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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

Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability

Background: The role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes. Methods: We isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds. Results: We analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs. Conclusions: This process appears similar to genetic shifts seen with swine influenza where a stable "triple reassortant internal gene" core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the need for continued surveillance in gull and waterfowl populations, both on the Pacific and especially Atlantic coasts of North America, to document virus intercontinental movement and the role of gull species in the evolution and epidemiology of AI.

Virology Journal

Applying the scientific method when assessing the influence of migratory birds on the dispersal of H5N1

Background The role of wild birds in the dispersal of highly pathogenic avian influenza virus H5N1 continues to be the subject of considerable debate. However, some researchers functionally examining the same question are applying opposing null hypotheses when examining this issue. Discussion I describe the correct method for establishing a null hypothesis under the scientific method. I suggest that the correct null hypothesis is that migratory birds can disperse this virus during migration and encourage researchers to design studies to falsify this null. Finally, I provide several examples where statements made during this debate, while strictly true, are not generally informative or are speculative. Summary By adhering to the scientific method, definitive answers regarding the role of wild birds in the dispersal of highly pathogenic viruses will be reached more effectively.

Virology Journal

Wild bird surveillance for highly pathogenic avian influenza H5 in North America

It is unknown how the current Asian origin highly pathogenic avian influenza H5 viruses arrived, but these viruses are now poised to become endemic in North America. Wild birds harbor these viruses and have dispersed them at regional scales. What is unclear is how the viruses may be moving from the wild bird reservoir into poultry holdings. Active surveillance of live wild birds is likely the best way to determine the true distribution of these viruses. We also suggest that sampling be focused on regions with the greatest risk for poultry losses and attempt to define the mechanisms of transfer to enhance biosecurity. Responding to the recent outbreaks of highly pathogenic avian influenza in North America requires an efficient plan with clear objectives and potential management outcomes.

Virology Journal

Identification of two novel reassortant avian influenza a (H5N6) viruses in whooper swans in Korea, 2016

Background On November 20, 2016 two novel strains of H5N6 highly pathogenic avian influenza virus (HPAIVs) were isolated from three whooper swans ( Cygnus cygnus ) at Gangjin Bay in South Jeolla province, South Korea. Identification of HPAIVs in wild birds is significant as there is a potential risk of transmission of these viruses to poultry and humans. Results Phylogenetic analysis revealed that Gangjin H5N6 viruses classified into Asian H5 clade 2.3.4.4 lineage and were distinguishable from H5N8 and H5N1 HPAIVs previously isolated in Korea. With the exception of the polymerase acidic (PA) gene, the viruses were most closely related to A/duck/Guangdong/01.01SZSGXJK005-Y/2016 (H5N6) (98.90 ~ 99.74%). The PA genes of the two novel Gangjin H5N6 viruses were most closely related to AIV isolates previously characterized from Korea, A/hooded crane/Korea/1176/2016 (H1N1) (99.16%) and A/environment/Korea/W133/2006 (H7N7) (98.65%). The lack of more recent viruses to A/environment/Korea/W133/2006 (H7N7) indicates the need for analysis of recent wild bird AIVs isolated in Korea because they might provide further clues as to the origin of these novel reassortant H5N6 viruses. Conclusions Although research on the origins and epidemiology of these infections is ongoing, the most likely route of infection for the whooper swans was through direct or indirect contact with reassortant viruses shed by migratory wild birds in Korea. As H5N6 HPAIVs can potentially be transmitted to poultry and humans, continuous monitoring of AIVs among wild birds will help to mitigate this risk.

Virology Journal

Isolation and characterization of the fall Chinook aquareovirus

Background Salmon are paramount to the economy, ecology, and history of the Pacific Northwest. Viruses constitute one of the major threats to salmon health and well-being, with more than twenty known virus species that infect salmon. Here, we describe the isolation and characterization of the fall Chinook aquareovirus, a divergent member of the species Aquareovirus B within the family Reoviridae . Methods The virus was first found in 2014 as part of a routine adult broodstock screening program in which kidney and spleen tissue samples from healthy-appearing, adult fall Chinook salmon ( Oncorhynchus tshawytscha ) returning to a hatchery in Washington State produced cytopathic effects when inoculated onto a Chinook salmon embryo cell line (CHSE-214). The virus was not able to be confirmed by an RT-PCR assay using existing aquareovirus pan-species primers, and instead was identified by metagenomic next-generation sequencing. Metagenomic next-generation sequencing was used to recover the full genome and completed using 3′ RACE. Results The genome of the fall Chinook aquareovirus contains 11 segments of double-stranded RNA totaling 23.3 kb, with each segment flanked by the canonical sequence termini found in the aquareoviruses. Sequence comparisons and a phylogenetic analysis revealed a nucleotide identity of 63.2% in the VP7 gene with the Green River Chinook virus, placing the new isolate in the species Aquareovirus B . A qRT-PCR assay was developed targeting the VP2, which showed rapid growth of the isolate during the initial 5 days in culture using CHSE-214 cells. Conclusions This sequence represents the first complete genome of an Aquareovirus B species. Future studies will be required to understand the potential pathogenicity and epidemiology of the fall Chinook aquareovirus.

Virology Journal

Avian keratin disorder of Alaska black-capped chickadees is associated with Poecivirus infection

Background Avian keratin disorder (AKD) is an epizootic of debilitating beak deformities, first documented in black-capped chickadees ( Poecile atricapillus ) in Alaska during the late 1990s. Similar deformities have now been recorded in dozens of species of birds across multiple continents. Despite this, the etiology of AKD has remained elusive, making it difficult to assess the impacts of this disease on wild populations. We previously identified an association between infection with a novel picornavirus, Poecivirus, and AKD in a small cohort of black-capped chickadees. Methods To test if the association between Poecivirus and AKD holds in a larger study population, we used targeted PCR followed by Sanger sequencing to screen 124 symptomatic and asymptomatic black-capped chickadees for Poecivirus infection. We further compared the efficacy of multiple non-terminal field sampling methods (buccal swabs, cloacal swabs, fecal samples, and blood samples) for Poecivirus screening. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to determine if virus is actively replicating in beak tissue. Results Poecivirus was detected in 28/28 (100%) individuals with AKD, but only 9/96 (9.4%) asymptomatic individuals with apparently normal beaks (p < 0.0001). We found that cloacal swabs are the most sensitive of these sample types for detecting Poecivirus in birds with AKD, but that buccal swabs should be combined with cloacal swabs in evaluating the infection status of asymptomatic birds. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to provide evidence of active viral replication. Conclusion The data presented here show a strong, statistically significant relationship between Poecivirus infection and AKD, and provide evidence that Poecivirus is indeed an avian virus, infecting and actively replicating in beak tissue of AKD-affected BCCH. Taken together, these data corroborate and extend the evidence for a potential causal association between Poecivirus and AKD in the black-capped chickadee. Poecivirus continues to warrant further investigation as a candidate agent of AKD.

Alaska

The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence).

Virology Journal

Surveillance theory applied to virus detection: a case for targeted discovery

Virus detection and mathematical modeling have gone through rapid developments in the past decade. Both offer new insights into the epidemiology of infectious disease and characterization of future risk; however, modeling has not yet been applied to designing the best surveillance strategies for viral and pathogen discovery. We review recent developments and propose methods to integrate viral and pathogen discovery and mathematical modeling through optimal surveillance theory, arguing for a more targeted approach to novel virus detection guided by the principles of adaptive management and structured decision-making.

Future Virology