Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

Characterization of an avian rotavirus A strain isolated from a velvet scoter (Melanitta fusca): implication for the role of migratory birds in global spread of avian rotaviruses.

Avian G18P[17] rotaviruses with similar complete genome constellation, including strains that showed pathogenicity in mammals, have been detected worldwide. However, it remains unclear how these strains spread geographically. In this study, to investigate the role of migratory birds in the dispersion of avian rotaviruses, we analysed whole genetic characters of the rotavirus strain RK1 that was isolated from a migratory species of birds [velvet scoter (Melanitta fusca)] in Japan in 1989. Genetic analyses revealed that the genotype constellation of the RK1 strain, G18-P[17]-I4-R4-C4-M4-A21-N4-T4-E4-H4, was highly consistent with those of other G18P[17] strains detected in various parts of the world, supporting the possibility that the G18P[17] strains spread via migratory birds that move over a wide area. Furthermore, the RK1 strain induced diarrhoea in suckling mice after oral gastric inoculation, indicating that at least some of the rotaviruses that originated from migratory birds are infectious to and pathogenic in mammals. In conclusion, it was demonstrated that migratory birds may contribute to the global spread of avian rotaviruses that are pathogenic in mammalian species.

Evidence of a Possible Viral Host Switch Event in an Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi).

Avipoxviruses have been characterized from many avian species. Two recent studies have reported avipoxvirus-like viruses with varying pathogenicity in reptiles. Avipoxviruses are considered to be restricted to avian hosts. However, reports of avipoxvirus-like viruses from reptiles such as the green sea turtle (Chelonia mydas) and crocodile tegu (Crocodilurus amazonicus) suggest that cross-species transmission, within avian species and beyond, may be possible. Here we report evidence for a possible host switching event with a fowlpox-like virus recovered from an endangered northern royal albatross (Diomodea sanfordi)-a species of Procellariiformes, unrelated to Galliformes, not previously known to have been infected with fowlpox-like viruses. Complete genome sequencing of this virus, tentatively designated albatrosspox virus 2 (ALPV2), contained many fowlpox virus-like genes, but also 63 unique genes that are not reported in any other poxvirus. The ALPV2 genome contained 296 predicted genes homologous to different avipoxviruses, 260 of which were homologous to an American strain of fowlpox virus (FWPV). Subsequent phylogenetic analyses indicate that ALPV2 likely originated from a fowlpox virus-like progenitor. These findings highlight the importance of host-switching events where viruses cross species barriers with the risk of disease in close and distantly related host populations.

Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

Metagenomic detection and characterisation of multiple viruses in apparently healthy Australian Neophema birds.

Emerging viral pathogens are a significant concern, with potential consequences for human, animal and environmental health. Over the past several decades, many novel viruses have been found in animals, including birds, and often pose a significant threat to vulnerable species. However, despite enormous interest in virus research, little is known about virus communities (viromes) in Australian Neophema birds. Therefore, this study was designed to characterise the viromes of Neophema birds and track the evolutionary relationships of recently emerging psittacine siadenovirus F (PsSiAdV-F) circulating in the critically endangered, orange-bellied parrot (OBP, Neophema chrysogaster), using a viral metagenomic approach. This study identified 16 viruses belonging to the families Adenoviridae, Circoviridae, Endornaviridae, Picobirnaviridae and Picornaviridae. In addition, this study demonstrated a potential evolutionary relationship of a PsSiAdV-F sequenced previously from the critically endangered OBP. Strikingly, five adenoviral contigs identified in this study show the highest identities with human adenovirus 2 and human mastadenovirus C. This highlights an important and unexpected aspects of the avian virome and warrants further studies dedicated to this subject. Finally, the findings of this study emphasise the importance of testing birds used for trade or in experimental settings for potential pathogens to prevent the spread of infections.

Traceable surveillance and genetic diversity analysis of coronaviruses in poultry from China in 2019.

