信阳地区散养土鸡禽白血病病毒和马立克病病毒混合感染的分子生物学诊断

doi:10.16431/j.cnki.1671-7236.2025.02.034

摘要/Abstract

摘要： 【目的】了解信阳地区散养土鸡群中禽白血病病毒(Avian leukosis virus,ALV)与马立克病病毒(Marek’s disease virus，MDV)混合感染及其遗传进化情况。【方法】对送检的疑似ALV与MDV混合感染的病鸡进行病理解剖,使用特异性引物进行PCR扩增，并对J亚群ALV(ALV-J) gp85基因与MDV meq基因进行扩增测序，使用MegAlign软件进行核苷酸、氨基酸序列相似性比对以及氨基酸变异位点分析。利用Mega 11.0软件构建gp85、meq氨基酸序列系统进化树。【结果】送检发病鸡腺胃、脾脏肿大，肝脏出现弥漫性肿大并伴有灰白色结节。ALV-J gp85与MDV 132-bpr基因PCR扩增结果均为阳性，分别获得545、317 bp目的条带。测序比对结果显示，扩增序列分别与ALV-J分离株GX12NN04 gp85基因(登录号：KT598488)和MDV-1型分离株YLO40920 meq基因(登录号：DQ174459)高度相似，相似性分别为94.6%和99.9%，初步确定分离株为ALV-J和MDV，并分别命名为HN23XY01-ALV、HN23XY01-MDV。核苷酸序列分析结果显示,HN23XY01-ALV gp85基因与13株ALV-J参考毒株核苷酸序列相似性为92.1%～94.6%；HN23XY01-MDV meq基因与14株MDV参考毒株的核苷酸序列相似性为99.3%～99.9%。氨基酸序列分析结果显示，HN23XY01-ALV gp85与13株ALV-J参考毒株氨基酸序列相似性为87.4%～91.4%，HN23XY01-MDV与14株MDV参考毒株的氨基酸序列相似性为97.9%～99.7%。系统进化树结果显示，HN23XY01-ALV与ALV-J亲缘关系较近，处于同一进化分支；HN23XY01-MDV与广西分离株GXY2、YLO40920、GX20NN2和河南分离株HNSC105处于同一个分支，与疫苗株CVI988、814和CU-2亲缘关系较远。氨基酸变异位点分析结果显示，HN23XY01-ALV存在D(E)65Y、Q(K/R)75L、A(T)76S、R(T)119K、T(M/A)219K氨基酸位点突变；HN23XY01-MDV存在K77E、D80Y、T139A、P176R和P217A氨基酸位点突变，且含有疫苗株所缺失的第193位脯氨酸，与MDV-1型强毒株的特征相符合。【结论】本研究从信阳地区散养土鸡群中检测到ALV-J与MDV混合感染，结果为当地土鸡ALV-J与MDV协同致病机制研究以及混合感染的疫情防控提供重要参考。

关键词: 禽白血病病毒(ALV); 马立克病病毒(MDV); gp85基因; meq基因; 混合感染

Abstract: 【Objective】 The objective of this experiment was to investigate the co-infection and genetic evolution of Avian leukosis virus (ALV) and Marek’s disease virus (MDV) in free range local chickens in Xinyang area. 【Method】 Pathological autopsy was performed on the chickens suspected to be co-infected with ALV and MDV,and PCR amplification was performed using specific primers.The ALV-J subgroup (ALV-J) gp85 gene and MDV meq gene were amplified and sequenced,and the nucleotide and amino acid sequences similarity and amino acid variation sites were analyzed by MegAlign software.The phylogenetic tree of gp85 and meq amino acid sequences was constructed with Mega 11.0 software. 【Result】 The chickens were examined for enlargement of the gland,stomach and spleen,and diffuse enlargement of the liver with grayish white nodules.PCR results of ALV-J gp85 and MDV 132-bpr genes were positive, and 545 and 317 bp target bands were obtained, respectively. Sequencing results showed that the amplified sequences were highly similar to the gp85 gene of ALV-J strain GX12NN04 (accession number: KT598488) and the meq gene of MDV-1 strain YLO40920 (accession number: DQ174459), and the similarity was 94.6% and 99.9%, respectively. The isolates were initially identified as ALV-J and MDV,and named as HN23XY01-ALV and HN23XY01-MDV,respectively.Nucleotide sequence analysis showed that the nucleotide sequence similarity between HN23XY01-ALV gp85 gene and 13 ALV-J reference strains was 92.1%-94.6%，and the nucleotide sequence similarity between HN23XY01-MDV meq gene and 14 MDV reference strains was 99.3%-99.9%.The amino acid sequence analysis showed that the amino acid sequence similarity between HN23XY01-ALV gp85 and 13 ALV-J reference strains was 87.4%-91.4%,and the amino acid sequence similarity between HN23XY01-MDV and 14 MDV reference strains was 97.9%-99.7%.Phylogenetic tree results showed that HN23XY01-ALV and ALV-J were closely related and in the same evolutionary branch.HN23XY01-MDV was in the same branch with Guangxi isolates GXY2,YLO40920 and GX20NN2，and Henan isolate HNSC105,and was distantly related to vaccine strains CVI988,814 and CU-2.Analysis of amino acid variation sites showed that HN23XY01-ALV had mutations at D(E)65Y,Q(K/R)75L,A(T)76S,R(T)119K and T(M/A)219K.HN23XY01-MDV had K77E,D80Y,T139A,P176R and P217A amino acid mutations，and contained site 193 (proline) that was missing from the vaccine strains,which was consistent with the characteristics of MDV-1 virulent strains. 【Conclusion】 In this study,co-infection of ALV-J and MDV was detected in free range local chickens in Xinyang area，and the results provided important references for the study of the co-pathogenic mechanism of ALV-J and MDV in local chickens and the epidemic prevention and control of co-infection.

Key words: Avian leukosis virus (ALV); Marek’s disease virus (MDV); gp85 gene; meq gene; co-infection

中图分类号:

S855.3

李迎晓, 尹磊, 赵瑜, 董建国, 何书海, 曲哲会, 焦凤超. 信阳地区散养土鸡禽白血病病毒和马立克病病毒混合感染的分子生物学诊断[J]. 中国畜牧兽医, 2025, 52(2): 844-854.

LI Yingxiao, YIN Lei, ZHAO Yu, DONG Jianguo, HE Shuhai, QU Zhehui, JIAO Fengchao. Molecular Biological Diagnosis of Co-infection of ALV and MDV in Free Range Local Chickens in Xinyang Area[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 844-854.

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