新型鸭呼肠孤病毒分离鉴定及其σC基因序列分析

doi:10.16431/j.cnki.1671-7236.2022.09.026

Abstract

Abstract: 【Objective】 The aim of this study was to understand and master the epidemiological characteristics and biological characteristics of Novel duck reovirus (NDRV), and to provide theoretical basis and technical support for the prevention and treatment of NDRV.【Method】 The tissues suspected of Duck reovirus infection was collected from a duck farm in Hebei, and after aseptic treatment, it was inoculated with SPF chicken embryos to isolate the virus, and the third-generation allantoic fluid was harvested for hemagglutination test and in vitro cell culture.The virus was identified by observation and indirect immunofluorescence assay (IFA).The isolated virus was subjected to animal regression test, and its σC gene was analyzed by Mega 7.0.【Result】 The isolated virus couldn't agglutinate chicken erythrocytes, and could kill chicken embryos.The dead chicken embryos were hemorrhagic and congested.The virus was NDRV positive by PCR identification, and other pathogens (Avian reovirus, Duck hepatitis virus, Duck Tembusu virus, Duck plague virus and Fowl adenovirus 4) were all negative, and the isolated virus was named BD/CHN/2020 strain.Spherical virus particles with a diameter of 60-80 nm were observed by electron microscopy and no envelope could be seen after viruses were purificated.The strain could be proliferated stably on BHK cells and LMH cells and produced a cytopathic effect (CPE) of cell fusion.IFA results showed that specific green fluorescence could be observed under a laser confocal microscope after BD/CHN/2020 strain was inoculated in BHK cells.After the BD/CHN/2020 strain was subcutaneously inoculated, the diseased ducks showed clinical symptoms such as depression and white loose stools.The autopsy showed pathological changes such as spleen hemorrhage, enlargement, and white necrotic foci.Sequence alignment showed that the BD/CHN/2020 strain and NDRV (TH11, 091 and other strains) were in the same branch, and had the highest similarity of nucleotide and amino acid sequences to the NDRV SY strain, which were both 99.7%, and belonged to the NDRV.Compared with the inactivated vaccine TH11 strain (KC493571.1) and the attenuated vaccine JS01-105P strain (CCTCC No.:V202168), the amino acid site mutations of BD/CHN/2020 strain had amino acid site mutations at sites 93, 120, 132, 158, 253 and 298.【Conclusion】 One NDRV BD/CHN/2020 strain was successfully isolated.The isolated strain had strong pathogenicity to Peking duck.Compared with the domestic vaccine strain, the σC protein of the BD/CHN/2020 strain had already undergone amino acid changes.The results laid the foundation for the epidemiology and vaccine development of NDRV disease.

Key words: Novel duck reovirus (NDRV); isolation and identification; σC gene

CLC Number:

S858.32

ZHANG Wei, WU Hua, YIN Yajie, LI Songli, HOU Shaohua. Isolation and Identification of Novel Duck Reovirus and Its σC Gene Sequence Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3520-3529.

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Isolation and Identification of Novel Duck Reovirus and Its σC Gene Sequence Analysis

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