Detection and characterization of viruses of the genus Megalocytivirus in ornamental fish imported into an Australian border quarantine premises: an emerging risk to national biosecurity

This report documents an emerging trend of identification of Megalocytivirus-like inclusions in a range of ornamental fish species intercepted during quarantine detention at the Australian border. From September 2012 to February 2013, 5 species of fish that had suffered mortality levels in excess of 25% whilst in the post-entry quarantine and had Megalocytivirus-like inclusion bodies in histological sections were examined by PCR. The fish had been imported from Singapore, Malaysia and Sri Lanka. Ninety-seven of 111 individual fish from affected tanks of fish tested were positive for the presence of Megalocytivirus by PCR. Sequence analysis of representative PCR products revealed an identical sequence of 621 bp in all cases which was identical to a previously characterized Megalocytivirus (Sabah/RAA1/2012 strain BMGIV48). Phylogenetic analysis of available Megalocytivirus major capsid protein (MCP) sequences confirmed the existence of 3 major clades of Megalocytivirus. The virus detected in this study was identified as a member of Genotype II. The broad host range and pathogenicity of megalocytiviruses, coupled to the documented spread of ornamental fish into the environment, render this a significant and emerging biosecurity threat to Australia.     

真鲷虹彩病毒引起养殖斑石鲷大规模死亡的研究

2017年8月，山东省烟台市某养殖场网箱养殖的斑石鲷(Oplegnathus punctatus)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20 μm的肿大细胞，肿大细胞内含有大量直径约为145 nm、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14 d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。     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.     

鱼类细胞肿大虹彩病毒病研究进展

鱼类细胞肿大虹彩病毒病在东亚和东南亚的真鲷、石斑鱼、大菱鲆、大黄鱼、鳜鱼等多种养殖鱼类中的频繁流行,致使感染鱼类出现大面积死亡,已成为危害养殖鱼类最严重的病毒病之一.文章综述了近年来鱼类细胞肿大虹彩病毒病的研究现状,重点介绍了该类疾病的流行病学、临床症状、组织病理学、分子生物学检测技术、病毒的传播途径、致病机制以及病毒的免疫防控等方面的研究进展,并对当前鱼类细胞肿大虹彩病毒研究中存在的问题进行了分析和展望.     

大口黑鲈虹彩病毒双重PCR检测方法的建立

为同时检测患病大口黑鲈中不同属虹彩病毒感染和带毒情况,针对蛙病毒属和肿大细胞病毒属大口黑鲈虹彩病毒主衣壳蛋白（major capsid protein,MCP）基因的序列,分别设计1对特异性引物Rana-mcp F/R和Mega-mcp F/R,扩增产物片段大小分别为475 bp和262 bp。通过PCR反应体系的优化、反应的特异性和灵敏度试验,建立一种可以同时检测大口黑鲈蛙病毒属和肿大细胞病毒属虹彩病毒感染的双重PCR检测方法,最低DNA检测量分别为6.5 pg和14.5 pg。用此方法,对临床获得的15个大口黑鲈样品进行双重PCR检测和序列测定,获得5个蛙病毒属和1个肿大细胞病毒属虹彩病毒检测阳性结果。本研究中建立的基于MCP基因的大口黑鲈虹彩病毒双重PCR检测方法,可用于养殖大口黑鲈虹彩病毒病快速鉴别诊断和分子流行病学调查。     

Development of primary cell culture from spleen of giant gourami Osphronemus goramy for propagation of giant gourami iridovirus (GGIV)

The severe mortality of fish due to the infection of megalocytivirus caused significant economic losses. Since 2011, megalocytivirus (giant gourami iridovirus (GGIV)) has become the main pathogen in giant gourami (Osphronemus goramy), particularly in West Java, Central Java and Bali. This study aimed to develop primary cell culture from spleen as the target organ for propagating megalocytivirus in vitro, which was developed by explant method with enzymatic dissociation. Optimization was carried out at incubation temperature, medium and serum concentrations. The origin of the primary cell, cell susceptibility and GGIV pathogenicity were observed. The results showed that the primary cell (GP cells) can grow well in 10% foetal bovine serum L-15 medium at 27°C, which was sufficient for cell growth. PCR and BLAST analyses showed the primary cell was originated from giant gourami. In infected GP cells, cell enlargement and cell rounding were observed. Virus propagated in GP cells was highly virulent when injecting giant gourami in an artificial infection experiment. Intraperitoneal injection of diluted virus supernatant showed 100% mortality in 7–11 days post-injection and 97% mortality in 21 days post-cohabitation, with abnormalities observed in spleen and kidney. In conclusion, GP cell was successfully subcultured for more than 30 passages and susceptible to GGIV.