Coronavirus disease 2019 (COVID-19), caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China, and rapidly spread throughout the world. This newly emerging pathogen is highly transmittable and can cause fatal disease. More than 35 million cases have been confirmed, with a fatality rate of about 2.9% to October 9, 2020. However, the original and intermediate hosts of SARS-CoV-2 remain unknown. Here, 3160 poultry samples collected from 14 provinces of China between September and December 2019 were tested for SARS-CoV-2 infection. All the samples were SARS-CoV-2 negative, but 593 avian coronaviruses were detected, including 485 avian infectious bronchitis viruses, 72 duck coronaviruses, and 36 pigeon coronaviruses, with positivity rates of 15.35%, 2.28%, and 1.14%, respectively. Our surveillance demonstrates the diversity of avian coronaviruses in China, with higher prevalence rates in some regions. Furthermore, the possibility that SARS-CoV-2 originated from a known avian-origin coronavirus can be preliminarily ruled out. More surveillance of and research into avian coronaviruses are required to better understand the diversity, distribution, cross-species transmission, and clinical significance of these viruses.

Glycosylation on envelope glycoprotein of duck Tembusu virus affects virus replication in vitro and contributes to the neurovirulence and pathogenicity in vivo.

Duck Tembusu virus (DTMUV), an emergent flavivirus, causes domestic waterfowls to suffer from severe egg-drop syndrome and fatal encephalitis, greatly threatens duck production globally. Like other mosquito-borne flaviviruses, the envelope (E) protein of all DTMUV strains was N-glycosylated at the amino acid position 154. Thus far, the biological roles of DTMUV E glycosylation have remained largely unexplored. Herein, we demonstrated the key roles of E glycosylation in the replication and pathogenicity of DTMUV in ducks by characterizing the reverse-genetics-derived DTMUV wild-type MC strain and MC bearing mutations (N154Q and N154I) that abolish the E glycosylation. Our data showed that the disruption of E glycosylation could substantially impair virus attachment, entry, and infectivity in DEFs and C6/36 cells. Notably, ducks inoculated intracerebrally with the wild-type virus exhibited severe disease onset. In contrast, those inoculated with mutant viruses were mildly affected as manifested by minimal weight loss, no mortality, lower viral loads in the various tissues, and reduced brain lesions. Attenuated phenotypes of the mutant viruses might be partly associated with lower inflammatory cytokines expression in the brains of infected ducks. Our study offers the first evidence that E glycosylation is vital for DTMUV replication, pathogenicity, and neurovirulence in vivo.

Ocurrence of rotavirus and picobirnavirus in wild and exotic avian from amazon forest.

The present study reports the occurrence of rotavirus A (RVA), rotavirus D (RVD), rotavirus F (RVF), rotavirus G (RVG), and picobirnavirus (PBV) in fecal specimens of wild (n = 22), and exotic birds (n = 1) from different cities of Pará state. These animals were hospitalized at Veterinary Hospital of the Federal University of Pará, Brazil, in a period from January 2018 to June 2019. The animals exhibited different clinical signs, such as diarrhea, malnutrition, dehydration, and fractures. The results showed 39.1% (9/23) of positivity for RVA by RT-qPCR. Among these, one sample (1/9) for the NSP3 gene of T2 genotype was characterized. About 88.9% (8/9) for the VP7 gene belonging to G1, G3 equine like and G6 genotypes, and 55.5% (5/9) for the VP4 gene of P[2] genotype were obtained. In the current study, approximately 4.5% of the samples (1/23) revealed coinfection for the RVA, RVD and RVF groups. Furthermore, picobirnavirus (PBV) was detected in one of the 23 samples tested, and was classified in the Genogroup I. The findings represent the first report of RVA, RVD, RVF, RVG, and PBV genotypes in wild birds in Brazil, and due to wide distribution it can implies potential impacts of RVs, and PBVs on avian health, and other animals contributing to construction of new knowledge, and care perspectives.

