PAMs免疫粘附受体CR 1-like对PRRSV感染的影响

Effects of porcine alveolar macrophages immune adhesive receptor CR 1-like on PRRSV infection

[目的]本试验旨在探讨猪肺泡巨噬细胞(Porcine alveolar macrophages,PAMs)表面天然免疫粘附受体—猪I型补体受体(Porcine complement receptor 1⁃like,CR1⁃like)对猪繁殖与呼吸综合征病毒(Porcine reproductive and respi⁃ratory syndrome virus,PRRSV)感染PAMs的影响,为阐述CR1⁃like在PRRSV免疫应答中的生物学功能提供理论数据。[方法]采取PRRSV减毒活疫苗免疫接种PRRSV抗体为阴性的长白仔猪20~28 d后,提取分离抗病毒血清并检测抗PRRSV血清中和效价;将抗PRRSV血清进行不同倍比稀释,制备各类PRRSV抗原抗体复合物并接种于PAMs上,建立抗体依赖性增强(Antibody⁃dependent enhancement,ADE)模型,qRT⁃PCR技术检测各时间点PRRSV N基因拷贝数变化;PRRSV感染PAMs 9 h后,将抗血清和猪新鲜血清共处理细胞,运用ELISA测定不同时间点细胞上清液中猪新鲜血清C3b、C4b、CH50总活性;将PAMs分为试验组(PRRSV感染PAMs 9 h后同时加入抗血清和猪新鲜血清)和6个对照组,采用qRT⁃PCR技术检测各组不同时间点胞内和上清中PRRSV N基因的拷贝数,确定猪新鲜血清对PRRSV感染PAMs的作用;采用免疫阻断技术将PAMs分为FcR McAb阻断组、CR1⁃like McAb阻断组、共阻断组及3个对照组,qRT⁃PCR技术检测PRRSV N基因拷贝数变化,确定免疫粘附受体CR1⁃like对PRRSV感染PAMs的影响。[结果]抗PRRSV血清在PAMs上的中和抗体效价为1∶5.37;qRT⁃PCR结果成功筛选出1:20稀释度的抗PRRSV血清对病毒的复制具有促进作用;ELISA检测猪新鲜血清C3b、C4b和CH50浓度随PRRSV感染时间延长呈降低趋势;qRT⁃PCR结果显示,猪新鲜血清可以与1∶20稀释度抗PRRSV血清协同促进PRRSV的复制;qRT⁃PCR、免疫阻断技术得出1∶20稀释度抗PRRSV血清、猪新鲜血清协同促进PRRSV感染增强不只依赖于FcR,还与CR1⁃like有关。[结论]体外条件下,PAMs表面的CR1⁃like是介导PRRSV感染PAMs过程中产生ADE效应的分子之一,可与FcR协同促进PRRSV感染PAMs。

[Objective]The purpose of this study was to investigate the effect of porcine complement receptor 1-like(CR1-like),a natural immune adhesion receptor on the surface of porcine alveolar macrophages(PAMs),on the infection of PMAs by por⁃cine reproductive and respiratory syndrome virus(PRRSV).The study provided theoretical data to elucidate the biological function of CR1-like in the immune response of PRRSV.[Methods]PRRSV attenuated live vaccine was used to immunize PRRSV antibody-negative Landrace piglets aged 20~28 d.Antiviral serum was extracted and isolated,and the titer of anti-PRRSV antibody was detected.Different dilution of anti-PRRSV serum were used to prepare PRRSV antigen-antibody com⁃plexes and inoculated into PAMs to establish an antibody-dependent enhancement(ADE)model.qRT-PCR was used to detect changes in PRRSV N gene copy numbers at various time point.Nine hours after PRRSV infection of PAMs,the cells were cotreated with antiserum and fresh porcine serum.ELSA was used to measure the total activity of porcine fresh serum C3,C4b,and CH50 in the cell supernatant at different time points.PAMs were divided into the experimental group(PRRSV infection of PAMs followed by simultaneous addition of antiserum and porcine fresh serum)and six control groups.qRT-PCR was used to detect the copy numbers of PRRSV N gene at different time points in cells and supernatant of each group to determine the ef⁃fect of fresh porcine serum on PRRSV infection of PAMs.Immune blocking technology was used to divide PAMs into FcR McAb blocking group,CR1-like McAb blocking group,combined blocking group,and three control groups.qRT-PCR was used to detect changes in PRRSV N gene copy numbers to determine the effect of immune adhesion receptor CR1-like on PRRSV infection of PAMs.[Results]The neutralizing antibody potency of anti-PRRSV serum on PAMs was 1∶5.37.qRTPCR results successfully screened out 1∶20 dilution of anti-PRRSV serum that promoted virus replication.ELISA detected that the concentrations of porcine fresh serum C3b,C4b,and CH50 decreased with the prolongation of PRRSV infection time.qRT-PCR results showed that fresh porcine serum could synergistically promote PRRSV replication with 1∶20 dilution of anti-PRRSV serum.qRT-PCR and immune blocking technology revealed that the synergistic enhancement of PRRSV infec⁃tion by 1∶20 dilution of anti-PRRSV serum and fresh porcine serum not only depended on FcR but also on CR1-like.[Conclu⁃sion]Under in vitro conditions,CR1-like on the surface of PAMs is one of the molecules that mediates the ADE effect during PRRSV infection of PAMs and can synergistically promote PRRSV infection of PAMs with FcR.