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At least 55 records · Page 3Linked to original sources

Structural proteins of two salmonid rhabdoviruses

Purified infectious hematopoietic necrosis (IHN) virus and the virus of haemorrhagic septicaemia (VHS) (Egtved virus) each contain five structural proteins which were designated L, G, N, M-1, and M-2. The IHN viral polypeptides have molecular weights estimated to be 157,000, 72,000, 40,000, 25,000 and 20,000, respectively, whereas those of VHS viral polypeptides are estimated to be 157,000 74,000, 41,000, 21,500, and 19,000, respectively. The carbohydrate composition of the glycoprotein (G) was confirmed by demonstrating selective incorporation of [3H]glucosamine into the designated G protein of both viruses. Phosphoproteins were identified by incorporation of [32P]orthophosphate into the N and M-1 proteins of IHN virus and into the N protein of VHS virus. The glycoprotein of each virus was selectively solubilized by treatment with Triton X-100 in low salt buffer, whereas the M-1, and M-2 proteins along with the G protein were solubilized by Ttition X-100 in 0.43 M NaCl. The protein composition of the salmonid rhabdoviruses resembles that of the rabies virus group more closely than the vesicular stomatitis virus group.

Journal of Virology

Differential inhibition of host protein synthesis in L cells infected with RNA - temperature-sensitive mutants of vesicular stomatitis virus

The response of mouse L cells to infection with wild-type (wt) and temperature-sensitive (ts) mutants of vesicular stomatitis virus was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to delineate the synthesis of host cell and viral proteins. Experiments utilized transcriptase mutants of complementation group I (ts114 and ts13), a group IV mutant (ts44) that is restricted in total RNA synthesis (RNA-1) but not in primary transcription, and a group II mutant (ts52) variably restricted in RNA synthesis (RNA +/-). L cells infected with ts mutants at permissive temperature exhibited the wt response of progressive inhibition of host cell protein synthesis accompanied by accumulation of all five viral proteins. Mutant ts44 (IV) also switched off cell protein synthesis at restrictive temperature and accumulated all five viral proteins, but with disproportionate ratios of N and G proteins. At restrictive temperature, cells infected with group I ts mutants failed to accumulate any viral protein and did not exhibit significant reduction in host cell protein synthesis. These data suggest that vesicular stomatitis virus inhibits cell protein synthesis at a stage of viral infection after transcription and possibly translation but preceding replication of progeny viral RNA.

Journal of Virology

Comparative membrane microviscosity of fish and mammalian rhabdoviruses studied by fluorescence depolarization

The microviscosity of the hydrophobic region of the membrane of infectious hematopoietic necrosis virus was determined using fluorescence depolarization analysis of the probe 1,6-diphenyl-1,3,5-hexatriene and was found to be much lower at 37 C than that of another rhabdovirus, vesicular stomatitis virus. However, the microviscosity of this fish virus at 18 C, the temperature at which it was grown, corresponded to the microviscosity of vesicular stomatitis virus at 37 C. Data obtained with the fish virus host cell (chinook salmon embryo cells) grown at 18 C suggest that its membranes have a lower microviscosity than either L-929 or BHK-21 cells (the vesicular stomatitis virus host cells) grown at 37 C.

Journal of Virology

Ultrastructure of lymphocystis virus

Lymphocystis virus obtained from bluegills ( Lepomis macrochirus ) was cultured in the permanent bluegill cell line BF-2 and examined by electron microscopy in ultra-thin sections of cell cultures and in negative-contrast preparations from cells and from centrifuged culture medium. According to negative-contrast preparations, the icosahedral virions have an overall diameter close to but not exceeding 300 m μ . Delicate filaments seem to issue from the vertices. In collapsed virions, an ordered array of morphological units was seen. Positively contrasted virions in ultrathin sections show a shell with three dark (heavy metal-stained) layers alternating with and separated by two clear layers. The acquisition of an additional outer membrane during release from the cell, as found in African swine fever virus, was never seen. Morphologically, lymphocystis virus is considered to be closely related to Tipula iridescent virus.

Journal of Virology

Herpesvirus salmonis: Characterization of a new pathogen of rainbow trout

A new agent, provisionally designated Herpesvirus salmonis , was isolated from post-spawning rainbow trout ( Salmo gairdneri ) and studied primarily in the RTG-2 rainbow trout cell line. Infection of RTG-2 cells resulted in the formation of syncytia and Cowdry type A intranuclear inclusions. Replication occurred regularly at 5 and 10°C, but was inconsistent at 15°C, largely inhibited at 0°C, and completely inhibited at 20°C or higher. The virus was acid, heat, ether, and chloroform labile, but stable to freezing and thawing. It did not hemagglutinate. Viral DNA had a buoyant density of 1.709 g/cm 3 and a guanine-cytosine value of 50%. Hexagonal nucleocapsids had a diameter of 90 nm and were first seen in nuclei at 36 h. Enveloped forms measured about 150 nm and occurred both cytoplasmically and extracellularly. At 10°C, a one-step growth culture required about 96 h; cell-associated virus peaked at about 10 5 PFU/ml and exceeded released virus by a factor of about 10.

