| Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV,a member of the Arteriviridae family,has a positive-sense,single-stranded RNA genome of approximately 15 kb encoding at least 10 functional ORFs.There are two major genotypes of PRRSV,type1(EU-type),and type2(NA-Type,).Now different variants(JXA1?HuN4)of highly pathogenic PRRSV(HP-PRRSV)considered to be evolved from lineage 8 strains is predominant in China.Variants of Lineage1(NADC30-like)has been prevalent since 2013 and leads to PRRS pandemic again in China.Lineage1 and Lineage8 are both type 2 strains.Porcine circovirus 2(PCV2)is a single-stranded DNA virus in the family Circoviridae which contributes to a group of disease syndromes collectively termed porcine circovirus associated disease or PCVAD.PCV2 infection alone rarely results in clinical signs and co-infections involving with PRRSV are common in growing pigs contributing to a range of polymicrobial disease syndromes.PCV3 is a novel disovered single-stranded DNA virus and is also in the family Circoviridae.It was first reported in the United States in 2016 and subsequently detected in several other countries.PCV3 is considered to be associated with PDNS and is reported to have mixed infection with PCV2 clinically.The co-infeciton of PRRSV and PCV2 increased the difficulty of prevention and control of PRRSV.The newly discovered PCV3,if co-infected with the two viruses,will undoubtedly make it more difficult to control PRRSV.PRRSV is one of the most rapidly mutating RNA viruses leading to the occurrence of various kinds of new strains.Mixed infection of PRRSV with other viruses including porcine circoviruses are also common.The existing means of PRRSV prevention and control are far from enough to meet the demand.Exploration of other ways to control PRRSV is of great significance.Object:This study is aimed at investigating the situation of PRRSV infection,genetic evolution and co-infection with PCV2 and PCV3 in pig farms of nanning,qinzhou,fangchenggang,liuzhou and yulin in guangxi province.And,we also want to learn in this study how the swine interferon induced transmembrane protein(IFITMs)response to the PRRSV infection and its role on PRRSV replication.The research we made is to provide some helpful evidence for the local PRRS prevention and control on theoretical and technical level.Contents:1.The study was to detect the PRRSV infection rate of samples collected from Nanning,Qinzhou,Fangchenggang,Liuzhou and Yulin in Guangxi province.four positive samples were selected and the whole genome was amplified and sequcenced.The genetic evolution status of local PRRSV strains were analyzed by phylogenetic trees construction and amino acids alignment.The pathogenicity of the PRRSV GXBB16-1 was also detected.2.A real-time quantitative PCR assay was established,evaluated and was utilized to detect the PCV3 infection rate in samples from Nanning,Qinzhou,Fangchenggang,Liuzhou and Yulin.The results were compared with that by traditional RT-PCR detection.The local co-infection status of PRRSV,PCV2 and PCV3 as well as the genetic evolution characteristics of PCV3 were also investigated.3.Primary porcine alveolar macrophage(PAM)and peripheral blood lymph cells(PBMC)cells were isolated and infected by PRRSV GXBB16-1 to explore the response of pig IFITMs gene to PRRSV infection in vitro.Marc145-IFITMs stable cell lines were generated and were inoculated with GXBB16-1 to study the influence of IFITM on the replication of PRRSV.Methods:1.RT-PCR was used to detect PRRSV infection rate in samples in guangxi province.Four samples were selected for whole genome amplification and sequencing.The genetic evolution and amino acid variation characteristics of PRRSV were studied by phylogenetic tree analysis and amino acid sequence comparison.GXBB16-1 was isolated and inoculated into PRRSV negative piglets after identification.The pathogenicity of GXBB16-1 was determined by observing the clinical symptoms,temperature and lung pathological changes of the piglets after inoculation.2.A SYBR green-based qPCR assay to detect PCV3 was established and the sensitivity,specificity and repeatability were evaluated.The PCV3 infection status of 7 big pig farms and 3 big pig slaughter houses in 5 cities metioned above were detected.Three positive samples were selected for the whole genome amplification and sequencing.Construction of phylogenetic trees and amino acids alignment were made based on the whole genome to analyse the genetic characteristics of PCV3 in guangxi.3.Primary porcine alveolar macrophage(PAM)and peripheral blood lymph cells(PBMC)cells were isolated from 4 weeks old piglets that are PRRSV serological negative and then were infected by PRRSV GXBB16-1 isolated in this study.Total cell RNA was extracted at different time points after infection and the transcription levels of IFITMs were measured by Realtime qPCR and normalizaed to ?-actin.To evaluate the role of IFITMs in PRRSV infection,Marc145 stable cell lines expressing Flag-N-tagged IFITMs or vector were generated based on the the third generation lentivector expression system respectively.The stable expression cell lines were identified by RT-PCR and Western Blot.Based on these cell lines,the replication of the virus was analyzed by RT-qPCR,Western blot and IFA assay at different time points post-infection.To further confirm the role of IFITM3 in cellular antiviral response,siRNAs targeting to IFITM3 genes were designed,and siRNA silencing analysis was performed in Marc145-IFITM3 stable cell lines.Results:1.The positive rate of PRRSV was 37.28%(63/169)in the lung samples in Guangxi.Three of the 4 selected positive samples were PRRSV JXA1-like strains,and shared high amino acid homologous with PRRSV Hanvet1.vn and JXA1 related vaccine strains,well ORF5 of GXBB17-1 is more closely related to NADC30-like strains.The isolated GXBB16-1 could cause significant clinical symptoms and lung pathological damage in piglets.2.The PCV3 positive rate of samples from 7 large-scale pig farms and 3 large slaughter houses in 5 cities of guangxi province were 36.13%(56/155)and 32.26%(50/155)detected by qPCR and traditional RT-PCR repectively.The PCV3 strains in 3 positive samples were amplified for whole genome,sequenced and designed PCV3-China/GX2016-1 ? PCV3-China/GX2016-2 and PCV3-China/GX2016-3 respectively.Surprisingly,A single “G” deletion was found at 1155 nt in PCV3-China/GX2016-1.Genetic evolution analysis and amino acid sequence alignment revealed that the three PCV3 strains identified in this study were all belong to the 24A+27R cluster.3.PAM and PBMC cells was susceptible to the isolated GXBB16-1,and the transcription level of the IFITMs gene increased in the two cells significantly.The lentivirus containing the target gene was successfully packaged and was inoculated into Marc145 cells.The Marc145-IFITMs stable cell lines were selected in the cell medium with puromycin at the concentration of 4.5?g/ml.The replication of GXBB16-1 can be significantly inhibited by IFITM3 in transcriptional and translational levels detected by qPCR,western blot and IFA,and the restrict efforts can be reduced after knockdown of the IFITM3 expression in the Marc145-IFITM3 stable cell lines.Conlusion:In summry,this study revealed the PRRSV infection status in parts of guangxi province.The PRRSV strains identified in the studay are mainly PRRSV JXA1-like subtypes,but there are some amino acids mutations in part of the antigen epitope,which may affect the vaccine immune efficiency.In addition,GXBB16-1 might be revertant of the JXA1 related live vaccine strains that have regained virulence.The PRRSV,PCV2 and PCV3 mixed infection present in Guangxi makes it difficult to control PRRS.The research of interaction between IFITM and PRRSV showed that IFITM play roles in the immune response to PRRSV infection and IFITM3 can ristrict PRRSV replication at the level of transcription and protein expression,which provides a theoretical basis for PRRS control in guangxi province. |
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