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 T89K,G92D,S411T and V417I,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.
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