Global landscape of structural proteins of infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.     

Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV), belonging to the genus Megalocytivirus in the family Iridoviridae, is one of the major agents causing mortality and economic losses to the freshwater fish culture industry in Asian countries. Currently, little information regarding the antigenic properties of Megalocytivirus (especially ISKNV) is available. Our previous study using four different workflows with systematic and comprehensive proteomic approaches led to the identification of 38 ISKNV virion-associated proteins (J. Virol. 2869-2877, 2011). Thus, in this report, the antigenicity of 31 structural proteins from ISKNV virion was investigated. A one-dimensional gel electrophoresis immunoblot profile coupled with MALDI-TOF-TOF MS/MS was applied to identify six immunogenic viral proteins, namely, ORFs major capsid protein (006L), 054L, 055L, 101L, 117L, and 125L. Then, the antigenicity of 31 structural proteins was characterized by Western blot by using pooled sera from mandarin fish that survived ISKNV infection. Of the 31 viral proteins, 22 were recognized by the fish ISKNV antiserum. Furthermore, this antiserum neutralizes MFF-1 cells ISKNV infection. To our knowledge, this study is the first report on the immunogenicity of viral proteins and characterization of the proteome of megalocytivirus infective agents. Our findings are expected to promote the development of effective vaccine candidates.     

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.     

Formation and structure of infectious DNA of spleen necrosis virus.

The kinetics of formation and the structure of infectious DNA of spleen necrosis virus were determined. Nonintegrated infectious viral DNA first appeared 18 to 24 h after infection of dividing cells and persisted for more than 14 days. The nonintegrated infectious viral DNA was in the form of either a double-stranded linear DNA with a molecular weight of 6 X 10(6), detected in both the cytoplasm and nucleus, or a closed circular DNA of the same molecular weight, detected primarily in the nucleus. Integrated infectious viral DNA appeared soon after the nonintegrated infectious viral DNA and was the predominant form of infectious viral DNA late after infection. Integration of the spleen necrosis virus DNA into the chicken cell genome was demonstrated by three independent criteria. Nucleic acid hybridization indicated that the linear infectious viral DNA had a 5- to 10-fold higher specific infectivity than either the closed circular or integrated infectious viral DNA. Infectious viral DNA did not appear in infected stationary cells, indicating some cellular influence on the formation of infectious viral DNA.     

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.     

Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA.

Spleen necrosis virus productively infects avian and rat cells. The average number of molecules of unintegrated and integrated viral DNA in cells at different times after infection was determined by hybridization and transfection assays. Shortly after infection, there was a transient accumulation of an average of about 150 to 200 molecules of unintegrated linear spleen necrosis virus DNA per chicken, turkey, or pheasant cell. No such accumulation was seen in infected rat cells. Soon after infection there was in chicken cells, but not inturkey, pheasant, or rat cells, also a transient integration of an average of 35 copies of viral DNA per cell. By 10 days after infection, the majority of this integrated viral DNA was lost from the population of infected chicken cells. At the same time, the majority of the unintegrated viral DNA was also lost from infected chicken, turkey, and pheasant cells. The transient cytopathic effect seen in these infected cells also occurred at this time. Late after infection about five copies of apparently nondefective spleen necrosis proviruses were stably integrated at multiple sites in chicken, turkey, pheasant, and rat DNA. These results demonstrate a correlation between the transient accumulation of large numbers of spleen necrosis virus DNA molecules and the transient occurrence of cytopathic effects.     

The viral TRAF protein (ORF111L) from infectious spleen and kidney necrosis virus interacts with TRADD and induces caspase 8-mediated apoptosis

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus of the Iridoviridae family. It causes a serious and potentially pandemic disease in wild and cultured fishes. ISKNV infection induces evident apoptosis in mandarin fish (Siniperca chuatsi) and zebrafish (Danio renio). However, the mechanism is still unknown. After a genome-wide bioinformatics analysis of ISKNV-encoded proteins, the ISKNV open reading frame 111L (ORF111L) shows a high similarity to the tumour necrosis factor receptor-associated factor (TRAF) encoded by fish, mice and mammals, which is essential for apoptotic signal transduction. Moreover, ORF111L was verified to directly interact with the zebrafish TNF receptor type 1 associated death domain protein (TRADD). A recombinant plasmid containing the DNA sequence of ORF111L was constructed and microinjected into zebrafish embryos at the 1-2 cell stage to investigate its biological function in vivo. ORF111L overexpression in the embryos resulted in increased apoptosis. ORF111L-induced apoptosis was clearly associated with significant caspase 8 upregulation and activation. The knockdown of zebrafish caspase 8 expression effectively blocked the apoptosis induced by ORF111L overexpression. Significantly, ORF111L overexpression resulted in much stronger effect on caspase 8 and caspase 3 upregulation compared to zebrafish TRAF2. This is the first report of a viral protein similar to TRAF that interacts with TRADD and induces caspase 8-mediated apoptosis, which may provide novel insights into the pathogenesis of ISKNV infection.     

Propagule pressure and the invasion risks of non-native freshwater fishes: a case study in England

European countries in general, and England in particular, have a long history of introducing non-native fish species, but there exist no detailed studies of the introduction pathways and propagules pressure for any European country. Using the nine regions of England as a preliminary case study, the potential relationship between the occurrence in the wild of non-native freshwater fishes (from a recent audit of non-native species) and the intensity ( i.e. propagule pressure) and diversity of fish imports was investigated. The main pathways of introduction were via imports of fishes for ornamental use ( e.g. aquaria and garden ponds) and sport fishing, with no reported or suspected cases of ballast water or hull fouling introductions. The recorded occurrence of non-native fishes in the wild was found to be related to the time (number of years) since the decade of introduction. A shift in the establishment rate, however, was observed in the 1970s after which the ratio of established-to-introduced species declined. The number of established non-native fish species observed in the wild was found to increase significantly ( P < 0·05) with increasing import intensity (log₁₀ x + 1 of the numbers of fish imported for the years 2000–2004) and with increasing consignment diversity (log₁₀ x + 1 of the numbers of consignment types imported for the years 2000–2004). The implications for policy and management are discussed.     

