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At least 19 recordsLinked to original sources
USGS2013

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↗
USGS2007

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↗
USGS2015

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↗
USGS2017

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↗
USGS2017

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↗
USGS2018

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↗
USGS2019

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↗
USGS1977

Virion RNA polymerases of two salmonid rhabdoviruses

RNA-dependent RNA polymerases were found to be associated with two salmonid rhabdoviruses: infectious hematopoietic necrosis (IHN) virus and the virus of hemorrhagic septicemia (VHS). The protein composition of these rhabdoviruses closely resembles that of rabies virus rather than that of vesicular stomatitis virus (McAllister and Wagner, 1975). The optimal temperature for in vitro transcription was found to be approximately 18 degrees C for IHN virus and approximately 15 degrees for VHS, closely approximating optimal temperatures for growth of these viruses in salmonid cells. Unlike vesicular stomatitis virus, manganese ion (1 mM) could be used as a divalent cation substitute for magnesium ion (5 mM). The in vitro transcription products of IHN and VHS viruses hybridized completely to the homologous genome but not at all to the heterologous genome.

Journal of Virology↗
USGS2006

Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

Journal of Virological Methods↗
USGS2010

Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.

Journal of Virological Methods↗
USGS2014

Identification and characterization of Highlands J virus from a Mississippi sandhill crane using unbiased next-generation sequencing

Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequencing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus, NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the application of NGS for the rapid identification and characterization of a virus isolated from the brain of an endangered Mississippi sandhill crane. This bird was part of a population restoration effort and was found in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012. Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arboviral etiology was strongly suspected. Because individual molecular assays for several known arboviruses were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the causative viral agent. Whole genome sequencing and phylogenetic analysis revealed the viral isolate to be the Highlands J virus, a known avian pathogen. This study demonstrates the use of unbiased NGS for the rapid detection and characterization of an unidentified viral pathogen and the application of this technology to wildlife disease diagnostics and conservation medicine.

Journal of Virological Methods↗
USGS2017

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout ( Oncorhynchus mykiss ) and arctic char ( Salvelinus alpinus ). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Journal of Virological Methods↗
USGS2020

Field-based method for assessing duration of infectivity for influenza A viruses in the environment

Understanding influenza A virus (IAV) persistence in wetlands is limited by a paucity of field studies relating to the maintenance of infectivity over time. The duration of IAV infectivity in water has been assessed under variable laboratory conditions, but results are difficult to translate to more complex field conditions. We tested a field-based method to assess the viability of IAVs in an Alaska wetland during fall and winter which incorporated physical and chemical properties of the waterbody in which samples were held. Filtered pond water was inoculated with avian fecal samples collected from the environment, aliquoted into a series of duplicate sealed vials and submerged back in the wetland for up to 132 days (October 2018–March 2019). Sample aliquots were sequentially recovered and tested for IAVs by rRT-PCR and virus isolation. One sample remained rRT-PCR positive for the duration of the study and virus isolation positive for 118 days. The surrounding water temperature was 1°–6 °C with near neutral pH (6.6–7.3) for the duration of the study. This proof of concept study demonstrates a protocol for testing the persistence of infectious IAV naturally shed from waterfowl under ambient environmental conditions.

Alaska↗
USGS2000

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 5095 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Journal of Virological Methods↗
USGS2013

The role of virulence for in vivo superinfection fitness of a vertebrate RNA virus

We have developed a novel, in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural, vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a co-evolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 hours to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal virulence and unequal virulence genotype pairs, the frequency of superinfection was the same regardless of which genotype was used in the primary exposure. However, the ability to replicate in the context of superinfection did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection, while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV, and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural, vertebrate host.

Journal of Virology↗
USGS2019

Aerosol transmission of gull-origin Iceland subtype H10N7 influenza A virus in ferrets

Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian–origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals, and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.

Journal of Virology↗
USGS2019

The nucleoprotein and phosphoprotein are major determinants of the virulence of viral hemorrhagic septicemia virus in rainbow trout

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout ( Oncorhynchus mykiss ). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout.

Journal of Virology↗
USGS2017

In-vitro replication of Chelonid herpesvirus 5 in organotypic skin cultures from Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with Chelonid herpesvirus 5 (ChHV5) that has historically been refractory to growth in tissue culture. Here, we show for the first time de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative for active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included 1) either in-vitro culturing of ChHV5-positive tumor biopsies (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and 2) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies revealing intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegumentation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign for active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures where most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as model to culture other viruses that are resistant to replication in conventional cell culture.

Hawai'i↗