Novel Aquareovirus isolated from channel catfish (Ictalurus punctatus) used in mussel restoration efforts in Wisconsin.

Channel catfish (Ictalurus punctatus) are a food fish extensively reared in aquaculture facilities throughout the world and are also among the most abundant wild catfish species in North America, making them a popular target of anglers. Furthermore, channel catfish are important members of aquatic ecosystems; for example, they serve as a glochidial host for the endangered winged mapleleaf mussel (Quadrula fragosa), making them critical for conserving this species through hatchery-based restoration efforts. During a routine health inspection, a novel aquareovirus was isolated from channel catfish used in mussel propagation efforts at a fish hatchery in Wisconsin. This virus was isolated on brown bullhead cells (ATCC CCL-59) and identified through metagenomic sequencing as a novel member of the family Spinareoviridae, genus Aquareovirus. The virus genome consists of 11 segments, as is typical of the aquareoviruses, with phylogenetic relationships based on RNA-dependent RNA polymerase and major outer capsid protein amino acid sequences showing it to be most closely related to golden shiner virus (aquareovirus C) and aquareovirus C/American grass carp reovirus (aquareovirus G) respectively. The potential of the new virus, which we name genictpun virus 1 (GNIPV-1), to cause disease in channel catfish or other species remains unknown.

Novel picornavirus (family Picornaviridae) from freshwater fishes (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) in Hungary.

In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fish species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete genome of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5'UTRIRES-?/P1(VP0-VP3-VP1)/P2(2A1NPG↓P-2A2H-box/NC-2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3'UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fish-origin species in an unassigned genus in the family Picornaviridae.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

Edwardsiella ictaluri type III secretion system (T3SS) effector EseN is a phosphothreonine lyase that inactivates ERK1/2.

EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vivo in head kidney derived macrophages. Interaction of EseN with phosphorylated ERK1/2 (pERK1/2) was also confirmed using PLA, suggesting that MVP serves as a signaling scaffold for ERK1/2 and EseN. Channel catfish Ictalurus punctatus infected with E. ictaluri strains lacking the eseN gene had reduced numbers of E. ictaluri in the tissues following infection and reduced mortality compared to fish infected with the wild-type. Our results indicate that eseN encodes a PTL domain that interacts with MVP as a possible scaffold protein and inactivates pERK1/2 to ERK1/2, resulting in increased proliferation of E. ictaluri and, ultimately, death of the host.

Characterization and application of monoclonal antibodies against channel catfish virus (CCV).

Three monoclonal antibodies (MAbs) (27E4, 17H2, 8B6) against channel catfish virus (CCV) were developed by immunizing Balb/C mice. Using indirect ELISA, these MAbs reacted only with CCV and not with three other fish viruses or nine fish cell lines. During western blotting analysis, MAb 27E4 recognized 170 kDa and 47 kDa proteins, while MAb 17H2 and MAb 8B6 recognized 47 kDa and 56 kDa proteins, respectively. Furthermore, a sandwich ELISA was developed for detection of CCV. The detection limit of the test was 105 TCID50/mL.

Complete genome sequence and analysis of ictalurid herpesvirus 2.

Ictalurid herpesvirus 2 (IcHV-2) has been causing substantial losses in the black bullhead aquaculture industry since the 1990s. Using next-generation sequencing, the genome of IcHV-2 was completely sequenced and analysed in this study. The complete genome was found to be 142,925 bp in size, containing 77 predicted protein-coding regions, including 12 ORFs that appear to have a homologue in every alloherpesvirus genome sequenced to date. The genome organization of the IcHV-2 shows high similarity to that of IcHV-1, the founding member of the genus Ictalurivirus within the family Alloherpesviridae. A unique sequence region of 101 kbp is flanked by terminal direct repeats of 20 kbp. Thirteen of the 77 putative genes do not show homology to any known genes with sequences in public databases; six of them are found in the repeat regions. Analysis of the whole genome confirms the previously established taxonomic position of IcHV-2.

Whole genome sequencing and phylogenetic characterization of brown bullhead (Ameiurus nebulosus) origin ranavirus strains from independent disease outbreaks.

Ranaviruses are emerging pathogens associated with high mortality diseases in fish, amphibians and reptiles. Here we describe the whole genome sequence of two ranavirus isolates from brown bullhead (Ameiurus nebulosus) specimens collected in 2012 at two different locations in Hungary during independent mass mortality events. The two Hungarian isolates were highly similar to each other at the genome sequence level (99.9% nucleotide identity) and to a European sheatfish (Silurus glanis) origin ranavirus (ESV, 99.7%-99.9% nucleotide identity). The coding potential of the genomes of both Hungarian isolates, with 136 putative proteins, were shared with that of the ESV. The core genes commonly used in phylogenetic analysis of ranaviruses were not useful to differentiate the two brown bullhead ESV strains. However genome-wide distribution of point mutations and structural variations observed mainly in the non-coding regions of the genome suggested that the ranavirus disease outbreaks in Hungary were caused by different virus strains. At this moment, due to limited whole genome sequence data of ESV it is unclear whether these genomic changes are useful in molecular epidemiological monitoring of ranavirus disease outbreaks. Therefore, complete genome sequencing of further isolates will be needed to identify adequate genetic markers, if any, and demonstrate their utility in disease control and prevention.

