A rescued virus from the infectious clone of a PRRSV NADC34-like strain exhibits high pathogenicity for nursery pigs

doi:10.1016/j.jia.2024.08.015

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Zhenbang Zhu^1*, Zhengqin Ye^1*, Wenqiang Wang¹, Yanhua Li¹, Zhe Sun², Xiuling Yu², Kegong Tian^2,^3#, Xiangdong Li^1,^4#

¹Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

²National Research Center for Veterinary Medicine, Luoyang 471003, China

³College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

⁴Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

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摘要

猪繁殖与呼吸综合征病毒类NADC34毒株在我国的流行传播范围不断扩大，并在局部地区成为优势毒株。目前商品化疫苗不能对类NADC34毒株提供完全交叉保护，这给养猪业造成巨大的经济损失。临床病例中常见类NADC34 PRRSV毒株与其他PRRSV毒株的混合感染，这也给从临床样本中分离类NADC34 PRRSV毒株并鉴定其生物学特性和致病力带来了干扰与挑战。本研究基于前期分离到的未发生重组的类NADC34 PRRSV毒株JS2021NADC34，利用反向遗传技术拯救出病毒，并研究其在猪肺泡巨噬细胞（PAMs）上的复制能力及对仔猪的致病力。首先，根据JS2021NADC34 PRRSV毒株基因组序列设计特异性引物，将全长cDNA分成四段进行PCR扩增，通过同源重组的方法构建JS2021NADC34感染性克隆并拯救出病毒rNADC34。获得的拯救病毒在PAMs增殖良好，能够产生明显CPE效应，免疫荧光和免疫印迹试验都在PAMs上检测到了病毒的复制，其病毒拷贝数和病毒滴度与母本毒株相当。在致病性方面，本研究将15只仔猪分成三组，分别是空白组、NADC34攻毒组和rNADC34攻毒组。PRRSV rNADC34毒株感染仔猪后，仔猪表现出体温升高和体重下降，该指标与母本病毒攻毒组一致。而rNADC34攻毒后仔猪的存活率比母本病毒感染仔猪的存活率低。组织病理学结果显示，rNADC34毒株感染引起弥漫性间质性肺炎，肺脏、淋巴结、扁桃体中有出血以及大量中性粒细胞和淋巴细胞的浸润。免疫组化结果显示在rNADC34毒株感染组的肺脏、淋巴结和扁桃体中均检测到PRRSV病毒蛋白。此外，rNADC34感染仔猪产生PRRSV特异性抗体和病毒血症趋势与母本毒株的感染相似。这些数据表明，拯救出的rNADC34毒株具有较强的毒力，并对仔猪具有高致病性。本研究为PRRSV类NADC34毒株的生物学特性、分子致病机制、毒力基因等研究提供基础平台，也并为疫苗的研发和PRRSV防控提供理论依据。

Abstract

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) has been circulating in China for several years and became the dominant field strain in some provinces. Current commercial vaccines could not provide complete cross-protection to NADC34-like PRRSV infection, which led to huge economic losses on pig farms. Co-infections of NADC34-like PRRSV with some other PRRSV strains are commonly found in many clinical cases, and successful isolation of NADC34-like PRRSV strain from the clinical samples has been a challenge to study its biological characters and perform animal experiments to evaluate its pathogenicity. In this study, we constructed a NADC34-like PRRSV infectious clone derived from the isolated JS2021NADC34 PRRSV strain using the reverse genetics technique and investigated its virulence and pathogenicity for nursery pigs. The rescued (rNADC34) strain could proliferate well in porcine alveolar macrophages (PAMs), and the viral copy number and titers were comparable to parental strain. For pathogenicity, the rNADC34 strain-infected pigs showed high body temperature and body weight loss. The histopathological results presented interstitial pneumonia and severe hemorrhage, infiltration of neutrophils and lymphocyte in lungs, lymph nodes, and tonsils. The viral proteins were also detectable in rNADC34 strain-infected pigs using immunohistochemistry staining. Moreover, the trends of PRRSV-specific antibody and viremia in PRRSV rNADC34-infected pigs were similar with the parental strain-infected pigs. These data indicated that rNADC34 strain manifested strong virulence and high pathogenicity for nursery pigs.

Keywords: PRRSV NADC34-like strain infectious clone pathogenicity

Online: 22 August 2024

Fund:

This work was supported by the National Natural Science Foundation of China-CGIAR (32361143511), the National Key Research and Development Program of China (2022YFD1800301), the Natural Science Foundation of Jiangsu Province, China (BK20210804), the Jiangsu Innovative and Entrepreneurial Talent Team Project, China (JSSCTD202224), the 111 Project D18007, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Zhenbang Zhu is supported by the "LvYangJinfeng Program" of Yangzhou City, China.

About author: Zhenbang Zhu, E-mail: 007583@yzu.edu.cn; Zhengqin Ye, E-mail: yezhengqin@126.com; #Correspondence Xiangdong Li, E-mail: 007352@yzu.edu.cn; Kegong Tian, E-mail: tiankg@263.net *These authors contributed equally to this study.

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Zhenbang Zhu, Zhengqin Ye, Wenqiang Wang, Yanhua Li, Zhe Sun, Xiuling Yu, Kegong Tian, Xiangdong Li. 2024. A rescued virus from the infectious clone of a PRRSV NADC34-like strain exhibits high pathogenicity for nursery pigs. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.08.015

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