天津地区猪伪狂犬病毒变异株的分离鉴定及主要相关毒力基因分析

Isolation,identification and analysis of major virulence-related genes of a pseudorabies virus variant strain in Tianjin Region

为了鉴定猪伪狂犬病病毒(PRV)流行株及其携带毒力基因的变异情况,本研究采集1份天津地区某猪场免疫过Bartha-K61疫苗但患严重神经症状的仔猪脑组织样品,采用荧光定量PCR(q PCR)检测PRV g E基因,并将检测为PRV的阳性样品接种PK-15细胞分离培养,并传3代后采用Reed-Muench法测定分离病毒的TCID_(50),采用q PCR和间接免疫荧光试验(IFA)进一步鉴定分离病毒。结果显示,经q PCR检测仔猪脑组织样品出现扩增曲线,阳性样品分离培养后测得病毒滴度为10^(7.57)TCID_(50)/m L,分离病毒的q PCR结果出现扩增曲线,且其感染的细胞出现绿色荧光。表明分离到一株PRV,命名为TJbd2023株。采用PCR分别扩增TJbd2023株主要相关毒力基因g B、g C、g D、g E、g G、g I和TK,采用Meg Align软件分析分离病毒与Gen Bank登录的PRV参考株上述各毒力相关基因序列的同源性,通过Neighbor-Joining(NJ)法构建上述各毒力相关基因的进化树,采用Meg Align软件分析各毒力基因编码氨基酸主要位点的变异情况。结果显示,分别扩增到TJbd2023株的各相应毒力基因;各毒力基因的同源性分析结果显示,TJbd2023株与国内2012年后流行变异株的相似性高达98.4%~100.0%,与疫苗株(Bartha-K61)的相似性为89.6%~93.2%。进化树结果显示,这7个毒力基因均与国内GD0304株(MH582511)、BJYT株(KC981239)和DL1408株(KU360259)等PRV变异株位于同一分支,与疫苗株(Bartha-K61)未在同一分支。g B、g C、g E氨基酸序列除出现PRV变异株相同的突变位点外,g B还出现R^(223)H和E^(836)K的突变、g D出现R^(320)S和g E出现T^(242)A的突变。将TJbd2023株分别以5个剂量(10^(2.57)TCID_(50)/m L~10^(6.57)TCID_(50)/m L)感染6周龄KM小鼠,通过小鼠的临床症状、死亡率、死亡小鼠各组织的病理变化评估该病毒的致病性。结果显示,在5 d的观察期内,感染组小鼠陆续出现奇痒及神经症状,各实验组小鼠的死亡率分别为60%、80%、100%及100%,对照组小鼠均健活。死亡小鼠各组织病变观察可见肺组织浸润性出血、肝组织血管充血及淋巴细胞增多、脑胶质细胞增多及血管周围炎性细胞聚集形成血管套等病变,对照组小鼠各组织均无明显病变。上述结果表明,本研究在天津地区分离到一株PRV变异株,其毒力因子发生了独特的氨基酸位点突变,对小鼠的致病性较强,该结果丰富了PRV的致病性及其毒力因子变异的特征,为PR的综合防控提供参考。

To identify the circulating strains of pseudorabies virus (PRV) and analyze variations in their virulence genes,a brain tissue sample was collected from severely symptomatic piglets that had been immunized with the Bartha-K61 on a pig farm in the Tianjin area.The PRV g E gene was detected using a fluorescence quantitative PCR (q PCR).Positive samples were further isolated and cultured in PK-15 cells and then passaged three times,followed by virus purification using the limited dilution method.The TCID_(50)of the isolated virus was determined using the Reed-Muench method,and the isolated virus was further characterized by q PCR and indirect immunofluorescence assay (IFA).The q PCR detection of the piglet's brain tissue sample revealed an amplification curve,with a viral titer measured at 10^(7.57)TCID_(50)/m L.The q PCR result for the isolated virus also exhibited an amplification curve,and the infected cells showed green fluorescence,confirming the isolation of a strain named TJbd2023.PCR was employed to amplify the major virulence-related genes g B,g C,g D,g E,g G,g I,and TK from the TJbd2023 strain.The homology of the isolated virus with reference strains of PRV in Gen Bank was analyzed using Meg Align software for these virulence-related gene sequences.A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method,and the key amino acid variation in each virulence gene were analyzed using Meg Align software.The results showed that the corresponding virulence genes were successfully amplified from the TJbd2023 strain.Homology analysis indicated that the TJbd2023 strain shared a similarity of 98.4% to 100.0% with domestic strains that have circulated and mutated since 2012,and a similarity of 89.6%to 93.2%with the vaccine strain (Bartha-K61).The phylogenetic tree indicated that all seven virulence genes were all located in the same branch as PRV,GD0304,BJYT,and DL1408,and not with the vaccine strain (Bartha-K61).In addition to shared mutations in g B,g C,and g E,g B also displayed mutations R^(223)H and E^(836)K,g D showed mutation R^(320)S,and g E showed mutation T^(242)A.The TJbd2023 strain was used to infect 6-week-old KM mice at doses ranging from 10^(2.57)to 10^(6.57)TCID_(50)/m L.The pathogenicity of the virus was assessed based on the clinical symptoms,mortality rates,and pathological changes in the tissues of deceased mice.Over the 5-day observation period,the infected mice showed progressive itching and neurological symptoms,with mortality rates of 60%,80%,100%,and 100% in experimental group mice,and the control group mice were all survial.Pathological examination of tissues from deceased mice revealed severe multi-organ lesions,including infiltrative hemorrhaging in lung tissues,vascular congestion and lymphocyte proliferation in liver tissues,increased astrocytes in brains,and inflammatory cells surrounding blood vessels forming vascular cuffs;no lesions were observed in the control group mice.These findings indicate that a variant strain of PRV was isolated in Tianjin,exhibiting unique point mutations in virulence factors and strong pathogenicity in mice.This study enriches our understanding of PRV pathogenicity and variations in its virulence factors,providing insights for comprehensive prevention and control of PR.