Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Molecular identification of novel and genetically diverse adenoviruses in Passeriform birds.

Knowledge about adenoviruses in birds of the order Passeriformes is very scarce. Based on molecular characterizations, only two siadenoviruses, great tit adenovirus 1 and Gouldian finch adenovirus, have been described so far occurring in great tits and Gouldian finches, respectively. Assuming a broader occurrence of adenoviruses, various passeriform birds including pet, zoo, and wild birds were examined using a broad-range PCR targeting a fragment of the adenovirus DNA polymerase gene. Adenoviruses were detected in 25 individual birds belonging to 13 species and seven zoological families (Ploceidae, Fringillidae, Estrildidae, Paridae, Sylviidae, Turdidae, Muscicapidae). The putative viruses were further characterized by sequencing the PCR products and phylogenetic analyses. DNA of adenoviruses affiliating to 3 genera including aviadenovirus, siadenovirus, and atadenovirus was found. Viruses with sequences identical or closely related to great tit adenovirus 1 and Gouldian finch adenovirus 1 were detected in a great tit and in two zebra finches, respectively. Based on polymerase amino acid sequence comparisons, the viruses found in the remaining 22 birds revealed phylogenetic distances larger than 15% to adenoviruses known so far suggesting that they may belong to at least 14 different virus species. In some bird species (great tit, zebra finch, vitelline masked weaver) varying adenovirus genera were detected. These results suggest a broad variety of adenoviruses circulating in passeriform birds.

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.

Characterization of the genomic sequence of a novel CRESS DNA virus identified in Eurasian jay (Garrulus glandarius).

Circular replication associated protein (Rep)-encoding ssDNA (CRESS DNA) viruses have diverse genomic architecture and are widely distributed in different ecosystems. In this study we characterized the complete genomic sequence of a novel circovirus-like virus, Garrulus glandarius associated circular virus-1 (GgaCV-1). The genome size (1971 nt) and other features (the nonanucleotide, rolling circle replication motif and SF3 helicase motif) are also reminiscent of circoviruses. Similar genomes with uni-directionally localized and overlapping rep and cap genes are typical of type V CRESS DNA viruses that were identified in invertebrates and environmental samples of aquatic ecosystems. GgaCV-1 showed the highest aa identity with partial rep sequences detected in bat feces (77%) and with the rep (54%) and cap (42%) of Lake Sarah-associated circular virus-23 of New Zealand freshwater mussel origin. A dietary origin for GgaCV-1 could not be excluded as the virus was detected in the cloacal swab specimen of an Eurasian jay. Further studies may help to reveal the linkage among variable organisms regarding virus transmission.

Complete coding sequence of a novel picorna-like virus in a blackbird infected with Usutu virus.

Using random high-throughput RNA sequencing, the complete coding sequence of a novel picorna-like virus (a 9,228-nt contig containing 212,202 reads) was determined from a blackbird (Turdus merula) infected with Usutu virus. This sequence shares only 36% amino acid sequence identity with its closest homolog, arivirus 1, (an unclassified member of the order Picornavirales), and shares its dicistronic genome arrangement. The new virus was therefore tentatively named "blackbird arilivirus" (ari-like virus). The nearly complete genome sequence consists of at least 9,228 nt and contains two open reading frames (ORFs) encoding the nonstructural polyprotein (2235 amino acids) and structural polyprotein (769 amino acids). Two TaqMan RT-qPCR assays specific for ORF1 confirmed the presence of high levels of this novel virus in the original sample. Nucleotide composition analysis suggests that blackbird arilivirus is of dietary (plant) origin.

Identification and genetic characterization of polyomaviruses in estrildid and fringillid finches.

Polyomavirus infections were detected in 40 companion bird individuals belonging to a broad species range of estrildid and fringillid finches and originating from 21 different bird aviaries. Based on partial virus protein 1 (VP1) sequences, the viruses were identified as Serinus canaria polyomavirus 1 and Pyrrhula pyrrhula polyomavirus 1. Serinus canaria polyomavirus 1 was found in 18 birds belonging to one estrildid and four fringillid species. Pyrrhula pyrrhula polyomavirus 1 was detected in 22 birds of six estrildid and three fringillid species. There was a large overlap in host range. Increased mortality was frequently found in the affected bird aviaries while clinical signs were diverse. Co-infections with other viruses, bacteria or fungal pathogens were common and might have influenced the clinical signs. Sequence analyses, including partial VP1 sequences of the 40 virus strains, and full genome sequences of selected strains revealed a high genetic heterogeneity among virus subgroups of Serinus canaria polyomavirus 1 and Pyrrhula pyrrhula polyomavirus 1, indicating the existence of two virus variants for both virus species. For Pyrrhula pyrrhula polyomavirus 1, two genotypes were found that associated with the family of the finches, Estrildidae or Fringillidae.