Class II, major histocompatibility complex, transactivator (CIITA) in channel catfish: identification and expression patterns responding to different pathogens.

NLRs are a large family belonging to pattern recognition receptors which could recognize pathogen associated molecular patterns. Class II, major histocompatibility complex, transactivator (CIITA) is a member of NLR family. It is a critical transcription factor which could regulate the expression of MHC class II. In this study, a full-length cDNA of CIITA was cloned from channel catfish according to ten sequenced ESTs. This cDNA contains a 5'-UTR of 71 bp, a 3'-UTR of 238 bp and an ORF of 3,210 bp encoding 1,069 amino acids. Phylogenetic analysis showed that catfish CIITA was conserved with other CIITAs. Quantitative real-time PCR was conducted to detect the expression profiles of CIITA in normal tissues and responding to different pathogens (Edwardsiella tarda, Streptococcus iniae and channel catfish Hemorrhage Reovirus (CCRV)). The expression profile in blood was the highest (53.879-fold) in normal tissues. E. tarda and S. iniae could induce catfish CIITA in head kidney, liver and spleen. CCRV virus could also induce CIITA in head kidney and liver but reduce it in spleen. And S. iniae could induce the expression of CIITA to the highest extent and contrarily CCRV virus to the lowest extent. The expression data showed the tissue-specific and pathogen-specific expression patterns of CIITA responding to different pathogens. These expression data indicated the immune-related functions of CIITA. The data obtained in this study provide a basis for further research aimed at explore the precise immune-related molecular mechanism of CIITA in catfish.

Generation and characterization of monoclonal antibodies against channel catfish virus.

Three monoclonal antibodies (MAbs) against channel catfish virus (CCV) were generated from mice immunized with purified CCV. Western blot analysis revealed that the MAb 3G12 reacted with three CCV proteins of 94 kDa, 130 kDa, and 170 kDa; the MAb 4C4 reacted with two CCV proteins of 130 kDa and 170 kDa; and the MAb 4D4 reacted with two CCV proteins of 94 kDa and 98 kDa. Indirect immunofluorescence assay showed intense fluorescence in the CCV-infected channel catfish ovary (CCO) cells in areas corresponding to the location of granular structures. In addition, the three MAbs could completely neutralize CCV at a dilution of 1:500. This study demonstrated that these MAbs could recognize CCV specifically and will be useful in the development of diagnostic methods for the detection of fish CCV infection.

Microfibrillar-associated protein 4 (MFAP4) genes in catfish play a novel role in innate immune responses.

The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and direct pathogen killing. While MBLs have been reported in many fish species, ficolins do not appear to be present in the teleost lineage, despite their importance in invertebrate and higher vertebrate innate immunity. A protein with a similar fibrinogen-like domain, microfibrillar-associated protein 4, MFAP4, is present in fish, albeit with no described immune function. We examined whether MFAP4 genes in fish may potentially act as pathogen receptors in the absence of ficolin. We isolated and characterized five MFAP4 genes from channel catfish. Linkage mapping and phylogenetic analysis indicated that at least three of the catfish MFAP4 genes are tightly clustered on a single chromosome, suggesting that they may have arisen through tandem duplication. Divergent, duplicated families of MFAP4 genes are also present in other teleost species. Expression analysis of the catfish MFAP4 transcripts revealed unique patterns of homeostatic expression among the genes in gill, spleen, skin, liver, and muscle. Expression of the five MFAP4 transcripts showed significant changes in expression as soon as 4h after infection with either Edwardsiella ictaluri or Flavobacterium columnare with modulation of expression continuing up to 7 d following pathogen exposure. Several different tissues and gene-specific patterns were captured and transcript expression changes of >30-fold were observed over the course of the bacterial challenges. Our results suggest a novel role for MFAP4 in teleost immune responses.

Inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides.

The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene.

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.

Channel catfish virus gene expression in experimentally infected channel catfish, Ictalurus punctatus (Rafinesque).

Channel catfish virus (CCV) produces an acute haemorrhagic disease in fingerling channel catfish and establishes latent infection in fish that survive the primary infection. This study investigated CCV gene expression in tissues of experimentally infected fish. Reverse transcriptase polymerase chain reaction assays were developed for detection of transcripts expressed by each of the CCV direct repeat region genes in CCV-infected channel catfish ovary cells and in tissues of infected fish. Immediate-early, early and late gene transcripts were detected in the blood, brain, kidney and liver tissues of acutely infected catfish demonstrating active viral replication in multiple tissues during the early stages of CCV infection. However, there was no evidence for viral replication by 24 days post-infection in tissues of fish that survived the acute disease. Viral latency-associated transcripts encoded by CCV direct repeat genes were not detected in latently infected catfish. The results of this study provide a foundation for further studies to investigate the molecular basis of CCV pathogenesis and latency.

