【作者】 王建新；

【导师】 李彬； 刘涛；

【作者基本信息】 河北农业大学 ， 兽医硕士（专业学位）， 2024， 硕士

【摘要】 猪轮状病毒病是由猪轮状病毒(Porcine rotavirus,Po RV)引起的以急性腹泻为主的急性肠道传染病。以厌食、呕吐、腹泻和排糊状或水样粪便等症状为主要特征,潜伏期一般为16-24 h,且能使宿主产生与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)及猪δ冠状病毒(PDCo V)等病原类似的临床症状,有一定的致死率,对我国畜牧业造成了巨大的经济损失。目前暂无特效治疗Po RV感染的药物,市售商品化疫苗仅有1种,猪传染性胃肠炎、猪流行性腹泻、猪轮状病毒(G5型)三联活疫苗,用于预防Po RV G5型的感染。当前,Po RV基因型较多,伴随临床G9型的流行,G5型疫苗临床免疫保护效果不佳。因此,了解G9型猪轮状病毒的体外复制特性,探究致病性以及研发一款行之有效的疫苗是非常必要的。本研究将临床采集的仔猪腹泻样品,利用RT-PCR(反转录-聚合酶链式反应)进行PEDV、TGEV、Po RV和PDCo V检测,只有Po RV呈阳性,首先在易感细胞MA 104分离培养,并对分离毒株进行间接免疫荧光、PCR鉴定、电镜观察、RNA-PAGE、全基因组测序等试验进行鉴定;然后通过建立分离毒株对不同日龄仔猪的攻毒模型,分析分离毒株的致病性;最后以分离毒株制备灭活疫苗,并评价疫苗的安全性及有效性。结果显示,仔猪腹泻样品经终浓度为10μg/m L的胰酶在37℃孵育1 h能够在MA104细胞中稳定增殖传代,第3代开始出现稳定的细胞病变,间接免疫荧光试验表明该病毒可被猪轮状病毒保守的群抗原VP6蛋白的特异性单抗识别;RT-PCR检测可见与预期大小一致的目的条带。电镜观察发现,病毒粒子直径约70 nm。RNA-PAGE(RNA的聚丙烯酰胺电泳)鉴定其电泳带型为4∶2∶3∶2;全基因组测序分析确定了完整基因型为G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1,命名为AHFY 2022。通过建立的仔猪攻毒模型发现,毒株可以感染0、5和27日龄的仔猪,并引起不同程度的腹泻,测定其肛拭子的病毒排出情况也可以发现,所有日龄的仔猪在感染后均出现了不同程度的排毒,剖检可见,仔猪的肠壁有不同程度的变薄,肠绒毛脱落变短的情况,且在空肠和回肠中定殖;灭活疫苗的研制结果显示,以Po RV AHFY 2022(G9型)为靶病毒检测母猪、5日龄仔猪血清的中和抗体滴度(NT),母猪一免后NT略有升高,加强免疫后NT升高明显;初乳和新生5日龄仔猪血清NT基本一致。与之相比,商品化疫苗诱导的抗G9型的中和抗体滴度较低。5日龄仔猪的攻毒保护试验,灭活疫苗可以100%保护同型病毒的感染,仔猪无腹泻、粪便肛拭子排毒少、肠道组织带毒量低,相比对照组减少2～3个数量级;而商品化疫苗(G5)无法提供有效保护,全部感染发生腹泻,高载量排毒和带毒,与未免疫对照组基本无差异。综上,本研究成功分离获得一株G9P[23]基因型的猪轮状病毒,其中VP7、VP1、VP3、NSP3、NSP4基因与人源轮状病毒同源性高,介于65.84%～99.06%,表明可能发生了一定程度的基因种间重配;并能引起0、5和27日龄的仔猪病变;制备的灭活疫苗可以诱导同型病毒高抗体水平,且100%保护同型病毒的感染。更多还原

