Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Characterization of a Novel Megalocytivirus Isolated from European Chub (Squalius cephalus).

A novel virus from moribund European chub (Squalius cephalus) was isolated on epithelioma papulosum cyprini (EPC) cells. Transmission electron microscopic examination revealed abundant non-enveloped, hexagonal virus particles in the cytoplasm of infected EPC cells consistent with an iridovirus. Illumina MiSeq sequence data enabled the assembly and annotation of the full genome (128,216 bp encoding 108 open reading frames) of the suspected iridovirus. Maximum Likelihood phylogenetic analyses based on 25 iridovirus core genes supported the European chub iridovirus (ECIV) as being the sister species to the recently-discovered scale drop disease virus (SDDV), which together form the most basal megalocytivirus clade. Genetic analyses of the ECIV major capsid protein and ATPase genes revealed the greatest nucleotide identity to members of the genus Megalocytivirus including SDDV. These data support ECIV as a novel member within the genus Megalocytivirus. Experimental challenge studies are needed to fulfill River's postulates and determine whether ECIV induces the pathognomonic microscopic lesions (i.e., megalocytes with basophilic cytoplasmic inclusions) observed in megalocytivirus infections.

Complete genome sequence and phylogenetic analysis of megalocytivirus RSIV-Ku: A natural recombination infectious spleen and kidney necrosis virus.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981-66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.

Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei).

A newly discovered iridescent virus that causes severe disease and high mortality in farmed Litopenaeus vannamei in Zhejiang, China, has been verified and temporarily specified as shrimp hemocyte iridescent virus (SHIV). Histopathological examination revealed basophilic inclusions and pyknosis in hematopoietic tissue and hemocytes in gills, hepatopancreas, periopods and muscle. Using viral metagenomics sequencing, we obtained partial sequences annotated as potential iridoviridae. Phylogenetic analyses using amino acid sequences of major capsid protein (MCP) and ATPase revealed that it is a new iridescent virus but does not belong to the five known genera of Iridoviridae. Transmission electron microscopy showed that the virus exhibited a typical icosahedral structure with a mean diameter of 158.6 ± 12.5 nm (n = 30)(v-v) and 143.6 ± 10.8 nm (n = 30)(f-f), and an 85.8 ± 6.0 nm (n = 30) nucleoid. Challenge tests of L. vannamei via intermuscular injection, per os and reverse gavage all exhibited 100% cumulative mortality rates. The in situ hybridization showed that hemopoietic tissue, gills, and hepatopancreatic sinus were the positively reacting tissues. Additionally, a specific nested PCR assay was developed. PCR results revealed that L. vannamei, Fenneropenaeus chinensis, and Macrobrachium rosenbergii were SHIV-positive, indicating a new threat existing in the shrimp farming industry in China.

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

UNLABELLED: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus.

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.

Shift of phylogenic position in megalocytiviruses based on three different genes.

Major capsid protein (MCP), the adenosine triphosphatase (ATPase), and the PstI fragment genes from five Japanese and three Korean megalocytivirus isolates were sequenced and phylogenetically analyzed with known megalocytiviruses. Phylogenetic trees formed three major clusters (M1, M2, and M3 or P1, P2, and P3), and genogroup I was divided into two minor clusters (M1a/M1b and P1a/P1b) using three target genes. Sequence identity was >97% within each cluster, except cluster II of the PstI fragment (>94% of sequence identity). Interestingly, different genotyping patterns were observed for the same isolates depending on the gene analyzed. The JPN-YelTail and JPN-BfTuna isolates located in the minor M1a cluster, based on MCP and ATPase nucleotide sequences, appeared in the minor P1b cluster based on the PstI fragment, suggesting a shift of phylogenic position in megalocytiviruses. Further study will be conducted to compare the viral antigenicity and pathogenicity between the two isolates showing the shift of phylogenic position and the other isolates clustered within genogroup I.

Complete genome sequence of a Megalocytivirus (family Iridoviridae) associated with turbot mortality in China.

BACKGROUND: Turbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China. RESULTS: The genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae). CONCLUSIONS: The determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.

A new marine megalocytivirus from spotted knifejaw, Oplegnathus punctatus, and its pathogenicity to freshwater mandarinfish, Siniperca chuatsi.

Megalocytivirus is a newly defined piscine iridovirus and has been shown to be an important causative agent of viral diseases in fish. Here, a new megalocytivirus strain, designated SKIV-ZJ07, was isolated from spotted knifejaw (Oplegnathus punctatus) using a mandarinfish fry cell line (MFF-1). Phylogenetic analysis of the major capsid protein and ATPase genes showed that SKIV-ZJ07 was most similar to the orange-spotted grouper iridovirus (OSGIV) from China and a U1 strain red sea bream iridovirus (RSIV-U1) from Japan. SKIV-ZJ07 was purified and the major viral proteins were identified using one-dimensional gel electrophoresis mass spectrometry (1-DE-MS) analysis. Twenty proteins were found to match proteins derived from rock sea bream iridovirus (RBIV), OSGIV and infectious spleen and kidney necrosis virus (ISKNV). Among these, 19 proteins had not been previously identified as virion-associated proteins in megalocytivirus. Challenge tests showed that SKIV-ZJ07 was highly virulent in mandarinfish. Infected fish displayed typical histopathological symptoms of ISKNV-infected fish and died, indicating that the mandarinfish is an ideal model for further study of megalocytivirus-host interactions, molecular mechanisms of viral infection and pathogenesis. Interestingly, large numbers of regular paracrystalline SKIV-ZJ07 virion arrays were observed in both SKIV-infected MFF-1 cells and mandarinfish tissues by transmission electron microscopy (TEM), which is unusual for megalocytivirus under artificial infection conditions. Taken together, the results presented here provide new insight into the pathology of megalocytivirus infection.

