Juvenile Wels Catfish (Silurus glanis) Display Age-Related Mortality to European Catfish Virus (ECV) under Experimental Conditions.

We have limited knowledge about the course of the European catfish virus (ECV) infection in different age groups of wels catfish (Silurus glanis). The results of this study demonstrate that an ECV strain isolated from the brown bullhead (Ameiurus nebulosus) in Hungary could cause devastating losses among juvenile wels catfish. Furthermore, the age-related mortality rate following ECV infection was investigated in three virus challenge experiments at two different virus dosages. Eight-week-old (ca. 3 g), ten-week-old (ca. 8 g), and sixteen-week-old (ca. 55 g) catfish were infected with ECV at 21°C. In the youngest age group, 96% (at a 106 TCID50/mL dosage) and 100% (at 105 TCID50/mL) mortality rates were observed, while these rates were reduced to 56% and 68% in the ten-week-old groups, respectively. The mortality was significantly higher in the virus-exposed groups than in the control ones. In the sixteen-week-old group, 23% mortality was detected at a 105 TCID50/mL concentration of ECV. Here, a significant difference was not found between the exposed and control groups. The performed experiments show that different age groups of wels catfish may have various susceptibility to ECV. These findings draw attention to the importance of the prevention of/protection against virus infections in juvenile (up to 3-month-old) wels catfish in aquaculture.

Characterization of Ictalurid herpesvirus 1 Glycoprotein ORF59 and Its Potential Role on Virus Entry into the Host Cells.

The channel catfish virus (CCV, Ictalurid herpesvirus 1) has caused sustained economic losses in the fish industry because of its strong infectivity and pathogenicity. Thus, it is necessary to determine the function of viral proteins in the CCV infection process. The present study aimed to characterize CCV glycoprotein ORF59 and explore its impact on virus infection in host cells. Firstly, its exclusive presence in the membrane fraction of the cell lysate and subcellular localization verified that CCV ORF59 is a viral membrane protein expressed at late-stage infection. A protein blocking assay using purified His6 tagged ORF59, expressed in sf9 insect cells using a baculovirus expression system, indicated a dose-dependent inhibitory effect of recombinant ORF59 protein on virus invasion. Knockdown of the ORF59 using a short hairpin (shRNA) showed that ORF59 silencing decreased the production of infectious virus particles in channel catfish ovary cells. The results of this study suggest that recombinant ORF59 protein might inhibit CCV entry into the host cells. These findings will promote future studies of the key functions of glycoprotein ORF59 during CCV infection.

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.

Complete genome sequence of a novel fish papillomavirus detected in farmed wels catfish (Silurus glanis).

A novel papillomavirus (PV) was detected in farmed wels catfish (Silurus glanis) in Hungary showing clinical signs resembling those of wels catfish herpesvirus disease. The whole genome of Silurus glanis papillomavirus 1 (SgPV1) was identified using next-generation sequencing. The 5,612-bp complete genome contains four predicted protein coding regions (E1, E2, L1, and L2), which seem to have homologues in every PV genome sequenced to date. Five complete fish PV genome sequences are available in the GenBank database. Their genomes range between 5,748 and 6,086 bp and contain the minimal PV backbone genes E1, E2, L2, and L1, unlike PVs of higher vertebrates, which have larger genomes (6.8-8.6 kbp) and additional (onco)genes. Considering the current species demarcation criteria for the family Papillomaviridae, the establishment of a novel species named "Nunpapillomavirus siluri" is proposed for the SgPV1 in a novel genus, "Nunpapillomavirus", in the subfamily Secondpapillomavirinae.

Acyclovir inhibits channel catfish virus replication and protects channel catfish ovary cells from apoptosis.

The channel catfish virus (CCV) can cause lethal hemorrhagic infection in channel catfish, resulting in significant economic losses in the fish industry. Effective drugs for the virus are still lacking. Acyclovir is known as a potent antiviral agent against human herpes viruses and some animal DNA viruses. The present study was undertaken to explore the antiviral response and mechanism of acyclovir against CCV in channel catfish ovary (CCO) cells. Acyclovir was able to significantly inhibit the expression of viral genes related to CCV viral DNA synthesis and suppress viral replication at a safe concentration. Furthermore, acyclovir blocked the cytopathic effects and apoptosis induced by CCV, thereby maintaining the normal cellular morphological structure, as shown by the protection of CCO cells from the formation of apoptotic bodies or nuclear fragmentation. Moreover, reverse transcript quantitative polymerase chain reaction (RT-qPCR) demonstrated that acyclovir suppressed the expression of caspase 3, caspase 8 and caspase 9, while there was no significant impact on the expression of the apoptosis-inhibiting gene bcl-2 in CCV-infected cells. In addition, acyclovir did not promote the expression of immune-related genes such as MyD88, Mx1, IRF3, IRF7, IFN-I, NF-kB and IL-1ß, suggesting that the antiviral activity of acyclovir to CCV infection is not achieved by facilitating the expression of immune-related genes in CCO cells. Taken together, the results from this study suggest that acyclovir could effectively regulate CCV-induced infection, and thus is a promising therapeutic agent against CCV. Our results will aid our understanding of the pharmacological mechanisms of antiviral agents.

Discovery of a novel Piscanivirus in yellow catfish (Pelteobagrus fulvidraco) in China.