【Abstract】 Porcine rotavirus disease is an acute intestinal infectious disease mainly caused by porcine rotavirus(Porcine rotavirus,Po RV),featuring acute diarrhea.It is mainly characterized by symptoms such as anorexia,vomiting,diarrhea,and the discharge of pasty or watery feces.The incubation period is generally 16-24 hours and can cause hosts to present similar clinical symptoms to those caused by pathogens such as porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),and porcineδcoronavirus(PDCo V),with a certain fatality rate,causing significant economic losses to China’s animal husbandry.Currently,there are no specific drugs for treating Po RV infections,and there is only one commercialized vaccine available on the market,namely the trivalent live vaccine for porcine transmissible gastroenteritis,porcine epidemic diarrhea,and porcine rotavirus(G5 type),which is used to prevent infections of Po RV G5type.At present,there are many genotypes of Po RV.With the prevalence of the clinical G9type,the clinical immune protection effect of the G5 type vaccine is not satisfactory.Therefore,it is extremely necessary to understand the in vitro replication characteristics of the G9 type porcine rotavirus,explore its pathogenicity,and develop an effective vaccine.In this study,piglet diarrhea samples collected clinically were tested for PEDV,TGEV,Po RV and PDCo V using RT-PCR(reverse transcription-polymerase chain reaction).Only Po RV was positive.First,in susceptible cells MA 104 was isolated and cultured,and the isolated strains were identified through indirect immunofluorescence,PCR identification,electron microscopy,RNA-PAGE,whole-genome sequencing and other tests;then,the isolation was analyzed by establishing a challenge model of the isolated strains on piglets of different ages.The pathogenicity of the virus strain;finally,an inactivated vaccine is prepared from the isolated virus strain,and the safety and effectiveness of the vaccine are evaluated.The results showed that piglet diarrhea samples incubated with trypsin at a final concentration of 10μg/m L for 1 hour at 37℃were able to stably proliferate and passage in MA 104 cells,and stable cytopathic changes began to appear in the third passage.Indirect immunofluorescence tests showed that the virus it can be recognized by the specific monoclonal antibody of the conserved group antigen VP6 protein of porcine rotavirus;RT-PCR detection showed a target band consistent with the expected size.Electron microscopic observation found that the diameter of the virus particles was approximately70 nm.RNA-PAGE(polyacrylamide electrophoresis of RNA)identified the electrophoretic band pattern as 4∶2∶3∶2;whole-genome sequencing analysis determined that the complete genotype was G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1,named AHFY2022.Through the established piglet challenge model,it was found that the virus strain can infect piglets aged 0,5 and 27 days,and cause varying degrees of diarrhea.Measuring the virus shedding in their anal swabs also revealed that piglets of all ages were infected.Afterwards,various degrees of detoxification occurred.Necropsy showed that the intestinal wall of the piglets was thinned to varying degrees,the intestinal villi fell off and became shorter,and colonized in the jejunum and ileum.The results of the development of the inactivated vaccine showed that Po RV AHFY 2022(G9 type)is the target virus to detect the neutralizing antibody titer(NT)of the serum of sows and 5-day-old piglets.The NT of sows increased slightly after the first immunization,and the NT increased significantly after the booster immunization;colostrum and newborn The serum NT of5-day-old piglets was basically the same.In contrast,commercial vaccines induce lower neutralizing antibody titers against the G9 type.In the challenge protection test of5-day-old piglets,the inactivated vaccine can 100%protect against infection by the same type of virus.The piglets have no diarrhea,less fecal anal swabs toxins,and a low amount of virus in the intestinal tissue.Compared with the control group,there are 2 to 3 fewer viruses.orders of magnitude;while the commercial vaccine(G5)was unable to provide effective protection,all infections resulted in diarrhea,high-load virus shedding and virus transmission,and there was basically no difference from the unimmunized control group.In summary,this study successfully isolated a porcine rotavirus of the G9P[23]genotype,in which the VP7,VP1,VP3,NSP3,and NSP4 genes have high homology with human rotavirus,ranging from 65.84%-9 9.06%,indicating that a certain degree of interspecies reassortment may have occurred;and can cause pathological changes in piglets aged 0,5,and 27 days;the prepared inactivated vaccine can induce high antibodies to the same type of virus and is 100%protective against the same type of virus of infection.更多还原