一株鸡传染性贫血病毒的分离鉴定及致病性分析

Isolation,identification and pathogenicity analysis of a chicken infectious anemia virus

为鉴定来自山东某种鸡场196日龄疑似鸡传染性贫血病毒(CAV)感染的2份种鸡肝脏样品的致病原,本实验利用马立克病肿瘤淋巴母细胞(MDCC-MSB1细胞)对2份肝脏样品进行病毒的分离培养与传代,每代均观察细胞是否出现细胞病变(CPE)。将传至5代的病毒液分别采用PCR和间接免疫荧光试验(IFA)鉴定,PCR结果显示,扩增到与CAV JL17株阳性对照一致的约600 bp的目的条带;IFA结果显示,感染分离病毒与JL17株阳性对照的细胞均出现绿色荧光,而阴性对照既未扩增到目的条带也无绿色荧光。上述结果表明分离到一株CAV,命名为SDNN2022。为分析SDNN2022株的分子特征,采用PCR扩增其VP1基因并测序,采用DNAStar软件对SDNN2022株VP1基因与GenBank登录的39株CAV参考株VP1基因序列进行同源性分析;采用Megalign分析SDNN2022株VP1的氨基酸序列,构建SDNN2022株VP1基因的进化树。VP1基因的同源性结果显示,SDNN2022株与CAV参考株的VP1基因序列的同源性为95.0%~99.8%,其中与CAV参考株JS2020-PFV的同源性最高;SDNN2022株VP1高变区的氨基酸位点(aa139~aa151)与大多数强毒株一致。但在aa89、aa92、aa411和aa417与大多数CAV参考株不同,均发生了氨基酸突变,分别为T^(89)K、G^(92)D、S^(411)T和V^(417)I,其中后两个位点的突变为本研究首次发现。SDNN2022株与CAV强毒参考株CUX-1的aa394均为谷氨酰胺,符合CAV强毒的分子特征,表明SDNN2022株为一株CAV强毒株。VP1基因的进化树结果显示,分离株SDNN2022与CAV参考株CUX-1、JS2020-PFV、CAV-JL190130等聚为一支,且与JS2020-PFV株的遗传距离最近,均属于A群,进一步表明SDNN2022株为一株强毒株。将该株病毒分别感染6胚龄SPF鸡胚和14日龄SPF雏鸡进行致病性试验,结果显示,SDNN2022株对鸡胚的致死率为30%(3/10),病死鸡胚体的肝脏黄化;该株病毒感染14日龄SPF雏鸡后7 d出现精神萎靡、呆滞、羽毛凌乱等临床症状,发病率为83.3%(5/6),但无死亡;感染后14 d,与空白对照组鸡相比,感染组鸡体重显著降低(P<0.05),胸腺明显萎缩,且胸腺指数极显著降低(P<0.01)。本研究分离到一株强毒力CAV,且对SPF鸡胚和雏鸡均具有较强的致病性,丰富了国内CAV的病毒资料,为深入研究CAV的致病机制提供了数据支持。

In order to identify the pathogen in the two liver samples of 196-day-old breeder chickens suspected of CAV infection from a chicken farm in Shandong,the Marek's disease tumor lymphoblastoid cell line(MDCC-MSB1 cells)was used to isolate and passage the virus from the samples,and the cells were observed for cytopathic effects(CPE)in each passage.The virus fluid passed to the 5th generation was identified by PCR and indirect immunofluorescence assay(IFA).The PCR results showed that the target band of about 600bp was amplified,which was consistent with that of the CAV JL17 strain positive control;the IFA results showed that both cells infected with the isolated virus and the JL17 strain showed green fluorescence,while the negative control neither amplified the target band nor had green fluorescence.The above results indicated that a CAV strain was isolated and named as SDNN2022.In order to analyze the molecular characteristics of the SDNN2022 strain,its VP1 gene was amplified by PCR and sequenced.DNAStar software was used to analyze the similarity between the VP1 gene of the SDNN2022 strain and the VP1 gene sequences of 39 CAV reference strains registered in GenBank.Megalign was used to analyze the amino acid sequence of VP1 of the SDNN2022 strain,and the evolutionary tree of the SDNN2022 strain VP1 gene was constructed.The results showed that the similarity of the VP1 gene sequence between the SDNN2022 strain and the CAV reference strain was 95.0%to 99.8%,with the highest similarity between the SDNN2022 strain and the reference strain JS2020-PFV;the amino acid position(aa139-aa151)in the hypervariable region of VP1 of the SDNN2022 strain were consistent with most virulent strains.However,aa89,aa92,aa411 and aa417 were different from most reference strains.These mutations were T^(89)K,G^(92)D,S^(411)T and V^(417)I,respectively.The mutations at the latter two sites had not been reported before.The aa394 of the SDNN2022 strain and the virulent reference strain CUX-1 were both glutamine,which was consistent with the molecular characteristics of CAV virulent strain,indicating that the SDNN2022 strain was a virulent strain.The evolutionary tree showed the isolated SDNN2022 together with the reference strains CUX-1,JS2020-PFV,CAV-JL190130,etc.,were clustered into one branch,and had the closest genetic distance to the JS2020-PFV strain,and both belonged to group A.These results further indicated that the SDNN2022 strain was a virulent strain.For pathogenicity analysis,6-day-old SPF chicken embryos and 14-day-old SPF chicks were infected with the SDNN2022 strain.The results showed that the lethality rate of SDNN2022 strain to chicken embryos was 30%(3/10),and the liver of dead chicken embryos was yellowed;7 days after infection,the 14-day-old SPF chicks showed clinical symptoms such as listlessness,sluggishness,and messy feathers appeared,with an incidence rate of 83.3%(5/6),but no deaths;14 days after infection,compared with the chickens in the control group,the weight of the chickens in the infected group was significantly reduced(P<0.05),the thymus atrophied significantly,and the thymus index was extremely significantly reduced(P<0.01).In this study,a highly virulent CAV was isolated,which was highly pathogenic to SPF chicken embryos and chicks,enriching the domestic CAV virus data and providing data support for in-depth research on the pathogenic mechanism of CAV.