广东地方品种鸡ALV-K分子流行病学及gag基因12 bp缺失对病毒体外复制能力影响的研究

doi:10.11843/j.issn.0366-6964.2022.11.021

Abstract

Abstract: To investigate the molecular characteristics of ALV-K isolates from local breed chickens in Guangdong, 8 042 anticoagulant samples were collected from core flocks of healthy appearance in seven large-scale breeding poultry farms (A-G) in Guangdong that are undergoing eradication of exogenous ALV during 2020-2021. Among them, 357 positive samples were identified by DF-1 cell culture and p27 antigen ELISA test. PCR identification and env gene sequencing were performed on 150 positive samples with S/P values of 0.2 to 0.6 by ELISA, and 32 ALV-K strains were isolated. Sixteen isolates were further selected for whole-genome sequencing analysis. The results showed that the total genome length of the 16 ALV-K isolates ranged from 7 481 to 7 496 bp, and the genome conformed to the typical retrovirus structure of 5'-LTR-UTR-gag-pol-env-UTR-LTR-3', without known oncogenes. The gp85 gene of the isolates and ALV-K reference strains were located in the same evolutionary branch and had the highest similarity (>94.0%). The pol and gp37 genes were relatively conserved, with the similarity more than 94.0% to other ALV subgroups. The LTR showed highest similarity with endogenous ALV and most ALV-K reference strains (91.4%-99.5%), and the LTR U3 region had the same transcriptional regulatory elements as the endogenous ALV LTR. Additionally, 31.3% (5/16) of isolates were observed to have a 12 bp deletion at nucleotides 373-384 of the gag gene. Further, the full-length cDNA clones of the deletion strain (rGD20 JM10) and the complementary strain (rGD20 JM10 A12) were respectively constructed by PCR segmental amplification and homologous recombination based on the deletion strain GD20 JM10, and the infectious clones were transfected into DF-1 cells. The ELISA and IFA results showed the two viruses were successfully rescued, and the in vitro replication curves showed no significant difference between the two viruses. The epidemic ALV-K strains from seven breeding poultry farms in Guangdong investigated in this study all carried endogenous LTR, so it is particularly important to use a more sensitive detection technology when carrying out exogenous ALV eradication. Additionally, the growth curves showed that such gag gene with the 12 bp deletion had no evident effect on the virus replication in vitro.

Key words: ALV-K, local breed chickens, molecular epidemiology, gag gene, infectious clone

CLC Number:

S852.659.3

GUO Yanyan, LIANG Canxin, LI Jinqun, DONG Xinyi, LIAO Ming, CAO Weisheng. Study on Molecular Epidemiology of ALV-K in Guangdong Local Breed Chickens and the Effect of 12 bp Deletion of gag Gene on Virus Replication Ability in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3956-3966.

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Study on Molecular Epidemiology of ALV-K in Guangdong Local Breed Chickens and the Effect of 12 bp Deletion of gag Gene on Virus Replication Ability in vitro

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