Isolation And Antiviral Activities Analysis Of Porcine Peproductive And Respiratory Syndrome Virus Nsp9- Specific Nanobodies

Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failure in sows and respiratory diseases in all ages of pigs. The causative agent, PRRS virus(PRRSV) is currently one of the most economically important viral pathogens threatening the swine industry worldwide. Current vaccines cannot provid complete protection to the vaccinated pigs, mainly due to the high antigenic heterogeneity and antibody-dependent enhancement, thus, it is necessary to develop novel anti-PRRSV strategies to control this important viral disease. PRRSV nonstructural protein 9(Nsp9) possesses RNA-dependent RNA polymerase(Rd Rp) activity, which is essential for virus life cycle, and its sequence is relatively conserved, making it a logical antiviral target for control PRRSV infection. Nanobodies possess many attractive features, such as high specificity and solubility, as well as stability, small size and can recognize structures not recognized by conventional antibodies. Recently nanobodies against many human viruses have been isolated and shown to exhibit potent antiviral activity and a good prospect in clinical application,however nanobodies against animal viruses have been rarely reported.The objective of this study was to generate Nsp9-specific nanobodies via phage display and analyze the antiviral activities of the nanobodies, which may pave the way for developing novel antiviral reagents for controlling PRRSV infection. The main works and results of this study were as follows:1. Full length Nsp9 gene sequence was cloned into p ET-28 a vector to generate the p ET-28a-Nsp9 plasmid. Soluble Nsp9 recombinant protein was expressed in Transetta(DE3) cells transformed with p ET-28a-Nsp9 plasmid after induced with IPTG. The Nsp9 protein was purified to near homogeneity using a Ni-NTA affinity chromatography column and subsequently a gel chromatography column. BALB/C mice were immunized with Nsp9 recombinant protein to test the immunogenicity of Nsp9. The murine antisera titers reached 1: 1,000,000, which suggests that the Nsp9 recombinant protein has good immunogenicity.2. Bactrian camel was immunized with the purified Nsp9 recombinant protein. The total Ig G titer of the serum raised against Nsp9 recombinant protein reached 1: 128,000 after 6 times of immunization. A phage display VHH library, consisting of approximately 4×108 individual colonies, was constructed from B lymphocyte c DNA encoding VHHs. Eight Nsp9-specific nanobodies were isolated via phage display, in which Nb6 and Nb7 showed the highest binding activity to Nsp9.3. Co-immunoprecipitation experiments were carried out on lysates of cotransfected HEK293 T cells, the result showed that Nb6 and Nb7 interacted with Nsp9 specifically when they were expressed in cell cytoplasm. MARC-145 cell lines stably expressing nanobodies were established, cell proliferation data showed that intracellularly expressed nanobodies have no cytotoxicity or negative effect on the MARC-145 cells.4. The cell lines were infected with PRRSV, and the antiviral activities of the nanobodies were analyzed. The results showed that intracellularly expressed Nb6 suppressed the replication of multiple strains of PRRSV(SD16、JX-A1、GD-HD、VR-2332). More importantly, Nb6 could protect MARC-145 cells from PRRSV-induced cytopathic effect(CPE) and fully block virus replication at an MOI of 0.01 or lower. Further studies indicated that intracellularly expressed Nb6 could interact with PRRSV-encoded Nsp9 and inhibit virus genome replication and transcription.In summary, PRRSV Nsp9-specific nanobodies were isolated from a camel VHH library, which may be useful tools for further study of Nsp9. Nb6 was shown to significantly suppress the replication of multiple strains of PRRSV in MARC-145 cells, suggesting that Nb6 has the potential to be used for developing novel antiviral drugs for controlling PRRSV infection.