一株高病毒载量 PCV2 毒株的基因组特征及序列分析

【目的】了解广东省某猪群中猪圆环病毒 2 型（Porcine circovirus type 2，PCV2）流行毒株的遗传进化情况，丰富 PCV2 分子流行病学数据，为当地 PCV2 疫苗候选株的选用和研发提供参考。【方法】使用 qPCR 方法对疑似 PCV2 的样品进行检测，发现 1 株具有高病毒载量的 PCV2 毒株，命名为 GD222858。通过 PCR 方法进行全基因组分子克隆及遗传进化分析。使用 MegAlign 软件将该毒株 ORF1、ORF2 基因编码的氨基酸序列与 PCV2 同亚型参考毒株进行比对，分析氨基酸序列的相似性；采用 DNAStar 预测该毒株的 Cap 蛋白二级结构及 B 细胞表位，并与 4 株疫苗株 DBN-SX07-2（HM641752）、LG（HM038034）、SH（HM038027）、ZJ（AY686764）的 Cap 蛋白抗原指数进行比对分析。【结果】GD222858 毒株基因组长度为 1 767 bp。遗传进化分析表明该毒株属于 PCV2d 亚型。与国内外 82 株参考毒株的核苷酸相似性为 91.4%~99.6%，与越南毒株 Han8（GenBank 登录号：JQ181600）的亲缘关系最近。在 ORF1 编码的 Rep 蛋白处发现多个特异性突变位点 F70Y、F77L、W202R、N256S；ORF2 编码的Cap 蛋白相对保守。Protean 预测 Cap 蛋白的氨基酸第 5~18、24~25、39~41、48~49、57~65、99、101、112~114、139~140、145~150、162~165、175~181、188~189、205~211、227~232 位 置 处 均 可 能 存 在 潜 在 的 B 细 胞 表 位。GD222858 毒株的 Cap 蛋白抗原指数与 4 株疫苗株均有差异，在氨基酸 45~57、124~132、223~233 位置处抗原指数明显高于 4 株疫苗株，且与疫苗株 HM038034 差异最大。【结论】GD222858 毒株感染猪群的原因可能是 Rep 蛋白多个位点发生特异性突变及疫苗株选用不当所致。

Genome Characterization and Sequence Analysis of Porcine Circovirus Type 2 Isolated with High Viral Load

【Objective】The study was carried out to understand the genetic evolution of Porcine Circovirus Type 2 (PCV2) strains on a swine farm in Guangdong Province, and enrich molecular epidemiological data of PCV2, in order to provide reference for the selection and development of local PCV2 vaccine candidate strains.【Method】Samples suspected of PCV2 infection were detected by using qPCR methods. A PCV2-positive isolate with a high viral load was found, named GD222858. Genome-wide molecular cloning and genetic evolution analysis were performed with PCR methods. MegAlign software was used to compare the amino acid sequences encoded by the ORF1 and ORF2 genes of the strain with the PCV2 isotype reference strain, and the similarity of the amino acid sequences was analyzed. The DNAStar was used to predicte the Cap protein secondary structure and B-cell epitope of this strain and compare it with the Cap protein antigen index of four vaccine strains DBN-SX07-2 (HM641752), LG (HM038034), SH (HM038027) and ZJ (AY686764). 【Result】Sequencing results showed that the genome length of GD222858 strain was 1 767 bp. The genetic evolution analysis indicated that the strain belonged to the PCV2d subtype. The nucleotide similarity with 82 reference strains at home and abroad ranged from 91.4% to 99.6%. It was closest to the Vietnamese strain Han8 (GenBank accession No.: JQ181600). Multiple specific mutation sites F70Y, F77L, W202R and N256S were found at the Rep protein encoded by ORF1. The Cap protein encoded by ORF2 was relatively conserved. Protean predicted that the potential B cell epitopes presented at amino acid positions 5-18, 24-25, 39-41, 48-49, 57-65, 99, 101, 112-114, 139-140, 145-150, 162-165, 175-181, 188-189, 205-211 and 227-232 of Cap proteins. The Cap protein antigen index of the GD222858 strain was different from that of the four vaccine strains, and the antigen index at the amino acids 45-57, 124-132 and 223-233 was significantly higher than that of the other four vaccine strains, and the difference with the LG vaccine strain (HM038034) was greatest.【Conclusion】The reason for the infection of GD222858 strain in pig herds may be due to specific mutations in multiple sites of the Rep protein and improper selection of vaccine strains.