Isolation And Identification Of Clinical Strains Of PRRSV And The Effect Of Tachyplesin On PRRSV Infection

Porcine reproductive and respiratory syndrome(PRRS),also called blue-ear disease,is a kind of infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is mainly characterized by reproductive disorder and respiratory tract infection,and bring huge economic loss to pig industry.PRRSV is a highly mutable RNA virus and has higher genetic diversity.In clinical practice,PRRSV infection often causes to secondary infection in.PRRSV has been identified for more than 30 years,but there is no effective prevention and control measures for PRRS so far,restricting the economic benefits of breeding industry.Tachyplesin is a 2.3 kDa cationic antimicrobial peptide isolated from Tachypleus tridentatus Leach.It is composed of 17 amino acids,and it is an important part of the natural defense system.Tachyplesin has anti-bacterial,antifungal,and antiviral effects.Tachyplesin was found to have an inhibitory effect on stomatitis virus,influenza A(H1N1)virus,reovirus,poliovirus and neuronecrosis virus.1.Isolation and identification of PRRSV clinical:Seven clinical samples from swines with suspected PRRSV infection was detected with ORF7-specific primers by RT-PCR.One of the positive samples was inoculated into Marc-145 cells and PAMs.The isolated strains were named SD-LY.ORF5 gene of SD-LY was amplified by RT-PCR.Sequencingand genetic evolution analysis of ORF5 gene of SD-LY was performed.The results showed that the ORF5 gene sequence of SD-LY is highly homologous to BJ-4 strain,and the homology is 96.0%.According to the phylogenetic tree analysis of ORF5 gene sequence of SD-LY,it belongs to Lineage 5.2.Measurement of cytotoxicity of tachypleusin:CCK-8 kit was used to evaluate the cytotoxicity of Tachyplesin to CRL2843^CD163 cells and PAMs.The results showed that there was no cytotoxicity at the concentration of 0?75 ng/mL,and cytotoxicity would occur when the concentration was 100 ng/mL.3.Effect of Tachyplesin on PRRSV replication:CRL2843^CD163 cells and PAMs were inoculated with GD-HD,and incubated with Tachyplesin.The results showed that compared with the control group,the expression level of PRRSV N protein and the titer of progeny virus were not significantly changed when the concentration of Tachyplesin was 25 ng/mL,and the expression level of PRRSV N protein and the titer of progeny virus were significantly decreased when the concentration of Tachyplesin was 50 ng/mL and 75 ng/mL,in a concentration dependent manner.4.Effect of Tachyplesin on PRRSV infection activity:After co-incubation of GD-HD with Tachyplesin,and the mixture was co-incubated with CRL2843^CD163 cells or PAM.The results showed that the expression level of PRRSV N protein and the titer of the progeny virus in the cell supernatant were significantly decreased when the concentration of Tachyplesin was 25 ng/mL,50 ng/mL and 75 ng/mL,in a concentration-dependent manner.5.Effect of Tachyplesin on PRRSV susceptibility:After co-incubation with CRL2843^CD163 cells or PAM,the PRRSV GD-HD was inoculated.The results showed that the expression level of PRRSV N protein and the titer of the progeny virus in the cell supernatant were not significantly changed when Tachyplesin was 25 ng/mL,50 ng/mL and75 ng/mL.6.Effect of Tachyplesin on replication of clinical isolates of PRRSV:CRL2843^CD163cells were inoculated with SD-LY of PRRSV and incubated with different concentrations of Tachyplesin.The results showed that the expression level of PRRSV N protein and the progeny virus titer were significantly decreased when the concentration of Tachyplesin was25 ng/mL,50 ng/mL and 75 ng/mL,in a concentration dependent manner.In summary,a clinical strain of PRRSV was isolated and identified in this study and named as SD-LY.According to genetic evolution analysis of ORF5 gene sequence,SD-LY is belongs to the lineage 5 lineage.Tachyplesin inhibited the replication of PRRSV in CRL2843^CD163 cells or PAMs in a concentration-dependent manner,mainly by inhibiting the infection activity of the virus,which laid a theoretical and experimental foundation for the research and development of Tachyplesin as an anti-PRRSV treatment drug.