Channel Catfish Herpesvirus Research Articles

Identification of envelope protein ORF10 of channel catfish herpesvirus

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.

Experimental annotation of channel catfish virus by probabilistic proteogenomic mapping

Experimental identification of expressed proteins by proteomics constitutes the most reliable approach to identify genomic location and structure of protein-coding genes and substantially complements computational genome annotation. Channel catfish herpesvirus (CCV) is a simple comparative model for understanding herpesvirus biology and the evolution of the Herpesviridae. The canonical CCV genome has 76 predicted ORF and only 12 of these have been confirmed experimentally. We describe a modification of a statistical method, which assigns significance measures, q-values, to peptide identifications based on 2-D LC ESI MS/MS, real-decoy database searches and SEQUEST XCorr and DeltaC(n) scores. We used this approach to identify CCV proteins expressed during its replication in cell culture, to determine protein composition of mature virions and, consequently, to refine the canonical CCV genome annotation. To complement trypsin, we used partial proteinase K digestion, which yielded greater proteome coverage. At FDR <5%, for peptide identifications, we identified 25/76 previously predicted ORF using trypsin and 31/76 using proteinase K. Furthermore, we identified 17 novel protein-coding regions (7 potential ATG-initiated ORF). Most of these novel ORF encode small proteins (<100 amino acids). Directed, strand-specific reverse transcription real-time PCR confirmed RNA expression from 6/7 novel ATG-initiated ORF investigated.

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.

An Overlapping Bacterial Artificial Chromosome System That Generates Vectorless Progeny for Channel Catfish Herpesvirus

Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.

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.

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.

Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

S5 11:00 Inhibition of channel catfish herpesvirus by catfish reovirus is mediated by intra-cellular competition and induction of an anti-viral factor

S5 11:00 Inhibition of channel catfish herpesvirus by catfish reovirus is mediated by intra-cellular competition and induction of an anti-viral factor

Deletion of Thymidine Kinase Gene Attenuates Channel Catfish Herpesvirus While Maintaining Infectivity

A recombinant thymidine kinase (TK) gene deletion mutant of channel catfish herpesvirus (CCV) was constructed and compared to the parent virus for replicative ability in cell culture, for the ability to infect, replicate, and cause lethal disease in the channel catfish host, and for the ability to induce protective immunity in channel catfish fingerlings. There was no difference between parent CCV and the TK-negative mutant (CCVTK-) in vital production in cell culture. However, in immersion challenge trials, 100-fold more CCVTK- than parent CCV was required to kill similar numbers of channel catfish fingerlings. The attenuation was confirmed as a TK-associated characteristic in marker rescue; the revertant regained the pathogenic characteristic of the parent virus. When catfish were immersion challenged with equal amounts of CCVTK- or CCV, the levels of virus isolated from the posterior kidney were similar through the peak production period which occurred 4 days postinfection. Subsequently, CCVTK- levels decreased dramatically when compared to CCV levels. Exposure to CCVTK- induced protective immunity against challenge with a lethal dose of wild-type CCV. The degree of protection provided by CCVTK- was related to of dose of CCVTK- given. This study demonstrates that the genetically distant CCV retains recombination properties and TK-associated attenuation properties of herpesviruses of higher vertebrates. Additionally, the unique characteristic of CCV containing two copies of the TK gene did act prevent the use of this gene as a selectable site of homologous recombination.

Channel Catfish Herpesvirus (CCV) Encodes a Functional Thymidine Kinase Gene: Elucidation of a Point Mutation That Confers Resistance to Ara-T

The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by marker rescue analysis using a TK-deficient channel catfish ovary cell line (CCO), a TK-negative CCV mutant, and a panel of cloned CCV genomic DNA fragments. The TK-deficient cell line (CCOBr) was isolated after repeated propagation of CCO cells in increasing concentrations of 5-bromo-2′-deoxyuridine. Infection of CCOBr cells with CCV produced high levels of TK activity. The TK- virus (CCVAr) was isolated after repeated propagation in the presence of the TK-activated antiherpetic agent, 1-β-d-arabinofuranosylthymine (Ara-T). A CCV genomic DNA library was constructed into cosmid pHC 79. Marker rescue analysis mapped the mutation within a 3.1-kb fragment located internal to the 18-kb repeat ends of the CCV genome. These genomic coordinates contained a putative TK gene identified by homology to other herpesvirus TK and cellular deoxycytidine kinase genes. DNA sequencing of the mapped coordinates identified the presence of a single mutation in the CCVAr mutant virus which resulted in a stop codon at amino acid position 97. These results functionally confirm that ORF 5 identified by Davison (Virology 186, 9-14, 1992) is the TK gene and show that CCV is amenable to marker rescue and marker transfer genetic analyses extensively used for investigations of the molecular biology of other herpesviruses.

A protein kinase-related gene within the Channel Catfish herpesvirus genome

A protein kinase-related gene within the Channel Catfish herpesvirus genome

Three new continuous cell lines from marine fishes of asia

Three new continuous cell lines were established from two species of marine fishes economically important in Asia. Perch (Lateolabrax japonicus) heart (PH), perch liver (PL), and grouper (Epinephelus amblycephalus) kidney (GK) lines were established in Eagle’s minimum essential medium with 10% fetal bovine serum and have been subcultured over 120 times. The optimum growth temperature was 25°C for the PK and GK lines and 30° C for the PH line. The modal chromosome numbers for each cell line are: PH (49), PK (50), and GK (65). None of the lines was susceptible to the rhabdovirus infectious hematopoietic necrosis virus (IHNV) or channel catfish herpesvirus (CCV); however, all three cell lines were susceptible to a variety of fish birnaviruses, including infectious pancreatic necrosis virus (IPNV), the EVE eel virus, and newly isolated birnaviruses from a variety of fish and shellfish in Taiwan.

Decreased Antiviral Effect of Phosphonoacetic Acid on the Poikilothermic Herpesvirus of Channel Catfish Disease

SummaryBoth the laboratory adapted Auburn strain and a recently isolated wild-type strain of channel catfish herpesvirus (CCV) were found to be inhibited by phosphonoacetic acid (PAA) when replicated in catfish cell cultures. The inhibition of virus cytopathic effect by PAA exhibited a direct relationship between the multiplicity of infection and amount of drug required. However, in this poikilothermic system up to 20 times the amount of PAA required for inhibition of homeothermic herpesvirus systems was found necessary to inhibit CCV cytopathology.