Genomic Characterisation of a Highly Divergent Siadenovirus (Psittacine Siadenovirus F) from the Critically Endangered Orange-Bellied Parrot (Neophema chrysogaster).

Siadenoviruses have been detected in wild and captive birds worldwide. Only nine siadenoviruses have been fully sequenced; however, partial sequences for 30 others, many of these from wild Australian birds, are also described. Some siadenoviruses, e.g., the turkey siadenovirus A, can cause disease; however, most cause subclinical infections. An example of a siadenovirus causing predominately subclinical infections is psittacine siadenovirus 2, proposed name psittacine siadenovirus F (PsSiAdV-F), which is enzootic in the captive breeding population of the critically endangered orange-bellied parrot (OBP, Neophema chrysogaster). Here, we have fully characterised PsSiAdV-F from an OBP. The PsSiAdV-F genome is 25,392 bp in length and contained 25 putative genes. The genome architecture of PsSiAdV-F exhibited characteristics similar to members within the genus Siadenovirus; however, the novel PsSiAdV-F genome was highly divergent, showing highest and lowest sequence similarity to skua siadenovirus A (57.1%) and psittacine siadenovirus D (31.1%), respectively. Subsequent phylogenetic analyses of the novel PsSiAdV-F genome positioned the virus into a phylogenetically distinct sub-clade with all other siadenoviruses and did not show any obvious close evolutionary relationship. Importantly, the resulted tress continually demonstrated that novel PsSiAdV-F evolved prior to all known members except the frog siadenovirus A in the evolution and possibly the ancestor of the avian siadenoviruses. To date, PsSiAdV-F has not been detected in wild parrots, so further studies screening PsSiAdV-F in wild Australian parrots and generating whole genome sequences of siadenoviruses of Australian native passerine species is recommended to fill the siadenovirus evolutionary gaps.

Viral gene expression profile of goose haemorrhagic polyomavirus in susceptible primary cells.

Routine culturing of goose haemorrhagic polyomavirus (GHPV) is cumbersome, and limited data are available about its replication and gene expression profile. In this study, goose embryo fibroblast cells were infected with GHPV for temporal measurement of the viral genome copy number and mRNA levels with quantitative PCR. Accumulation of small and large tumour antigen-encoding mRNAs was detected as early as 9 hours post-infection (hpi), while high level expression of the capsid protein encoding VP1-VP3, and ORF-X mRNAs was first detected at 24 hpi. Elevation of GHPV genome copy number was noted at 48 hpi. The results indicate that the gene expression profile of GHPV is similar to that described for mammalian polyomaviruses.RESEARCH HIGHLIGHTS GHPV was propagated in culture of primary goose embryo fibroblast cells.The transcription commenced before the onset of viral DNA replication.The transcription patterns of GHPV and mammalian polyomaviruses were comparable.

Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns.

Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, obscuring reassortment-specific epistasis and changes in substitution rates. To determine the co-segregation patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 complete RVA genomes isolated from mammalian hosts and compared the segments' geodesic distances. We found that segments 4 (VP4) and 9 (VP7) occupied significantly different tree spaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable tree spaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the weakest association with host species. Bayesian Skyride plots were generated for each segment to compare relative genetic diversity among segments over time. All segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices were calculated with respect to different host genomes. Codon usage varied by segment with segment 11 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that translational efficiency is not a barrier in RVA zoonosis.

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.

Diversity of Coronaviruses in Wild Representatives of the Aves Class in Poland.

