| Avian leukosis(AL)is a collective term for a variety of neoplastic diseases of poultry caused by avian leukosis virus(ALV).Guangxi is the major province of poultry production in China,and since the introduction of ALV into Guangxi,the J subgroup of ALV(ALV-J)has been become the most prevalent with the highest infectivity and pathogenicity in chickens.At present,no drug or vaccine,but only the measure of eradication carried out in the breeding flocks is available and effective for the prevent and control of AL.In the study,firstly,the culture and isolation of ALV with the samples from the eradication testing and the suspected clinical cases in Guangxi during the recent two years were performed,then the env gene of the isolated ALVs was sequenced and analyzed,and one of the cases that was co-infected with ALV-J and type II REV was further studied in the pathogens;Secondly,six commercial ELISA detection kits for the ALV-P27 antigen were used for the comparative detection on the egg-white and cell culture samples,and the further validation of the ALV-P27 antigen detection measure with the cell-culture supernatant samples was performed in the comparison with the PCR detection for the viral gene with the cell cultures,aiming to find a better procedure for the eradication program.1.The isolation and identification of ALVs from the eradication testing breeders and the clinical cases,and the sequencing and analysis of the env gene of the isolates in Guangxi during 2021～2022The plasma samples of the eradication testing breeders from the breeding companies and the tissue samples from the birds with the clinically suspected AL in Guangxi were performed,and the positive samples were further identified by the subgroup-specific PCR,immunofluorescence assay(IFA),and sequencing and analysis of the env gene of the isolates,respectively.The results showed that a total of 11 ALV-J isolates were obtained;BLAST analysis of the nucleotide sequences of the env gene of the isolates found that the similarities with the conference strains available in Gen Bank were95.71%～99.82%.Eight of the isolates had a similarity of higher than 95.00%with the Guangxi isolates previously published by our group,one ALV-J isolates had a similarity of 95.71% with the Guangdong isolate GD15MM01,and the 2 isolates had a similarity of higher than 97.00%,with the United States virus EAV-21-3;According to the classification method of ALV-J established by our group,six of the isolates belonged to Clade 1.1,one belonged to Clade 1.2 and four belonged to Clade 1.3,and the results were basically consistent with the results of the phylogenetic analysis based on the env gene sequences.Interestingly,an ALV-J of Clade 1.3 and type II REV were both detected in one of the clinical case samples,The ALV-J isolate GX20YLJ1 from the co-infection was further performed for the viral whole-genome sequencing and the comparative analysis of the nucleotide sequences of genes gag,pol and env with 65 reference strains.The results showed that the whole-genome of the isolate GX20YLJ1 was 7.5 kb in length.The nucleotide and amino acid sequences of the gag and pol genes had higher than 92.20% of the similarities with those of the reference strains,and those of the env gene gene had higher than 87.60% and 84.20%,respectively.The homology modeling analysis of the encoded ENV subunits by the Alpha Flod2 software revealed that the functional regions of the secondary structures of the gp37 subunit of GX20YLJ1 and the gp20 subunit of the REV isolate GX20YLR1 were both composed of α-Helix,whereas the secondary structures of the gp85 subunit of GX20YLR1 and the gp90 subunit of GX20YLR1 were diverse.The REV isolate GX20YLR1 had the most similar secondary structure of gp90 with the type II REV strain SNV from the United States,which was consistent with the result of the phylogenetic analysis.2.The comparisons of different ALV-P27 antigen detection kits for the detection of egg-white and cell culture samples490 egg-white samples and 385 cell culture samples were detected and compared by using six ALV-P27 antigen detection kits.And another 200 cell culture samples were used for the comparative detection with the ELISA detection of ALV-P27 antigen and the PCR detection of the viral gene.In the comparative detection on the egg-white samples,the detection results showed that kits B and F had the highest specificity,while kits C and E had the best sensitivity,accuracy and consistency.In the comparative detection on the cell culture samples,the comparison showed that kits A and F had the highest sensitivity,while kits B and F had the best specificity,accuracy and consistency.In the validation of the ALV-P27 antigen detection measure with the PCR detection on the viral gene on the cell culture samples,the positive detection rates for kits A,B and C were 12.00%(24/200),11.50%(23/200)and16.00%(32/200),respectively,while that of the PCR detection was 18.00%(36/200),and the consistency of the detection results with kits A,B and C was87.50%(21/24),86.95%(20/23)and 68.75%(22/32),respectively;The results showed that the accuracy of kits A and B was relatively high,while that of C kit was low.The results of the study demonstrated that ALV-J has been absolutely the predominant subgroup recently in Guangxi.The ALV-J isolates prevalent in Guangxi in the recent two years had high genetic homology with the strains previously isolated by our group,and the co-prevalence of Clades 1.1,1.2 and1.3 strains was found,among which Clades 1.1 and 1.3 are dominant.In addition,a case of co-infection of Clade 1.3 ALV-J with type II REV was identified for the first time.The comparative detection results of six different commercial ALV-P27 antigen detection kits proved that Kit C and E were better in egg white sample detection.While Kit B and F were better in cell culture sample detection.Results of the PCR validation detection showed that kits A and B were better accuracy than others.and for the detection of cell culture sample,the PCR method was more accurate than the ELISA detection. |
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