Molecular identification of iridoviruses infecting various sturgeon species in Europe

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low‐to‐high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74–88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus‐European (AcIV‐E). Moreover, three samples infected with AcIV‐E showed genetic heterogeneity, with the co‐existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV‐E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV‐E and an NV‐related virus.     

大黄鱼幼鱼虹彩病毒感染的电镜研究

目的:探索大黄鱼幼鱼夏季高温病流行的病毒性病原体。方法:应用电子显微镜观察大黄鱼幼鱼内脏组织超薄切片。结果:发现大黄鱼幼鱼脾脏细胞肿胀,细胞内存在大量虹彩病毒颗粒。病毒颗粒直径约120nm,呈六角形,有一层蛋白质外壳,核心为核酸。病毒感染主要位于细胞质内。结论:大黄鱼幼鱼脾细胞感染的病毒是虹彩病毒,虹彩病毒感染及环境因素可能是导致大黄鱼幼鱼夏季高温病暴发流行并致死的主要原因。     

Ultrastructural analysis of sequential cyprinid herpesvirus 3 morphogenesis in vitro

Cyprinid herpesvirus 3 (CyHV‐3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV‐3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF‐1 and CCB) infected with CyHV‐3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1–4 h post‐infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post‐infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5–7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non‐enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non‐infectious particles, particularly in KF‐1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV‐3 propagation.     

Isolation and characterization of an atypical Siberian sturgeon herpesvirus strain in Russia: novel North American Acipenserid herpesvirus 2 strain in Europe?

Siberian sturgeon herpesvirus (SbSHV) was isolated in Russia for the first time in 2006. Nine SbSHV isolates were recovered from different fish hatcheries producing the same cytopathic effect in cell cultures, the same clinical signs and mortality kinetics in virus‐infected fish and the same virus neutralization pattern and shared identical nucleotide sequences. In 2011, a new isolate was recovered from juvenile sturgeon, which caused completely different cytopathic effect. That isolate was not readily neutralized by Siberian sturgeon hyperimmune antisera, and its DNA was not recognized by the routine PCR developed for SbSHV detection. Molecular study of the novel isolate revealed that it was more closely related to North American Acipenserid herpesvirus 2 (AciHV‐2) isolates from white sturgeon, while the genome sequences of the former SbSHV isolates showed high similarity to the AciHV‐2 isolated from shortnose sturgeon. While clinical signs and mortality caused by the novel isolate in infected Siberian sturgeon were similar to those of the formerly described SbSHV isolates, the incubation period and mean time to death produced by the novel isolate were twice as long. The differences between the former isolates and the recent one suggest that a novel SbSHV strain emerged in Europe and the molecular findings imply its North American origin.     

一种虹彩病毒感染大菱鲆的病理学研究

对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。     

A natural infection by the red sea bream iridovirus‐type Megalocytivirus in the golden mandarin fish Siniperca scherzeri

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 10² to 7.95 × 10⁶ copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.     

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection

Cyprinid herpesvirus 2 (CyHV‐2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV‐2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non‐permissive temperature for 4 days, initiated at three different time points post‐infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post‐infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR‐positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV‐2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.     

Viral susceptibility,transfection and growth of SPB – a fish neural progenitor cell line from the brain of snubnose pompano,Trachinotus blochii (Lacépède)

This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV‐K1), red sea bream iridovirus (RSIV‐Ku), grouper nervous necrosis virus (GNNV‐K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV‐K1, RSIV‐Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP‐B1 was predominantly localized in the nuclei, EFPF‐B2 was distributed throughout the cytoplasm and nucleus, and granular 35L‐DsRed was localized with secreted vesicles. The expression of EFPF‐B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP‐B1 or 35L‐DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum‐dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.     

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post‐mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus‐like particles, subsequently identified as Cyprinid herpesvirus‐2 (CyHV‐2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV‐2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.     

Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.     

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.     

Outbreak of hirame rhabdovirus infection in cultured spotted sea bass Lateolabrax maculatus on the western coast of Korea

In this study, we determined the cause of a disease outbreak in spotted sea bass, Lateolabrax maculatus reared in culture cages on the western coast of Korea in 2013. The major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. No external morphological signs of infection were visible, except for a darkening in colour. No parasites or pathological bacteria were isolated from the diseased fish; however, epithelioma papulosum cyprini (EPC) cells inoculated with tissue homogenates from the diseased fish showed cytopathic effects (CPEs). Virus particles in the EPC cells were bullet‐shaped, 185–225 nm long and 70–80 nm wide, characteristic of Rhabdoviridae. Polymerase chain reaction analyses of homogenized tissues from the diseased fish and supernatants of cell cultures with CPEs indicated specific, 553‐bp‐long fragments corresponding to the matrix protein gene of the hirame rhabdovirus (HIRRV). Phylogenetically, the HIRRV phosphoprotein gene of spotted sea bass was more closely related to phosphoproteins from Chinese and Polish HIRRV strains than from other Korean strains. To our knowledge, this is the first report of HIRRV infection in cultured spotted sea bass.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Change in Sox9 protein localization through gonad development in Russian sturgeon (Acipenser gueldenstaedtii)

The Russian sturgeon is a highly prized species reared in aquaculture. The process of gonad development in this critically endangered species is poorly understood. The aim of this study was to describe the localization of Sox9 protein during gonad development of the Russian sturgeon from the day of hatching to the 1440 day post hatching (dph). The larvae at age 1, 10, 25 dph and prepared gonads of 300, 720, 1440 dph individuals were immunohistochemistry‐stained for Sox9 detection. Sox9‐positive regions were detected in larvae in primordial germ cells cytoplasm. Analysis of 300 dph sturgeon gonads revealed the presence of the Sox9 protein in cytoplasm of some oocytes in the chromatin nucleus stage. In testes at 720 dph, Sox9 was observed in the cytoplasm of type A and early B spermatogonias. In the ovaries, Sox9 was observed in the cytoplasm of diplotene oocytes and prefollicular cells. In testes of 1440 dph sturgeon, Sox9 was present in the nucleus of the spermatocytes and in types A and B spermatogonias cytoplasm. Analysis of ovaries at 1440 dph reveals multiple diplotene oocytes with a Sox9‐positive cytoplasm. Furthermore, in 720 and 1440 dph, sturgeon presence of intersexual gonads was detected. In intersex gonads, Sox9 was observed in the cytoplasm of diplotene oocytes and type A spermatogonias. This study may be the first attempt to determine Sox9 protein localization during ontogenesis of the Russian sturgeon. Localization of Sox9 protein may become a useful marker of the maturation level in testis of the Russian sturgeon.     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.     

Ultrastructural morphogenesis of salmonid alphavirus 1

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

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.