猪繁殖与呼吸综合征病毒湖南分离株ORF5基因的遗传进化分析

doi:10.16431/j.cnki.1671-7236.2021.10.032

摘要/Abstract

摘要： 为了解猪繁殖与呼吸综合征病毒（PRRSV）在湖南省地方猪保种场的感染情况，本研究在2019－2020年间从湖南省2个地方猪保种场采集287份全血样品。首先将血样混合成41份，采用RT-PCR或PCR法进行PRRSV病原检测，进一步通过高保真PCR扩增从PRRSV阳性样品中扩增PRRSV ORF5基因；测序后利用DNAStar软件分析获得的ORF5基因及其编码的GP5氨基酸与国内外不同PRRSV毒株的遗传进化关系；最后用PRRSV阳性血清接种Marc-145细胞，经盲传分离毒株，并用Reed-Muench法测定病毒滴度。结果显示，检测的41份混样中有3份PRRSV病原核酸呈阳性；从PRRSV阳性混样中单独扩增获得6条PRRSV ORF5基因序列，均属于PRRSV-2型的lineage 8分支，相似性为99.2%~99.8%；6条ORF5基因编码的GP5蛋白氨基酸序列在信号肽区域（第23位）、潜在的N-糖基化位点（第33位）和表位C （第59位）存在差异；PRRSV阳性血清接种Marc-145细胞盲传5代后出现明显的细胞病变，获得1株PRRSV毒株，命名为NX-1，病毒TCID₅₀为4×10⁵/mL。本研究表明，湖南省地方猪保种场存在PRRSV感染，感染的PRRSV属于PRRSV-2型的lineage 8，其GP5氨基酸序列存在的多处变异可能是造成疫苗免疫失败的原因之一，以上结果可为湖南省地方猪保种场的免疫防控提供一定参考。

关键词: 猪繁殖与呼吸障碍综合征病毒(PRRSV); ORF5基因; 遗传进化分析

Abstract: In order to explore infection of Porcine reproductive and respiratory syndrome virus (PRRSV) in native pig breeding farms of Hunan province, 287 blood samples were collected from two native pig breeding farms during 2019 to 2020. PRRSV were detected by RT-PCR in 41 mixed seral samples. Open reading frame 5 (ORF5) gene of PRRSV were amplified from PRRSV positive samples by high fidelity PCR. DNAStar software was performed to analyze the genetic evolution of ORF5 gene and GP5 amino acid between these PRRSV strains and different PRRSV strains from China and abroad after sequencing. Finally, PRRSV positive seral were inoculated in Marc-145 cells followed by blind passaging. The titer of the isolated PRRSV was determined by Reed-Muench method. The results showed that 3 samples from the 41 mixed samples were positive for PRRSV. Six ORF5 sequences were amplified from PRRSV positive mixed samples, all of which belonged to branch lineage 8 of PRRSV-2 strain, and the nucleotide homology was 99.2% to 99.8%. The corresponding GP5 protein acid sequences encoded by six ORF5 gene sequences were different in the signal peptide region (position 23), potential N-glycosylation site (position 33) and epitope C (position 59). Obvious cytopathic effect (CPE) was observed in the Marc-145 cells inoculated the PRRSV positive sera after five blind passages. A stain of PRRSV designated as NX-1 was obtained with a TCID₅₀ of 4×10⁵/mL. The data indicated that there existed infection of PRRSV in Hunan native pig breeding farms. The prevalent PRRSV isolate belonged to lineage 8 of PRRSV-2 with several variations in the amino acid sequence of GP5, which might be associated with immune failure. The present study could supply a foundation for immune prevention and control in Hunan native pig breeding farms.

Key words: porcine reproductive and respiratory syndrome (PRRSV); ORF5 gene; genetic evolution analysis

中图分类号:

S852.65⁺1

邓吉平, 陈指龙, 郭松扬念, 张坤, 许道军, 杨青, 袁安文. 猪繁殖与呼吸综合征病毒湖南分离株ORF5基因的遗传进化分析[J]. 中国畜牧兽医, 2021, 48(10): 3795-3802.

DENG Jiping, CHEN Zhilong, GUO Songyangnian, ZHANG Kun, XU Daojun, YANG Qing, YUAN Anwen. Genetic Evolution Analysis of ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Isolated from Hunan Province[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3795-3802.

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