The revealed prevalence of coronaviruses in wild bird populations in Poland was 4.15% and the main reservoirs were birds from orders Anseriformes and Charadriiformes, with a prevalence of 3.51% and 5.59%, respectively. Gammacoronaviruses were detected more often than deltacoronaviruses, with detection rates of 3.5% and 0.7%, respectively. Gammacoronaviruses were detected in birds belonging to six orders, including Anseriformes, Charadriiformes, Columbiformes, Galliformes, Gruiformes, and Passeriformes, indicating a relatively wide host range. Interestingly, this was the only coronavirus detected in Anseriformes (3.51%), while in Charadriiformes, the prevalence was 3.1%. The identified gammacoronaviruses belonged to the Igacovirus and Brangacovirus subgeneras. Most of these were igacoviruses and formed a common phylogenetic group with a Duck Coronavirus 2714 and two with an Avian Coronavirus/Avian Coronavirus9203, while the viruses from the pigeons formed a distinct "pigeon-like" group, not yet officially represented. The presence of deltacoronaviruses was detected in birds belonging to three orders, Charadriiformes, Galliformes, and Suliformes indicating a narrower host range. Most identified deltacoronaviruses belonged to the Buldecovirus subgenus, while only one belonged to Herdecovirus. Interestingly, the majority of buldecoviruses were identified in gulls, and they formed a distinct phylogenetic lineage not represented by any officially ratified virus species. Another separate group of buldecoviruses, also not represented by the official species, was formed by a virus identified in a common snipe. Only one identified buldecovirus (from common pheasant) formed a group with the ratified species Coronavirus HKU15. The results obtained indicate the high diversity of detected coronaviruses, and thus also the need to update their taxonomy (establishing new representative virus species). The serological studies performed revealed antibodies against an infectious bronchitis virus in the sera of white storks and mallards.

A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses.

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

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.

Very virulent infectious bursal disease virus infection caused changes in cloacal temperature and clinical manifestations in pigeons (Columba livia domestica) and is transmitted to sentinel chickens.

In this study, changes in cloacal temperature and clinical manifestations due to very virulent infectious bursal disease virus (vvIBDV) infection in pigeons (Columba livia domestica) and transmission to chickens were demonstrated. Thirty pigeons (3-6 weeks old) and thirty chickens (3 weeks old) divided into 4 groups (I-IV) were used for this study. Group I comprised of 10 uninoculated pigeons only; II comprised of 10 inoculated pigeons and 10 sentinel chickens; III comprised of 10 sentinel pigeons and 10 inoculated chickens, while IV comprised of 10 uninoculated chickens only. Pigeons in group II and chickens in group III were each inoculated with 0.20 mL (titre of 109.76CID50/mL) of vvIBDV (Nigerian strain). Cloacal temperature was monitored and clinical manifestations scored post-inoculation (pi). Results indicated significant (P < 0.05) pyrexia at 2 days pi (dpi), mild clinical signs and no mortality in inoculated pigeons. Significant (P < 0.05) pyrexia at 2-4 dpi, severe clinical signs and mortality (50%; 60%) were observed in inoculated and sentinel chickens. IBDV antigen and antibody were detected in pigeons and chickens. Pigeons showed response to vvIBDV infection thus suggesting susceptibility of pigeons to IBD. Sentinel chickens presented clinical manifestations of IBD and this suggests transmission from pigeons to chickens. This study therefore documents pyrexia and clinical manifestations due to vvIBDV infection in pigeons and successful transmission of the virus between pigeons and chickens.

Usutu virus infection in aviary birds during the cold season.

The mosquito-borne flavivirus Usutu virus (USUV) is responsible for countless deaths in both resident populations and birds kept in outdoor aviaries. Since 2001, USUV outbreaks have attracted increased attention due to the rapid geographical spread of the virus and its close relationship to West Nile virus (WNV), an emerging pathogen in humans and animals. Similar to WNV, the USUV enzootic transmission cycle predominantly involves Culex spp. as vectors, whereas birds serve as amplifying reservoir hosts. In Europe, USUV-associated disease outbreaks in birds are almost exclusively described during late spring and early autumn (early April to late October). Contagiousness of virus particles excreted by infected birds has not yet been proven, so that the role of non-vector-borne transmission, as it is known for the closely related WNV, remains unclear. Here we report the diagnosis of USUV infection in 15 of 24 birds from mortality outbreaks that occurred during the cold season between late October 2018 and early April 2019, in eight different aviaries located in Germany. Detection of USUV was performed using standardized molecular biological methods and immunohistochemistry for verification of the infection. USUV infection in a parrot species, a tropical finch and two estrildid finches are reported for the first time. Further research on the occurrence of USUV infection during the cold season is key to understanding the dynamics of viral transmission as well as for a profound health risk assessment for aviary birds as well as humans.

