV infection. RT-PCR and sequence alignment showed that the B7 multi-epitope gene was inserted in viral genome correctly. Recombinant PRRSV was passaged on PAM cells and the FMDV B7 multi-epitope gene remained stable for at least five passages. This work established the reverse genetic operation platform for PRRSV, laying a good foundation for the development of recombinant PRRSV vector vaccines expressing foreign genes. |
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