Systemic herpes-like virus in catfish Ictalurus melas (Italy) differs from Ictalurid herpesvirus 1 (North America).

A herpesvirus was isolated during 2 occurrences of mass mortality among adult catfish Ictalurus melas raised in different farms in northern Italy. The agent replicated in the channel catfish ovary (CCO) cell line from channel catfish I. punctatus, inducing a cytopathic effect similar to that caused by Ictalurid herpesvirus 1 (also referred to as channel catfish herpesvirus, CCV). The new herpesvirus, designated I. melas herpesvirus (IcmHV) did not react with polyclonal rabbit or monoclonal antibodies directed to CCV in either neutralization or indirect immunofluorescence assays. The virions of IcmHV possessed a hexagonal nucleocapsid of 107 nm in diameter surrounded by an envelope with a diameter of 227 nm (n = 20) typical for members of the family Herpesviridae. Virions of IcmHV purified from infected CCO cells contained 17 polypeptides ranging in size from 17.5 to 175 kDa and most differed in molecular weight from those found for CCV. The IcmHV was also distinct from CCV when compared by restriction fragment length polymorphisms (RFLP) of genomic DNA following digestions with the endonucleases Kpn I and Sac I. Lastly, the virulence of IcmHV for channel catfish fry and juveniles, respectively, was demonstrated by experimental infections induced by bath exposure or intraperitoneal injection that resulted in 78 to 96% cumulative mortality in groups of exposed fish. Preventing the introduction of this agent into geographic regions where significant channel catfish production occurs should be a high priority.

Influence of methisoprinol on the replication of rhabdoviruses isolated from carp (Cyprinus carpio) and catfish (Ictalurus melas): in vitro study.

Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.

Protective immunity induced by DNA vaccination of channel catfish with early and late transcripts of the channel catfish herpesvirus (IHV-1).

Seven full-length transcripts encoding four early and three late genes of the channel catfish virus (CCV), ictalurid herpesvirus I (IHV-1), have been cloned following rt-PCR amplification and DNA sequencing. Transcripts were selected based on their predicted association with membrane structures, identification as an envelope glycoprotein, or as a viral capsid protein. The transcripts derived from ORF 6, ORF 7, ORF 8a, ORF 10, ORF 51, ORF 53, and ORF 59 were all shown to be complete and unspliced. Each of the seven ORFs was cloned into a vaccine expression vector designed to support high levels of expression of the inserted sequence in catfish tissues. Solutions of DNA containing one each of the seven CCV ORFs, vector alone or PBS were injected intramuscularly into 4-8 cm catfish. Four to 6 weeks after injection each experimental group was challenged with one LD(50) of CCV. Single injections of DNA expression constructs containing ORF 59, encoding the envelope glycoprotein, or ORF 6, encoding a presumptive membrane protein, were found to elicit the strongest resistance to challenge compared to uninjected, PBS injected or vector injected groups. Even more effective was a combination vaccine pair in which both ORF 59 and ORF 6 expression constructs were injected. Other ORFs did not provide consistent protection to challenge above that observed in control fish. Both percent survival and kinetics of cumulative deaths were improved using the combination DNA vaccine encoding ORF 6 and ORF 59. Both ORF 6 and ORF 59 were able to elicit virus neutralizing antibodies capable of an anamnestic response on viral challenge. We believe this evidence provides adequate proof of principle for the use of DNA vaccines in channel catfish and the effectiveness of the resistance to viral infection they elicit.

Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin.

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

Transcriptional regulation of the channel catfish virus genome direct repeat region.

Channel catfish virus (CCV), a member of the herpesvirus family, causes a severe haemorrhagic disease in juvenile channel catfish. In this report, we confirm that CCV gene expression is temporally regulated into immediate-early (IE), early and late phases, similar to that of other herpesviruses. The transcriptional regulation of the 14 genes within the direct repeat region of the CCV genome was determined by Northern hybridization analysis of RNA isolated from infected cells in the presence or absence of metabolic inhibitors. Two CCV genes within the direct repeat, ORFs 1 and 3, expressed IE transcripts. Early RNAs were encoded by ORFs 2-9 and 11-14. ORFs 4, 7 and 10-13 expressed late transcripts after the onset of viral DNA replication. A time-course study conducted without metabolic inhibitors confirmed that CCV direct repeat transcription is temporally regulated. The characterization of CCV transcription during cytolytic infection in vitro will provide a foundation for the analysis of CCV gene expression in tissues of acutely and latently infected catfish.