真鲷虹彩病毒引起养殖斑石鲷大规模死亡的研究

Red Seabream Iridovirus Causing Mass Mortality in Farmed Spotted Knifejaw,Oplegnathus punctatus

2017年8月,山东省烟台市某养殖场网箱养殖的斑石鲷(Oplegnathuspunctatus)幼鱼突然发病并大量急性死亡。疾病调查显示,养殖海域水温为26℃~28℃;病鱼为4~5月龄,全长为(16.3±1.6) cm,体重为(156.9±37.0) g;80万尾斑石鲷幼鱼2周内累积死亡率达90%以上,经济损失惨重。临诊检查发现,病鱼体表无明显损伤,但活力差、呼吸急促。剖检可见病鱼脾肿大、质地脆、易碎,肾糜烂,肝有出血点。组织切片观察发现,病鱼脾、肾造血组织中可见许多直径约为20μm的肿大细胞,肿大细胞内含有大量直径约为145nm、呈六边形的病毒颗粒。用过滤除菌的病鱼脾组织匀浆液,腹腔注射感染健康斑石鲷,感染组14d内累积死亡率达95%。人工感染病鱼表现出与自然发病鱼类似的外观症状,且在脾、肾组织切片中也可观察到大量的肿大细胞及相似的病毒粒子。使用特异性PCR引物,从自然发病鱼和人工感染病鱼的肝、脾和肾组织中均检测到鱼类虹彩病毒的高强度感染。克隆、测序得到了1362 bp的病毒主要衣壳蛋白基因(MCP),序列比对显示,该病毒的MCP序列与真鲷虹彩病毒(RSIV)RIE12-1的相应序列完全相同。构建的虹彩病毒系统发育树也显示,该病毒属于虹彩病毒科肿大细胞病毒属RSIV类群,是RSIV的一个分离株。本研究首次证实RSIV可以导致斑石鲷大规模死亡,研究结果为诊断和防治斑石鲷病毒病提供了重要参考。

Spotted knifejaw(Oplegnathus punctatus)is a new species of mariculture fish with high economic value in China.In August 2017,an outbreak of unknown etiology occurred in spotted knifejaw juveniles cultured in the offshore cages of a marine fish farm.Investigation showed that the water temperature was 26℃~28℃and that the infected fish were mainly juveniles at the age of 4~5 months.The total length of the diseased fish was(16.3±1.6)cm and their body weight was(156.9±37.0)g.The cumulative mortality of 800000 juveniles was more than 90%within 2 weeks of disease onset.Clinical examination showed that the diseased juveniles had no obvious apparent lesions but had poor vitality and rapid breathing.Necropsy findings included a swollen and brittle spleen,kidney erosion,and hemorrhagic spots in the liver.No parasites were detected,and no pathogenic bacteria were isolated from the liver,spleen,and kidney of the diseased fish.Histopathological sections showed a large number of enlarged cells(about 20μm in diameter)in the spleen and kidney tissues of diseased fish.In addition,a large number of hexagonal virus particles with a diameter of about 145 nm were observed in the electron microscope sections of the spleen and the body kidney.The spleen homogenate of naturally infected fish was filtered and injected intraperitoneally into healthy fish at a water temperature of 26℃.The fish in the infected group began to die after 10 days post infection(dpi),and the cumulative mortality reached 95%within 14 dpi.Artificially infected fish showed symptoms similar to those in naturally infected fish,and a large number of enlarged cells and similar virus particles could be observed in the spleen and kidney tissue sections.Severe iridovirus infection was detected in the spleen and kidney tissues of naturally and artificially infected fish using specific PCR primers.The major capsid protein gene(MCP)of the iridovirus,at 1362 bp in length was cloned and sequenced,and the phylogenetic tree of the family Iridoviridae was constructed.The results showed that the virus belonged to the red sea bream iridovirus(RSIV)of genus Megalocytivirus in the family Iridoviridae.Based on the results of epidemiological investigation,clinical symptom observation,etiological detection,histopathological observation,artificial infection experiments,molecular biology diagnosis,and viral phylogenetic analysis,it was confirmed that the disease causing the mass death of spotted knifejaw was Megalocytivirus disease,and that the pathogen was a strain of RSIV.This study demonstrates for the first time that RSIV can cause large-scale death in the spotted knifejaw.Furthermore,the research results provide an important reference to diagnose and control viral diseases in the spotted knifejaw.