Mosquito blood-feeding patterns and nesting behavior of American crows, an amplifying host of West Nile virus.

BACKGROUND: Although American crows are a key indicator species for West Nile virus (WNV) and mount among the highest viremias reported for any host, the importance of crows in the WNV transmission cycle has been called into question because of their consistent underrepresentation in studies of Culex blood meal sources. Here, we test the hypothesis that this apparent underrepresentation could be due, in part, to underrepresentation of crow nesting habitat from mosquito sampling designs. Specifically, we examine how the likelihood of a crow blood meal changes with distance to and timing of active crow nests in a Davis, California, population. METHODS: Sixty artificial mosquito resting sites were deployed from May to September 2014 in varying proximity to known crow nesting sites, and Culex blood meal hosts were identified by DNA barcoding. Genotypes from crow blood meals and local crows (72 nestlings from 30 broods and 389 local breeders and helpers) were used to match mosquito blood meals to specific local crows. RESULTS: Among the 297 identified Culex blood meals, 20 (6.7%) were attributable to crows. The mean percentage of blood meals of crow origin was 19% in the nesting period (1 May-18 June 2014), but 0% in the weeks after fledging (19 June-1 September 2014), and the likelihood of a crow blood meal increased with proximity to an active nest: the odds that crows hosted a Culex blood meal were 38.07 times greater within 10 m of an active nest than > 10 m from an active nest. Nine of ten crow blood meals that could be matched to a genotype of a specific crow belonged to either nestlings in these nests or their mothers. Six of the seven genotypes that could not be attributed to sampled birds belonged to females, a sex bias likely due to mosquitoes targeting incubating or brooding females. CONCLUSION: Data herein indicate that breeding crows serve as hosts for Culex in the initial stages of the WNV spring enzootic cycle. Given their high viremia, infected crows could thereby contribute to the re-initiation and early amplification of the virus, increasing its availability as mosquitoes shift to other moderately competent later-breeding avian hosts.

A Pilot Study Investigating the Dynamics of Pigeon Circovirus Recombination in Domesticated Pigeons Housed in a Single Loft.

Pigeon circovirus (PiCV) infects pigeon populations worldwide and has been associated with immunosuppression in younger pigeons. Recombination is a common mechanism of evolution that has previously been shown in various members of the Circoviridae family, including PiCV. In this study, three groups of pigeons acquired from separate lofts were screened for PiCV, and their genome sequence was determined. Following this, they were housed in a single loft for 22 days, during which blood and cloacal swab samples were taken. From these blood and cloacal swabs, PiCV genomes were determined with the aim to study the spread and recombination dynamics of PiCV in the birds. Genome sequences of PiCV were determined from seven pigeons (seven tested PiCV positive) before they were housed together in a loft (n = 58 sequences) and thereafter from the ten pigeons from blood and cloacal swabs (n = 120). These 178 PiCV genome sequences represent seven genotypes (98% pairwise identity genotype demarcation), and they share >88% genome-wide pairwise identity. Recombination analysis revealed 13 recombination events, and a recombination hotspot spanning the 3' prime region, the replication-associated protein (rep) gene and the intergenic region. A cold spot in the capsid protein-coding region of the genome was also identified. The majority of the recombinant regions were identified in the rep coding region. This study provides insights into the evolutionary dynamics of PiCV in pigeons kept under closed rearing systems.