北京地区猪圆环病毒3型感染的检测及基因组遗传变异分析

doi:10.11843/j.issn.0366-6964.2018.10.022

摘要/Abstract

摘要：

旨在了解北京地区猪圆环病毒3型（porcine circovirus type 3，PCV3）的流行现状、分子生物学特征和遗传变异规律，为PCV3的防控提供参考依据。采集北京地区规模化养殖场73份临床表现为繁殖障碍和呼吸系统疾病的组织样品，对其中的PCV3进行检测和测序。根据PCV3全基因组序列设计3对特异性引物，从患PDNS的断奶仔猪内脏组织中扩增PCV3全基因序列片段，克隆到pMD19-T载体中，经测序、拼接获得PCV3全长cDNA序列，利用DNAStar和DNAMAN生物软件对全基因组进行遗传变异分析。结果：北京地区PCV3感染率为30.1%（22/73），PCV3全基因组长2 000 bp，GenBank序列登录号为MG546667，将该病毒命名为PCV3/CN/BJ-YH2016。其基因组有3个开放阅读框（ORF），其中两个ORF编码的蛋白与圆环病毒的复制相关蛋白（replication-associated protein，Rep）和衣壳蛋白（capsid protein，Cap）同源。PCV3/CN/BJ-YH2016与NCBI上30株PCV3基因组序列相似性为98.6%~99.3%，Rep蛋白氨基酸序列与美国毒株PCV3-US/MO2015（KX778720）的相似性最高（为100%），与中国毒株PCV3/CN/Guangdong-SG1/2016（MF589105）相似性最低（为95%），有16个氨基酸变异，全部集中在5-23位氨基酸。Cap蛋白氨基酸序列与美国毒株2164（KX458235）的相似性为100%，与其他毒株均有1~4个氨基酸发生变异，其中与PCV3-US/MO2015毒株相比变异位点分别位于24、26、56、100 aa。以上试验结果表明，成功从患断奶仔猪多系统衰竭综合征仔猪体内克隆了PCV3全基因，并完成了序列分析，这些数据将为研究这种新型病毒——PCV3的遗传变异和流行病学特征提供分子生物学依据。

Abstract:

A porcine circovirus type 3 (PCV3) was detected from a commercial swine farm in Beijing with an outbreak of postweaning multisystemic wasting syndrome (PMWS) in nursery pigs. This study was conducted to investigate the genetic variation of prevalent PCV3 in China and provide the clues for better control of the spread of disease, we identified its complete genome sequence. A total of 73 clinical samples from pigs with reproductive failure and respiratory disease were collected from pigs farms in Fujian province from 2016 to 2017 and these samples were used to detect PCV3 by PCR. Based on PCV3 genome sequences obtained from NCBI GenBank, three pairs of specific primers were designed and synthesized. The nucleotide sequences were amplified from eye and nasal secretions through reverse transcription-PCR (RT-PCR). The cDNA fragments were inserted into pMD19-T vector and the complete genome sequence was obtained through sequencing. Sequence analysis were run on DNAStar and DNAMAN biosoftwares. Results were as follows:The discovered PCV3 strain was named as PCV3/CN/BJ-YH2016 (GenBank accession number:MG546667). The length of its genome was 2 000 bp and the coding region includes three open reading frames (ORFs). Two of them encode replication-associated protein (Rep) and capsid protein (Cap), respectively. The homology between BJ-YH2016 and other 30 submitted PCV3 genomic sequences in GenBank is from 98.6% to 99.3%. The Rep amino acid sequence has the highest homology (100%) with an American strain PCV3-US/MO2015 (KX778720) and the lowest homology (95%) with a Chinese strain PCV3/CN/Guangdong-SG1/2016 (MF589105) where there are 16 mutations mainly located between positions 5 to 23. The Cap amino acid sequence has 100% homology with an American strain 2164. There are a few mutations comparing to other PCV3 strains but at different positions, amongst which four mutations at position 24, 26, 56, 100 comparing to PCV3-US/MO2015. In conclusion, we identified the complete PCV3 genome sequence from a sick piglet with PMWS. The genomic sequence analysis would help us to come to a better understanding on PCV3 genetic variation and its epidemiologic feature.

中图分类号:

S852.659.2

孙明, 马君, 乔明明, 刘巧荣, 田新生, 杨欣艳, 孙胜永, 陈西钊. 北京地区猪圆环病毒3型感染的检测及基因组遗传变异分析[J]. 畜牧兽医学报, 2018, 49(10): 2261-2267.

SUN Ming, MA Jun, QIAO Ming-ming, LIU Qiao-rong, TIAN Xin-sheng, YANG Xin-yan, SUN Sheng-yong, CHEN Xi-zhao. Detection and Genetic Variation Analysis of a Porcine Circovirus Type 3 in Beijing[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2261-2267.

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