Molecular epidemiology and phylogenetic analysis of a marine fish infectious spleen and kidney necrosis virus-like (ISKNV-like) virus.

Infectious spleen and kidney necrosis virus-like (ISKNV-like) virus causes a serious systemic disease with high morbidity and mortality of freshwater and marine fishes. Based on the ISKNV putative major capsid protein (MCP), the vascular endothelial growth factor (VEGF), the mRNA capping enzyme (Capping), and the tumor necrosis factor receptor-associated protein (TNFR) genes, primers were designed and used in PCR to determine the host range of ISKNV-like viruses. From the sampling of >1600 marine fishes representing 6 orders, 25 families, and 86 species collected in the South China Sea, 13 cultured fish species (141 fish) and 39 wild fish species (102 fish) were confirmed hosts of ISKNV-like viruses. The average percentage of infection of ISKNV-like viruses was 14.6%. The results from phylogenetic analysis of these genes revealed that ISKNV-like viruses could be placed into two clusters: cluster I was more related to ISKNV; cluster II included OSGIV (orange-spotted grouper iridovirus) and RBIV (rock bream iridovirus), and was quite different from ISKNV. The results of this study can contribute to the prediction and prevention of ISKNV disease outbreaks.

Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread.

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.

Comparison of European systemic piscine and amphibian iridoviruses with epizootic haematopoietic necrosis virus and frog virus 3.

Iridovirus-like agents isolated from systemic infected fish (Silurus glanis, SFIR; Ictalurus melas, CFIR I, CFIR II, CFIR III) and from frogs (Rana esculenta, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Perca fluviatilis), and Frog Virus 3 (FV 3) isolated from frogs (Rana pipiens) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. All virus isolates proved to be similar in morphology and in size and reacted with EHNV polyclonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates cleaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3. Southern-blot analysis of viral BamH I digested DNA using an EHNV DNA probe revealed cross-hybridization with DNA of the investigated iridoviruses. Using a set of primers designed for an open reading frame of the EHNV genome, PCR products of about 250 bp were obtained with the DNA of systemic piscine and amphibian iridoviruses. The data suggest that the systemic piscine and amphibian iridoviruses should be regarded as members of the the genus Ranavirus within the family Iridoviridae.

Molecular biology of fish viruses: a review.

The goal of this review is to present some of the recent molecular aspects in the fish virus studies. Although more than 50 different fish virus have been isolated and tissue-culture adapted, very few of them have been molecularly cloned and sequenced. Five virus families have been mostly studied: Birnaviridae, the prototype being the infectious pancreatic necrosis virus (IPNV), and the channel catfish virus (CCV) belonging to the Herpesviridae family. In the Iridoviridae family, the fish lymphocystis disease virus (FLDV) is the most studied. Retroviridae have been recently isolated and studied. The last family is the Rhabdoviridae, in which infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) have been extensively studied.

Proposals for a new classification of iridescent viruses.

The need for comparative studies of iridoviruses to elucidate the relationships between them has been well appreciated. Sixteen iridoviruses, including type species from each of the four recognized genera of the Iridoviridae, were compared by restriction endonuclease characterization, hybridization to the major structural protein (MSP) gene of an invertebrate iridescent virus (IV) isolate at various stringencies, PCR amplification of the MSP gene region and by dot-blot hybridization studies. The results broadly supported previous serological studies. The vertebrate iridoviruses, frog virus 3 (genus Ranavirus) and flounder lymphocystivirus (genus Lymphocystivirus), appeared distinct from one another and from the invertebrate isolates. Naming and numbering invertebrate IV isolates according to history and host is no longer useful since IVs infect a number of species. A revised system, involving names based on the geographical origin of the isolate is proposed, in line with other virus families. The large IVs of invertebrates represented by Vero Beach IV (previously IV3 or mosquito IV; genus Chloriridovirus) showed little similarity to any other IVs. Members of the genus Iridovirus, the small invertebrate IVs, fell into three distinct groups of interrelated isolates. The largest group, containing the Plowden (IV1), Tia (IV2), Nelson (IV9, IV10 and IV18), Aberystwyth (IV22), Srinagar (IV24), Fort Collins (IV29) and Stoneville (IV30) iridoviruses, is named the Polyiridovirus complex. The Plowden iridovirus (IV1) is suggested as type species for this complex given the data available on its molecular biology. Based on previously published data, Timaru (IV16 and IV19) and Uitenhage (IV23) iridoviruses are also assigned to this complex. The second but smaller group is named the Oligoiridovirus complex, which includes Dazaifu (IV6) as the type species and contains Ntondwe (IV21 and IV28) on a tentative basis. Riverside IV (IV31) was distinct from both of the other groups, and is proposed as a third complex, Crustaceoiridovirus.

Amphibian and piscine iridoviruses proposal for nomenclature and taxonomy based on molecular and biological properties.

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.