Highly diverse RNA viruses and phage sequences concealed within birds.

The diversity of birds in most parts of the world is very high, and thus, they may carry different types of highly differentiated and unknown viruses. Thanks to advanced sequencing technologies, studies on the diversity of bird-associated viruses have increased over the past few years. In this study, a large-scale viral metagenomics survey was performed on cloacal swabs of 2,990 birds from nine provinces of the Chinese mainland. To detect undescribed RNA viruses in birds, more than 1,800 sequences sharing relatively low (<60%) amino acid sequence identity with the best match in the GenBank database were screened. Potentially novel viruses related to vertebrates have been identified, and several potential recombination signals were found. Additionally, hundreds of RNA viral sequences related to plants, fungi, and insects were detected, including previously unknown viruses. Furthermore, we investigated the novelty, functionality, and classification of the phages examined in this study. These viruses occupied topological positions on the evolutionary trees to a certain extent and might form novel putative families, genera, or species, thus providing information to fill the phylogenetic gaps of related viruses. These findings provided new insights into bird-associated viruses, but the interactions among these viruses remain unknown and require further investigation.IMPORTANCEStudying the diversity of RNA viruses in birds and mammals is crucial due to their potential impact on human health and the global ecosystem. Many RNA viruses, such as influenza and coronaviruses, have been shown to cross the species barrier and cause zoonotic diseases. In this metagenomics study involving 2,990 birds from at least 82 species, we identified over 1,800 RNA sequences with distant relationships to known viruses, some of which are rare in birds. The study highlights the scope and diversity of RNA viruses in birds, providing data to predict disease risks and monitor potential viral threats to wildlife, livestock, and human health. This information can aid in the development of strategies for disease prevention and control.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

RNA virome abundance and diversity is associated with host age in a bird species.

Despite the ongoing interest in virus discovery, little is known about the factors that shape communities of viruses within individual hosts. Here, we address how virus communities might be impacted by the age of the hosts they infect, using total RNA sequencing to reveal the RNA viromes of different age groups of Ruddy Turnstones (Arenaria interpres). From oropharyngeal and cloacal swabs we identified 14 viruses likely infecting birds, 11 of which were novel, including members of the Reoviridae, Astroviridae, and Picornaviridae. Strikingly, 12 viruses identified were from juvenile birds sampled in the first year of their life, compared to only two viruses in adult birds. Both viral abundance and alpha diversity were marginally higher in juvenile than adult birds. As well as informing studies of virus ecology, that host age might be associated with viral composition is an important consideration for the future surveillance of novel and emerging viruses.

POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.

Detection of Immunoreactivity to Psittaciform Bornavirus in the Serum of a Wild Cacatuid in Victoria, Australia.

An indirect immunofluorescence serologic assay, PCR assay, and histopathology were used to screen for psittaciform orthobornaviruses (PaBV) in wild Cacatuidae in Victoria, Australia. Anti-PaBV antibodies were detected, but PCR and histopathology did not detect PaBV. This study presents the first evidence of PaBV in wild birds in Australia.

Genetic and Antigenic Characterization of Avian Avulavirus Type 6 (AAvV-6) Circulating in Canadian Wild Birds (2005-2017).

We describe for the first time the genetic and antigenic characterization of 18 avian avulavirus type-6 viruses (AAvV-6) that were isolated from wild waterfowl in the Americas over the span of 12 years. Only one of the AAvV-6 viruses isolated failed to hemagglutinate chicken red blood cells. We were able to obtain full genome sequences of 16 and 2 fusion gene sequences from the remaining 2 isolates. This is more than double the number of full genome sequences available at the NCBI database. These AAvV-6 viruses phylogenetically grouped into the 2 existing AAvV-6 genotype subgroups indicating the existence of an intercontinental epidemiological link with other AAvV-6 viruses isolated from migratory waterfowl from different Eurasian countries. Antigenic maps made using HI assay data for these isolates showed that the two genetic groups were also antigenically distinct. An isolate representing each genotype was inoculated in specific pathogen free (SPF) chickens, however, no clinical symptoms were observed. A duplex fusion gene based real-time assay for the detection and genotyping of AAvV-6 to genotype 1 and 2 was developed. Using the developed assay, the viral shedding pattern in the infected chickens was examined. The chickens infected with both genotypes were able to shed the virus orally for about a week, however, no significant cloacal shedding was detected in chickens of both groups. Chickens in both groups developed detectable levels of anti-hemagglutinin antibodies 7 days after infection.