Distribution of Usutu Virus in Germany and Its Effect on Breeding Bird Populations.

Usutu virus (USUV) is an emerging mosquitoborne flavivirus with an increasing number of reports from several countries in Europe, where USUV infection has caused high avian mortality rates. However, 20 years after the first observed outbreak of USUV in Europe, there is still no reliable assessment of the large-scale impact of USUV outbreaks on bird populations. In this study, we identified the areas suitable for USUV circulation in Germany and analyzed the effects of USUV on breeding bird populations. We calculated the USUV-associated additional decline of common blackbird (Turdus merula) populations as 15.7% inside USUV-suitable areas but found no significant effect for the other 14 common bird species investigated. Our results show that the emergence of USUV is a further threat for birds in Europe and that the large-scale impact on population levels, at least for common blackbirds, must be considered.

Characterization of five unclassified orthobunyaviruses (Bunyaviridae) from Africa and the Americas.

The Bunyaviridae family is made up of a diverse range of viruses, some of which cause disease and are a cause for concern in human and veterinary health. Here, we report the genomic and antigenic characterization of five previously uncharacterized bunyaviruses. Based on their ultrastructure, antigenic relationships and phylogenomic relationships, the five viruses are classified as members of the Orthobunyavirus genus. Three are viruses in the California encephalitis virus serogroup and are related to Trivittatus virus; the two others are most similar to the Mermet virus in the Simbu serogroup, and to the Tataguine virus, which is not currently assigned to a serogroup. Each of these five viruses was pathogenic to newborn mice, indicating their potential to cause illness in humans and other animals.

Historical Biogeography and Extinction in the Hawaiian Honeycreepers.

Hawaiian honeycreepers, comprising an endemic radiation of passerine birds in the Hawaiian archipelago, have suffered losses of individual island populations and the extinction of many species as a result of colonization of the islands by Polynesians and, more recently, introduced avian pox virus and avian malaria. Here, I test the idea that populations have an intrinsic tendency toward extinction regardless of the cause. The distribution of each species before the arrival of humans in the archipelago was inferred from present distribution, historical records, and fossil remains. On the basis of these records, each species was placed in one of four stages of the taxon cycle: (1) expanding or recently expanded, (2) differentiating, (3) fragmenting, or (4) single-island endemic. Subsequent extinction of individual island populations was most frequent in stage 3 species, which had already suffered loss of individual island populations, suggesting commonality in vulnerability to extinction from anthropogenic and nonanthropogenic causes.

Molecular characterization of a recently identified circovirus in zebra finches (Taeniopygia guttata) associated with immunosuppression and opportunistic infections.

A recently identified circovirus (family Circoviridae) was detected in 14 zebra finches (Taeniopygia guttata) from seven aviaries and hobbyist breeders using polymerase chain reaction followed by sequencing. Full genome sequences of virus strains from six zebra finches consistently revealed characteristic circoviral genomic features such as a stem-loop structure and two major open reading frames (ORFs) encoding the replication-associated protein and the putative capsid protein. One further ORF encoding a protein of unknown function was additionally identified in all six genomes. Based on full genome nucleotide comparison, zebra finch circovirus was most similar to Finch circovirus originating from a Gouldian finch (Chloebia gouldiae) sharing 78% nucleotide identity. High genetic diversity was detected in the circoviruses from individual zebra finches. Comparison of the six full genome sequences revealed two genetic subgroups, which shared pairwise nucleotide identities between 91.4% and 92.7%. Analyses including partial sequences of the replication-associated protein gene of the zebra finch circovirus strains from all 14 birds supported the existence of two main clusters. Clinical diseases associated with circovirus infection were found in nestlings, fledglings and adult birds and varied from mild to severe with high mortality caused by secondary infections. Macrorhabdus ornithogaster was the most frequently detected opportunistic pathogen. Feathering disorders were seen in two birds. Lymphocytic depletion of the spleen and leukocytopaenia were detected in individual birds, suggesting immunosuppression and a pathogenesis common to circovirus infections in other birds.

Isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (Hypsipetes amaurotis) in Japan.

Five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified in the genus Orthoreovirus. Their genomes each consist of 10 dsRNA segments. A novel orthoreovirus was isolated from the haemorrhagic intestine of a dead brown-eared bulbul (Hypsipetes amaurotis) in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed non-enveloped capsids of ~70 nm diameter, which were characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and σC, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV, but was located on a phylogenetic branch different from that of ARV and NBV. The virus had the closest phylogenetic relationship to two reovirus strains: SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The 10 RNA segments had a 3' pentanucleotide sequence (UCAUC-3') conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal heptasequence (5'-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggested that the novel virus might form a new species with the two strains in the genus Orthoreovirus.

Detection of a novel astrovirus from a black-naped monarch (Hypothymis azurea) in Cambodia.

BACKGROUND: Astroviruses are comprised of two genera with Avastrovirus infecting birds and Mamastrovirus infecting mammals. Avastroviruses have primarily been associated with infections of poultry, especially chicken, turkey, duck, and guineafowl production systems, but also infect wading birds and doves. Outcomes result in a spectrum of disease, ranging from asymptomatic shedding to gastroenteritis with diarrhea, stunting, failure to thrive and death. FINDINGS: Virological surveillance was conducted in birds from two sites in Cambodia in 2010. Samples were screened for influenza, astroviruses, coronaviruses, flaviviruses, and paramyxoviruses. A total of 199 birds were tested and an astrovirus was detected in a black-naped monarch (Hypothymis azurea). CONCLUSIONS: This is the first astrovirus detection in a passerine bird. Phylogenetic analysis and nucleotide distances suggest that this avastrovirus forms a distinct lineage and may constitute a fourth avastrovirus group.

Molecular characterization of 3'UTRs of J subgroup avian leukosis virus in passerine birds in China.

To assess the status of avian leukosis virus subgroup J (ALV-J) infection in passerine birds in China, 365 passerine birds collected from northeast China from 2011 to 2013 were tested, and two ALV-J strains were isolated from yellow-browed warbler and marsh tit. The 3'untranslated regions (3'UTRs) of the two strains were amplified, cloned, and sequenced, with the results showing that the 3'UTRs of the two strains contained multiple mutations and deletions, which are similar to viral strains isolated from Chinese layer chickens. These results demonstrate the presence of ALV-J in passerine birds and reveal the molecular characteristics of the 3'UTRs of ALV-J from passerine birds.

Susceptibility of wild passerines to subtype H5N1 highly pathogenic avian influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have spread throughout many areas of Asia, Europe and Africa, and numerous cases of HPAI outbreaks in domestic and wild birds have been reported. Although recent studies suggest that the dissemination of H5N1 viruses is closely linked to the migration of wild birds, information on the potential for viral infection in species other than poultry and waterfowl is relatively limited. To investigate the susceptibility of terrestrial wild birds to infection with H5N1 HPAI viruses, common reed buntings (Emberiza schoeniclus), pale thrushes (Turdus pallidus) and brown-eared bulbuls (Hypsipetes amaurotis) were infected with A/mountain hawk-eagle/Kumamoto/1/07(H5N1) and A/whooper swan/Aomori/1/08(H5N1). The results showed that common reed buntings and brown-eared bulbuls were severely affected by both virus strains (100% mortality). While pale thrushes did not exhibit any clinical signs, seroconversion was confirmed. In common reed buntings, intraspecies-transmission of A/whooper swan/Aomori/1/08 to contact birds was also confirmed. The findings show that three passerine species; common reed buntings, brown-eared bulbuls and pale thrushes are susceptible to infection by H5N1 HPAI viruses, which emphasizes that continued surveillance of species other than waterfowl is crucial for effective monitoring of H5N1 HPAI virus outbreaks.

A continental risk assessment of West Nile virus under climate change.

Since first introduced to North America in 1999, West Nile virus (WNV) has spread rapidly across the continent, threatening wildlife populations and posing serious health risks to humans. While WNV incidence has been linked to environmental factors, particularly temperature and rainfall, little is known about how future climate change may affect the spread of the disease. Using available data on WNV infections in vectors and hosts collected from 2003-2011 and using a suite of 10 species distribution models, weighted according to their predictive performance, we modeled the incidence of WNV under current climate conditions at a continental scale. Models were found to accurately predict spatial patterns of WNV that were then used to examine how future climate may affect the spread of the disease. Predictions were accurate for cases of human WNV infection in the following year (2012), with areas reporting infections having significantly higher probability of presence as predicted by our models. Projected geographic distributions of WNV in North America under future climate for 2050 and 2080 show an expansion of suitable climate for the disease, driven by warmer temperatures and lower annual precipitation that will result in the exposure of new and naïve host populations to the virus with potentially serious consequences. Our risk assessment identifies current and future hotspots of West Nile virus where mitigation efforts should be focused and presents an important new approach for monitoring vector-borne disease under climate change.