Journal of Virology

Characterization of EV-2, a virus isolated from European eels (Anguilla anguilla) with stomatopapilloma

A virus designated EV-2 has been isolated from external tumor tissue and internal organs of European eels ( Anguilla anguilla ) with stomatopapilloma. It contains RNA and is ether, acid, and temperature labile above 4°C, and concentrated preparations agglutinate chicken and sheep erythrocytes. The addition of actinomycin D during the first 2.75 h of infection inhibits viral replication. As determined in sucrose gradients, the buoyant density of the virus is 1.19 g/cm 3 . EV-2 has a moderately pleomorphic spherical morphology; its diameter ranges from 80 to 140 nm. The virion has narrow, regularly spaced surface projections about 10 nm long. Replication in FHM cells at 15°C shows new infectivity appearing at 10 h postinfection and reaching a plateau at 20 h. Cytopathic effects consist of cell fusion, syncytia, and irregularly rounded cell masses. Viral antigen was detected in the cytoplasm of infected cells by specific immunofluorescence.

Journal of Virology

Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an acute disease in salmon and trout. In this study, a correlation between changes in tissue tropism and specific changes in the virus genome appeared to be made by examining four IHNV neutralization-resistant variants (RB-1, RB-2, RB-3, and RB-4) that had been selected with the glycoprotein (G)-specific monoclonal antibody RB/B5. These variants were compared with the parental strain (RB-76) for their virulence and pathogenicity in rainbow trout after waterborne challenge. Variants RB-2, RB-3, and RB-4 were only slightly attenuated and showed distributions of viral antigen in the livers and hematopoietic tissues of infected fish similar to those of the parental strain. Variant RB-1, however, was highly attenuated and the tissue distribution of viral antigen in RB-1-infected fish was markedly different, with more viral antigen in brain tissue. The sequences of the G genes of all four variants and RB-76 were determined. No significant changes were found for the slightly attenuated variants, but RB-1 G had two changes at amino acids 78 and 218 that dramatically altered its predicted secondary structure. These changes are thought to be responsible for the altered tissue tropism of the virus. Thus, IHNV G, like that of rabies virus and vesicular stomatitis virus, plays an integral part in the pathogenesis of viral infection.

Journal of Virology

Genomic variation of the fibropapilloma-associated marine turtle herpes virus across seven geographic areas and three host species

Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.

Hawai'i

Channel catfish virus: A new herpesvirus of ictalurid fish

Channel catfish virus was studied in ictalurid fish cell culture, the only system of fish, amphibian, avian, and mammalian cells found to be susceptible. Channel catfish virus infection resulted in intranuclear inclusions and extensive syncytium formation. Replication occurred from 10 to 33 C, but not higher. Best growth was from 25 to 33 C, and the amount of virus released nearly equalled the amount which remained cell-associated. The virus was labile to lipid solvents, and indirect determinations with labeled precursors and a metabolic inhibitor showed evidence of deoxyribonucleic acid. Electron microscopy showed progeny virus, about 100 nm in diameter, in various stages of development in cell nuclei by 4 hr. Present also were nuclear masses of exceptionally electron-dense lamellar material, with a unit dimension of 10 to 15 nm. Virus was enveloped at the nuclear membrane and in cytoplasmic vacuoles, resulting in virions having a diameter of 175 to 200 nm. Negative staining demonstrated icosehedral symmetry and 162 capsomeres. Our data indicate that channel catfish virus is a herpesvirus.

Journal of Virology

Comparison of in vitro growth characteristics of ten isolates of infectious hematopoietic necrosis virus

Ten isolates of infectious haematopoietic necrosis from salmonid fishes of different locations on the West Coast of North America from California to Alaska were compared by plaque size, single-step growth curves at 15 and 18 °C, rate of appearance of cytopathic effects in cell cultures, and growth over a range of temperatures. All isolates were distinguishable on the basis of each growth characteristic examined. The CO isolate from the Sacramento River drainage of California was the most singular of the 10 because of its diminutive plaque size and sensitivity to slightly elevated temperatures. The mean plaque diameter of the l0 isolates increased as the latitude of the geographic source of the isolate increased. Although the maximum titre obtained by all isolates was depressed at temperatures above approximately 18 °C, half of the isolates were not inhibited by temperatures as low as 0.5 °C. t.