A bacilliform virus was isolated from yellow catfish in China. This virus can directly adapt in cultures of EPC cells. The virus particles, which were rod-shaped approximately 120 nm long and 20 nm wide, were visible in the cytoplasm of EPC cells. The full-length genome of this virus is 26,985 nt. The genome contains four open reading frames that encode polyprotein1ab, spike glycoprotein, M protein, and N protein. There was a putative slippery sequence 14861UUUAAAC14867, which could be modeled into an RNA pseudoknot structure. The predicted amino acid sequence of pp1ab, S, M, and N genes shares 8.7%-40.2% homology with those of the two known Bafinivirus strains-WBV and FHMNV. Based on the viral morphology, genome organization, and sequence homology, this newly identified bacilliform virus appears to be Piscanivirus. To the best of our knowledge, this is the first report of Piscanivirus in yellow catfish and Piscanivirus in China.

An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish.

UNLABELLED: Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide. IMPORTANCE: Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens.

Complete genome sequence of the European sheatfish virus.

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.

Novel circovirus in European catfish (Silurus glanis).

Circular single-stranded DNA viral genomes had been identified worldwide in different species and in environmental samples. Among them, viruses belonging to the genus Circovirus of the family Circoviridae are present in birds and pigs, and recently, they were detected in barbels. The present study reports the identification of a new circovirus in fish. PCR amplification and sequencing were used to identify the novel circular DNA virus in European catfish (Silurus glanis). Full genome characterization and phylogenetic analysis showed that the virus belonged to the family Circoviridae and that it was distantly related to the previously described barbel circovirus.

Validation of a qPCR assay for the detection of Ictalurid herpesvirus-2 (IcHV-2) in fish tissues and cell culture supernatants.

Ictalurid herpesvirus-2 (IcHV-2) is a pathogen of cultured black bullhead, Ameiurus melas (Rafinesque), and has been shown to produce high mortality in experimental exposures of channel catfish, Ictalurus punctatus (Rafinesque). During acute infections, the virus grows readily in cell cultures but produces a cytopathic effect (CPE) similar to that of Ictalurid herpesvirus-1 (IcHV-1) and the channel catfish reovirus. We have developed a quantitative PCR assay that can be used to detect IcHV-2 in fish tissues and cell culture supernatants. The assay does not amplify other fish herpesviruses tested or host DNA. It is quantitative over a range of eight logs, and the limit of detection is <10 copies per reaction. In replicate assays carried out on different days, the coefficient of variability was 10%. The best organs for the detection of acute IcHV-2 infections by our assay are the spleen and kidney. This assay should be useful for the diagnosis of IcHV-2 disease, the identification of syncytial CPEs in cell cultures, and for the detection of latent infections in carrier fish.

A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny.

Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines.

Molecular confirmation of a new herpesvirus from catfish (Ameiurus melas) by testing the performance of a novel PCR method, designed to target the DNA polymerase gene of alloherpesviruses.

A PCR method with consensus degenerate primers was developed for the detection of herpesviruses (HVs) of anamnia. Compared to previously published PCRs, targeting the DNA polymerase gene of fish HVs, the size of PCR products was more than tripled. Although broad applicability of the method could not be proven, approximately 1,600-bp fragments from HVs of white sturgeon (Acipenser transmontanus) and black bullhead (Ameiurus melas) were obtained and sequenced. Phylogenetic tree reconstructions showed both HVs to be monophyletic with the single member (ictalurid HV-1) of the genus Ictalurivirus in the new family Alloherpesviridae.

Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae.

The sequences of four complete genes were analysed in order to determine the relatedness of koi herpesvirus (KHV) to three fish viruses in the family Herpesviridae: carp pox herpesvirus (Cyprinid herpesvirus 1, CyHV-1), haematopoietic necrosis herpesvirus of goldfish (Cyprinid herpesvirus 2, CyHV-2) and channel catfish virus (Ictalurid herpesvirus 1, IcHV-1). The genes were predicted to encode a helicase, an intercapsomeric triplex protein, the DNA polymerase and the major capsid protein. The results showed that KHV is related closely to CyHV-1 and CyHV-2, and that the three cyprinid viruses are related, albeit more distantly, to IcHV-1. Twelve KHV isolates from four diverse geographical areas yielded identical sequences for a region of the DNA polymerase gene. These findings, with previously published morphological and biological data, indicate that KHV should join the group of related lower-vertebrate viruses in the family Herpesviridae under the formal designation Cyprinid herpesvirus 3 (CyHV-3).

The attenuated V60 strain of channel catfish virus possesses a deletion in ORF50 coding for a potentially secreted glycoprotein.

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion (approximately 1200 bp) in ORF50 of the V60 strain. Cloning and sequencing of part of this ORF confirmed the presence of a 1164-bp deletion. It should result in a protein of 282 amino acids instead of 670. The predicted truncated protein lacks most of a threonine-rich, highly repetitive region in its central part. Since the protein encoded by ORF50 possesses a hydrophobic N-terminal leader sequence and no membrane anchor sequence, we suggest that it could be a secreted glycoprotein. This protein might be N-glycosylated (35 potential sites) and, given the repetitive arrangement of its residues (mainly threonines), also heavily O-glycosylated like the mucin-type glycoproteins. The deletion observed in ORF50 of the V60 strain implies the loss of 24 potential N-glycosylation sites and should considerably reduce the extent of O-glycosylation.