Genetic and evolutionary characterization of avian paramyxovirus type 4 in China.

As an economically important poultry pathogen, avian paramyxovirus serotype 4 (APMV-4) frequently reported and isolated from domestic and wild birds particularly waterfowls worldwide. However, evolutionary dynamics of APMV-4 based on genomic characteristics is lacking. In this study, APMV-4 strain designated JX-G13 was isolated from oropharyngeal and cloacal swab samples of wild birds in China. Phylogenetic analysis revealed APMV-4 strains were divided into four genetic genotypes and China isolates were mainly clustered into Genotype I. The MCMC tree indicated that APMV-4 diverged about 104 years ago with the evolutionary rate of 1.2927 × 10-3 substitutions/site/year. BSP analysis suggested that the effective population size of APMV-4 exhibited a steady state and decreased slowly after 2013. The F gene of APMV-4 was considered relatively conserved among isolates based on nucleotide diversity analysis. Although the F gene was under purifying selection, two positions (5 and 21) located in 3'-UTR were subject to positive selection. Our study firstly presented the evolutionary assessments on the genetic diversity of circulating APMV-4 from wild birds and domestic poultry.

Population genomic transformations induced by isolation of wild bird avian influenza viruses (Orthomyxoviridae) in embryonated chicken eggs.

Isolation and cultivation of wild-type viruses in model organism cells or tissues is standard practice in virology. Oftentimes, the virus host species is distantly related to the species from which the culture system was developed. Thus, virus culture in these tissues and cells basically constitutes a host jump, which can lead to genomic changes through genetic drift and/or adaptation to the culture system. We directly sequenced 70 avian influenza virus (Orthomyxoviridae) genomes from oropharyngeal/cloacal swabs collected from wild bird species and paired virus isolates propagated from the same samples following isolation in specific-pathogen-free embryonated chicken eggs. The data were analyzed using population genetic approaches including evaluation of single nucleotide polymorphism (SNP) frequencies and divergence with pooled-sequencing analyses, consensus sequence placement in neighbor-joining trees, and haplotype reconstruction and networks. We found that propagation of virus in eggs leads to skewed SNP mutation spectra with some SNPs going to fixation. Both synonymous and nonsynonmous SNP frequencies shifted. We found multiple consensus sequences that differed between the swabs and the isolates, with some sequences from the same sample falling into divergent genetic clusters. Twenty of 23 coinfections detected had different dominant subtypes following virus isolation, thus sequences from both the swab and isolate were needed to obtain full subtype data. Haplotype networks revealed haplotype frequency shifts and the appearance or loss of low-frequency haplotypes following isolation. The results from this study revealed that isolation of wild bird avian influenza viruses in chicken eggs leads to skewed populations that are different than the input populations. Consensus sequence changes from virus isolation can lead to flawed phylogenetic inferences, and subtype detection is biased. These results suggest that for genomic studies of wild bird influenza viruses the biological field should move away from chicken egg isolation towards directly sequencing the virus from host samples.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses.

Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by (fecal) contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds.

Clade 2.3.4.4, H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused devastating effects across wild and domestic bird populations. We investigated differences in the intensity and distribution of the hemagglutinin (HA) glycoprotein binding of a clade 2.3.4.4 H5 HPAIV compared to a H5 low pathogenic avian influenza virus (LPAIV). Recombinant HA from gene sequences from a HPAIV, A/Northern pintail/Washington/40964/2014(H5N2) and a LPAIV, A/mallard/MN/410/2000(H5N2) were generated and, via protein histochemistry, HA binding in respiratory, intestinal and cloacal bursal tissue was quantified as median area of binding (MAB). Poultry species, shorebirds, ducks and terrestrial birds were used. Differences in MAB were observed between the HPAIV and LPAIV H5 HAs. We demonstrate that clade 2.3.4.4 HPAIV H5 HA has a broader host cell binding across a variety of bird species compared to the LPAIV H5 HA. These findings support published results from experimental trials, and outcomes of natural disease outbreaks with these viruses.