Journal of General Virology

Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

Journal of General Virology

Distribution and variation of NV genes in fish rhabdoviruses

The fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) contains a non-virion (NV) gene between the glycoprotein (G) and polymerase (L) genes on its RNA genome. The present study investigated three other fish rhabdovirus genomes and found that the NV gene of hirame rhabdovirus is closely related to the NV of IHNV, whereas the viral haemorrhagic septicemia NV gene showed evidence of significant divergence. Most importantly, spring viraemia of carp virus, the only vesiculovirus-like fish rhabdovirus examined, did not have an NV gene at its genomic RNA G-L junction. These results suggest that the presence of an NV gene is characteristic of the unassigned fish rhabdovirus subgroup previously classified as lyssaviruses, and that the NV gene is not essential for replication in fish cells per se, since it is absent in a vesiculovirus-like fish rhabdovirus.

Journal of General Virology

Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral hemorrhagic septicemia virus, a fish rhabdovirus

To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245-300) accumulated substitutions and appeared to be highly variable. The genetic heterogeneity within a single isolate was high, with an apparent internal mutation frequency of 1.2 x 10(-3) per nucleotide site, attesting the quasispecies nature of the viral population. The phylogeny separated VHSV strains according to the major geographical area of isolation: genotype I for continental Europe, genotype II for the British Isles, and genotype III for North America. Isolates from continental Europe exhibited the highest genetic variability, with sub-groups correlated partially with the serological classification. Neither neutralizing polyclonal sera, nor monoclonal antibodies, were able to discriminate between the genotypes. The overall structure of the phylogenetic tree suggests that VHSV genetic diversity and evolution fit within the model of random change and positive selection operating on quasispecies.

Journal of General Virology

Genetic analyses reveal unusually high diversity of infectious haematopoietic necrosis virus in rainbow trout aquaculture

Infectious haematopoietic necrosis virus (IHNV) is the most significant virus pathogen of salmon and trout in North America. Previous studies have shown relatively low genetic diversity of IHNV within large geographical regions. In this study, the genetic heterogeneity of 84 IHNV isolates sampled from rainbow trout ( Oncorhynchus mykiss ) over a 20 year period at four aquaculture facilities within a 12 mile stretch of the Snake River in Idaho, USA was investigated. The virus isolates were characterized using an RNase protection assay (RPA) and nucleotide sequence analyses. Among the 84 isolates analysed, 46 RPA haplotypes were found and analyses revealed a high level of genetic heterogeneity relative to that detected in other regions. Sequence analyses revealed up to 7·6% nucleotide divergence, which is the highest level of diversity reported for IHNV to date. Phylogenetic analyses identified four distinct monophyletic clades representing four virus lineages. These lineages were distributed across facilities, and individual facilities contained multiple lineages. These results suggest that co-circulating IHNV lineages of relatively high genetic diversity are present in the IHNV populations in this rainbow trout culture study site. Three of the four lineages exhibited temporal trends consistent with rapid evolution.

Journal of General Virology

Local and systemic replicative fitness for viruses in specialist, generalist, and non-specialist interactions with salmonid hosts

Host tissues represent diverse resources or barriers for pathogen replicative fitness. We tested whether viruses in specialist, generalist, and non-specialist interactions replicate differently in local entry tissue (fin), and systemic target tissue (kidney) using infectious hematopoietic necrosis virus (IHNV) and three salmonid fish hosts. Virus tissue replication was host specific, but one feature was shared by specialists and the generalist which was uncommon in the non-specialist interactions: high host entry and replication capacity in the local tissue after contact. Moreover, specialists showed increased replication in systemic target tissues early after host contact. By comparing ancestral and derived IHNV viruses, we also characterized replication tradeoffs associated with specialist and generalist evolution. Compared with the ancestral virus, a derived specialist gained early local replicative fitness in the new host but lost replicative fitness in the ancestral host. By contrast, a derived generalist showed small replication losses relative to the ancestral virus in the ancestral host but increased early replication in the local tissue of novel hosts. This study shows that the mechanisms of specialism and generalism are host specific and that local and systemic replication can contribute differently to overall within host replicative fitness for specialist and generalist viruses.

Journal of General Virology

Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO 2 ) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrP TSE ) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO 2 suspensions degraded the PrP TSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrP TSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO 2 . Exposure to 5.6 mg MnO2 ml -1 (PrP TSE :MnO 2 =1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

Journal of General Virology

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

Hawai'i

Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response. ?? 2009 SGM.

Journal of General Virology