养殖银鲳虹彩病毒感染的组织病理学分析及分子检测

Histopathological analysis and molecular detection of iridovirus in cultured Pampus argenteus

摘要:

2018年7月，宁波象山某养殖场内银鲳出现集中死亡现象，累积死亡率高达80%以上，为探究此次疾病暴发的原因，本研究通过对患病银鲳进行组织病理学分析、透射电镜观察、病原的分子生物学检测及分析来对其病原进行鉴定，以便制定合理的防控措施。患病银鲳临床表现为厌食、身体失衡、脾脏肿大、肝脏颜色异常等病征。组织病理学观察发现，患病银鲳肝脏、脾脏和肾脏均出现直径10~15 μm、细胞质嗜碱性的肿大细胞。进一步的透射电镜观察则在脾脏、肾脏等组织细胞内发现了病毒包涵体结构以及大量的病毒粒子，直径为140~160 nm。通过虹彩病毒特异性PCR检测发现，患病组织样品为虹彩病毒阳性。此外，通过病毒主要衣壳蛋白基因(MCP)序列分析，发现银鲳源病毒与大黄鱼虹彩病毒(GenBank 登录号：AY779031.1)MCP同源性最高为99.76%，认为此分离病毒属于虹彩病毒科、肿大细胞病毒属、真鲷虹彩病毒(Red sea bream iridovirus, RSIV)类群。本实验首次报道了全人工养殖环境下银鲳感染虹彩病毒的病例，该研究将为养殖银鲳虹彩病毒病的诊断和防治提供重要的参考依据。

关键词:银鲳;养殖;肿大细胞病毒;虹彩病毒;分子诊断

Abstract:

In July 2018, an outbreak of unknown disease occurred in the silver pomfret (Pampus argenteus) cultured in a farm in Xiangshan, Ningbo, with a cumulative mortality rate of over 80%. To investigate the cause of disease outbreak, histopathological analysis, transmission electron microscope observation and molecular biological detection and analysis were carried out to identify the pathogen, so as to help us to understand and control the disease.The clinical manifestations of sick P. argenteus are anorexia, body imbalance, swelling of the spleen, and abnormal color of the liver. Histopathological analysis showed the presence of basophilic enlarged cells with a diameter of 10~15 μm in diseased fish spleen, liver and kidney. Further transmission electron microscopic observation revealed the presence of inclusion bodies and a large number of virus particles (a diameter of 140-160 nm) in cells from spleen and kidney tissues. Moreover, results of the iridovirus-specific PCR test showed that all these tissue samples collected from diseased fish were positive for iridovirus. To further detect the presence of iridovirus, the major capsid protein (MCP) gene of virus in P. argenteus was amplified, cloned and sequenced. Sequence similarity analysis showed that silver pomfret-derived virus (SPDV) shared the highest homology with the large yellow croaker iridovirus (LYCIV) (GenBank accession number: AY779031.1) MCP, with a sequence similarity of 99.76%. In a phylogenetic tree, SPDV and LYCIV are closely grouped with red sea bream iridovirus (RSIV) strains. Thus, we concluded that the virus that infected silver pomfret belongs to the Iridovirus family, Megalocytivirus genus, RSIV group. In summary, this paper reported Megalocytivirus infection in cultured P. argenteus for the first time. This study will provide an important reference for the diagnosis and prevention of P. argenteus iridescent virus disease.

Key words:Pampus argenteus;cultured;Megalocytivirus;iridovirus;molecular diagnosis