Ribonucleotides in unintegrated linear spleen necrosis virus DNA.

The structure of unintegrated spleen necrosis virus DNA was characterized by using various chemical and enzymatic treatments in conjunction with denaturing gels and nucleic acid hybridization probes. Throughout the course of the viral infection, the predominant species of viral DNA was that of a linear double-stranded molecule containing ribonucleotides covalently joined to the DNA. The majority of both - and + strands were continuous. The ribonucleotide linkages appeared to be relatively short, and the base composition and distribution of the ribonucleotide linkages were heterogeneous. On the average, the - strand had fewer of the ribonucleotide linkages than did the strand. Viral DNA containing ribonucleotide linkages was infectious in DNA transfection assays. The structure of spleen necrosis virus DNA was different from that of Schmidt-Ruppin Rous sarcoma virus-D, and mixed infections demonstrated that the observed differences are a result of cis-acting functions.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

Polypeptide composition of spleen necrosis virus, a reticuloendotheliosis virus.

The polypeptide composition of virions of spleen necrosis virus, a reticuloendotheliosis virus, was determined using electrophoresis on sodium dodecyl sulfate-containing, 10 percent polyacrylamide gels. Ten polypeptides were resolved. Four of these were present in minor and somewhat variable amounts. Two proteins, gp71 and gp22, contained D-glucosamine and were located on the outer surface of the lipid envelope, as demonstrated by lactoperoxidase-catalyzed iodination and by bromelain digestion. The results suggest that two of the minor proteins, p36 and p26, were also located on the outer surface, although they lacked D-glucosamine. Treatment of the virus with 0.25 percent Nonidet P-40 and 1 percent dithiothreitol produced a subparticle with a buoyant density of approximately 1.31 g/cm-3. This particle was relatively enriched with polypeptides p77, p62, and p50 and contained small amounts of three other polypeptides.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

Infectious spleen and kidney necrosis virus ORF48R functions as a new viral vascular endothelial growth factor

Infectious spleen and kidney necrosis virus (ISKNV) causes a pandemic and serious disease in fish. Infection by ISKNV causes epidermal lesions, in which petechial hemorrhages and abdominal edema are prominent features. ISKNV ORF48R contains a domain similar to that of the platelet-derived growth factor and vascular endothelial growth factor (VEGF) families of proteins. ISKNV ORF48R showed higher similarity to the VEGFs encoded by Megalocytivirus and Parapoxvirus than to those encoded in fish and mammals. We used zebrafish as a model and constructed a recombinant plasmid containing the DNA sequence of ISKNV ORF48R to study ISKNV infection. The plasmid was microinjected into zebrafish embryos at the one-cell stage. Overexpression of the ISKNV ORF48R gene results in pericardial edema and dilation at the tail region of zebrafish embryos, suggesting that ISKNV ORF48R induces vascular permeability. ISKNV ORF48R is also able to stimulate a striking expression of flk1 in the zebrafish dorsal aorta and the axial vein. Furthermore, ISKNV ORF48R, while cooperating with zebrafish VEGF(121), can stimulate more striking expression of flk1 than can either ISKNV ORF48R or zebrafish VEGF(121) alone. However, decreased expression of FLK-1 by gene knockdown results in the disappearance of pericardial edema and dilation at the tail region of zebrafish embryos induced by overexpression of ISKNV ORF48R in the early stages of embryonic development.     

Genetic diversity and epidemiology of infectious hematopoietic necrosis virus in Alaska

Forty-two infectious hematopoietic necrosis virus (IHNV) isolates from Alaska were analyzed using the ribonuclease protection assay (RPA) and nucleotide sequencing. RPA analyses, utilizing 4 probes, N5, N3 (N gene), GF (G gene), and NV (NV gene), determined that the haplotypes of all 3 genes demonstrated a consistent spatial pattern. Virus isolates belonging to the most common haplotype groups were distributed throughout Alaska, whereas isolates in small haplotype groups were obtained from only 1 site (hatchery, lake, etc.). The temporal pattern of the GF haplotypes suggested a 'genetic acclimation' of the G gene, possibly due to positive selection on the glycoprotein. A pairwise comparison of the sequence data determined that the maximum nucleotide diversity of the isolates was 2.75% (10 mismatches) for the NV gene, and 1.99% (6 mismatches) for a 301 base pair region of the G gene, indicating that the genetic diversity of IHNV within Alaska is notably lower than in the more southern portions of the IHNV North American range. Phylogenetic analysis of representative Alaskan sequences and sequences of 12 previously characterized IHNV strains from Washington, Oregon, Idaho, California (USA) and British Columbia (Canada) distinguished the isolates into clusters that correlated with geographic origin and indicated that the Alaskan and British Columbia isolates may have a common viral ancestral lineage. Comparisons of multiple isolates from the same site provided epidemiological insights into viral transmission patterns and indicated that viral evolution, viral introduction, and genetic stasis were the mechanisms involved with IHN virus population dynamics in Alaska. The examples of genetic stasis and the overall low sequence heterogeneity of the Alaskan isolates suggested that they are evolutionarily constrained. This study establishes a baseline of genetic fingerprint patterns and sequence groups representing the genetic diversity of Alaskan IHNV isolates. This information could be used to determine the source of an IHN outbreak and to facilitate decisions in fisheries management of Alaskan salmonid stocks.