中国广西地区分离的猪圆环病毒 2 型的分离与致病性。

Isolation and pathogenicity of porcine circovirus type 2 in mice from Guangxi province, China.

机构信息

National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, Hubei, 430070, PR China.

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, 430070, PR China.

出版信息

Virol J. 2023 Aug 29;20(1):195. doi: 10.1186/s12985-023-02161-5.

DOI:10.1186/s12985-023-02161-5

PMID:37644571

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10466715/

Abstract

BACKGROUND

Porcine circovirus type 2 (PCV2), a member of the genus Circovirus and family Circoviridae, is a closed, small, circular, and single-stranded DNA virus, and it is a crucial swine pathogen of porcine circovirus-associated diseases (PCVADs). PCV2 was first detected in PK-15(ATCC-CCL) cells in 1974, which has caused significant economic loss to the swine industry throughout the world. And the first case of PCV2 was reported in China in 2000. At present, PCV2d is the main genotype circulating widely in China.

METHODS

Lymph samples were obtained from piglets with emaciation and respiratory disease in Guangxi province, China. The main pathogens were detected via PCR from lymph samples, and then PCV2-single positive samples were used to inoculate with PK-15 cells. After successive generations, the isolate was subsequently identified by polymerase chain reaction (PCR), immunofluorescence assay (IFA), Western blot (WB), and transmission electron microscopic (TEM). The full-length genome and genetic characterization of isolates were analyzed by Sanger sequencing. The TCID of the PCV2-GX-6 was determined by IFA, and the pathogenicity of PCV2 in BALB/c mice was analyzed via the mouse model.

RESULTS

The isolates were successfully isolated from clinical samples. The complete genome of PCV2-GX-4, PCV2-GX-6, PCV2-GX-7, PCV2-GX-11 and PCV2-GX-16 have been amplified, sequenced, and deposited in GenBank (accession no.: OR133747, OQ803314, OR133748, OR133749, OR133750). Homology and phylogenetic analysis with reference strains showed that the isolates belonged to the PCV2d genotype. The PCV2-GX-6 could be stably passaged more than 30 times in PK-15 cells. PCV2-GX-6 was identified by PCR, IFA, WB and TEM. The results of homology showed that PCV2-GX-6 was closely related to the reference strains PCV2-JS17-8 (GenBank accession no.: MH211363). Pathogenicity studies in mice have shown that PCV2-GX-6 can lead to growth inhibition of mice. Meanwhile PCV2-GX-6 caused the typical lesions of spleen, lung and kidney. The results of qPCR showed that PCV2 can effectively proliferate in the liver, spleen, lung, and kidney.

CONCLUSION

PCV2-GX-6 can successfully infect BLAB/c mice, effectively proliferate in major organs, and possessed high pathogenicity. In conclusion, combined with the genotype and pathogenicity of PCV2d currently prevalent, PCV2-GX-6 can be used as a candidate vaccine strain.

摘要

背景

猪圆环病毒 2 型（PCV2）属于圆环病毒科圆环病毒属的单链环状 DNA 病毒，是一种重要的猪圆环病毒相关疾病（PCVADs）的病原体。PCV2 于 1974 年首次从 PK-15（ATCC-CCL）细胞中检测到，给全球的养猪业造成了巨大的经济损失。中国于 2000 年首次报告了 PCV2 病例。目前，PCV2d 是中国广泛流行的主要基因型。

方法

从广西消瘦和呼吸道疾病的仔猪中采集淋巴样本。从淋巴样本中通过 PCR 检测主要病原体，然后用 PK-15 细胞接种 PCV2-单阳性样本。经过连续几代传代，通过聚合酶链反应（PCR）、免疫荧光分析（IFA）、Western blot（WB）和透射电子显微镜（TEM）对分离株进行鉴定。通过 Sanger 测序分析分离株的全长基因组和遗传特征。通过 IFA 测定 PCV2-GX-6 的半数组织培养感染剂量（TCID），并通过小鼠模型分析 PCV2 在 BALB/c 小鼠中的致病性。

结果

从临床样本中成功分离出了分离株。成功扩增、测序并将 PCV2-GX-4、PCV2-GX-6、PCV2-GX-7、PCV2-GX-11 和 PCV2-GX-16 的全长基因组序列提交至 GenBank（登录号：OR133747、OR803314、OR133748、OR133749、OR133750）。与参考株的同源性和系统进化分析表明，分离株属于 PCV2d 基因型。PCV2-GX-6 可在 PK-15 细胞中稳定传代 30 余代。通过 PCR、IFA、WB 和 TEM 鉴定了 PCV2-GX-6。同源性结果表明，PCV2-GX-6 与参考株 PCV2-JS17-8（GenBank 登录号：MH211363）密切相关。在小鼠中的致病性研究表明，PCV2-GX-6 可导致小鼠生长抑制。同时，PCV2-GX-6 引起了脾脏、肺和肾脏的典型病变。qPCR 结果表明，PCV2 可在肝脏、脾脏、肺和肾脏中有效增殖。