West Nile virus infection of birds, Mexico.

West Nile virus (WNV) has caused disease in humans, equids, and birds at lower frequency in Mexico than in the United States. We hypothesized that the seemingly reduced virulence in Mexico was caused by attenuation of the Tabasco strain from southeastern Mexico, resulting in lower viremia than that caused by the Tecate strain from the more northern location of Baja California. During 2006-2008, we tested this hypothesis in candidate avian amplifying hosts: domestic chickens, rock pigeons, house sparrows, great-tailed grackles, and clay-colored thrushes. Only great-tailed grackles and house sparrows were competent amplifying hosts for both strains, and deaths occurred in each species. Tecate strain viremia levels were higher for thrushes. Both strains produced low-level viremia in pigeons and chickens. Our results suggest that certain avian hosts within Mexico are competent for efficient amplification of both northern and southern WNV strains and that both strains likely contribute to bird deaths.

Neurotropism in blackcaps (Sylvia atricapilla) and red-billed queleas (Quelea quelea) after highly pathogenic avian influenza virus H5N1 infection.

The epidemiologic role of passerine birds in the spread of highly pathogenic avian influenza virus (HPAIV) remains controversial. However, confirmed natural infections with HPAIV in Passeriformes, their close contact to poultry and humans, and their role as a human food source indicate a need for increased research on passerines. To date, there are only a few studies on viral shedding and pathomorphologic changes in songbirds infected with HPAIV. To investigate susceptibility, clinical outcome, virus spread, and pathomorphology, the authors inoculated oculo-oronasally 22 red-billed queleas (Quelea quelea) and 11 blackcaps (Sylvia atricapilla) with A/Cygnus cygnus/Germany/R65/2006 (H5N1) using 2 different doses of either 10(4) EID50 (50% egg infective dose) or 10(6) EID50 per animal. They monitored all birds for clinical signs and oropharyngeal and cloacal virus shedding. They also performed immunohistochemistry and obtained molecular virologic data by real-time reverse transcription polymerase chain reaction in tissue samples. In contrast to blackcaps, where 100% of the infected individuals died, queleas were much less susceptible, with a mortality of 82% and 18%, depending on the doses applied. In both species, the virus was shed within 3 to 6 days postinfection, mainly via the respiratory tract. Viral antigen was detected in 100% of the succumbed birds, particularly in the central nervous system. In blackcaps, the heart, lungs, and pancreas were mainly infected. In contrast, the pancreas was predominantly affected in queleas, whereas the heart and the lower respiratory tract were of minor relevance. The authors hypothesize that neurotropism should be considered a main factor for the fatal course of disease in Passeriformes after infection with HPAIV.

Pathology and Pathogenesis of Eurasian Blackbirds (Turdus merula) Naturally Infected with Usutu Virus.

The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission.

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.

Genomic characterisation of a novel avipoxvirus, magpiepox virus 2, from an Australian magpie (Gymnorhina tibicen terraereginae).

Avipoxviruses are large, double-stranded DNA viruses and are considered significant pathogens that may impact on the conservation of numerous bird species. The vast majority of avipoxviruses in wild birds remain uncharacterised and their genetic variability is unclear. Here, we fully sequenced a novel avipoxvirus, magpiepox virus 2 (MPPV2), which was isolated 62 years ago (in 1956) from an Australian black-backed magpie. The MPPV2 genome was 298,392 bp in length and contained 419 predicted open-reading frames (ORFs). While 43 ORFs were novel, a further 24 ORFs were absent compared with another magpiepox virus (MPPV) characterised in 2018. The MPPV2 genome contained an additional ten genes that were homologs to shearwaterpox virus 2 (SWPV2). Subsequent phylogenetic analyses showed that the novel MPPV2 was most closely related to other avipoxviruses isolated from passerine and shearwater bird species, and demonstrated a high degree of sequence similarity (95.0%) with MPPV.