Complete genome sequence analysis of Reticuloendotheliosis virus integrated in nonhomologous Avipoxvirus.

Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirus（APV） has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.

Isolation and identification of a pigeonpox virus strain and study on the integration of reticuloendotheliosis virus sequence.

In order to study the integration of reticuloendotheliosis virus (REV) in pigeonpox virus (PPV), we collected suspected pigeonpox disease material, amplified the 4b core protein gene of PPV, the gp90 gene of REV, and the integrated sequence fragments from the end of the ORF201 segment of PPV to the beginning of the LTR of REV, and sequenced these genes. The results showed that a 4b core protein fragment of 332 bp was amplified and identified as pigeonpox virus, which was named SX/TY/LTR 01/2023. Sequence analysis showed that the pigeonpox virus isolate belonged to genotype A2, which was the closest to the domestic CVL strain, with a identity of 99.4%. A band of 1191 bp was amplified from the gp90 gene of REV, named SX/TY/PPV-REV01/2023, and sequence analysis indicated that REV belonged to genotype III. The sequence analysis showed that REV belonged to genotype III, and belonged to the same large branch as the domestic isolates JSRD0701 and LNR0801, with 99.3% identity. The integrated sequence fragment was amplified to a band of 637 bp, which determined that the REV sequence was integrated in the PPV rather than a mixed infection of the two viruses. This indicates that REV was integrated in this isolation of PPV, suggesting that pigeon farms need to prevent reticuloendotheliosis at the same time when preventing pigeonpox.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

Systemic avian poxvirus infections associated with the B1 subclade of canarypox virus.

Avian poxvirus infections typically manifest as 2 forms: cutaneous ("dry") pox, characterized by proliferative nodules on the skin, and diphtheritic ("wet") pox, characterized by plaques of caseous exudate in the oropharynx and upper respiratory and gastrointestinal tracts. Systemic spread of virus to visceral organs beyond the skin and mucous membranes is rarely reported. Out of 151 cases diagnosed with avian poxvirus over a 20-year period at a zoological institution, 22 were characterized as having systemic involvement based on histopathology and molecular findings. Gross lesions in systemic cases included soft white nodules scattered throughout the liver, spleen, and kidneys. Two histopathologic patterns emerged: (1) widespread histiocytic inflammation in visceral organs with intrahistiocytic viral inclusions and (2) severe, localized dry or wet pox lesions with poxvirus-like inclusions within dermal and subepithelial histiocytes. In situ hybridization targeting the core P4b protein gene confirmed the presence of poxvirus DNA within histiocytes in both patterns. Polymerase chain reaction was performed targeting the reticuloendothelial virus long terminal repeat (REV LTR) flanking region and the core P4b protein gene. Sequences of the REV LTR flanking region from all systemic pox cases were identical to a previously described condorpox virus isolated from an Andean condor with systemic pox. Sequences of the core P4b protein gene from all systemic pox cases grouped into cluster 2 of the B1 subclade of canarypox viruses. Systemic involvement of avian poxvirus likely occurs as a result of infection with certain strain variations in combination with various possible host and environmental factors.

Vertebrate-tropism of a cressdnavirus lineage implicated by poxvirus gene capture.

Among cressdnaviruses, only the family Circoviridae is recognized to infect vertebrates, while many others have unknown hosts. Detection of virus-to-host horizontal gene transfer is useful for solving such virus-host relationships. Here, we extend this utility to an unusual case of virus-to-virus horizontal transfer, showing multiple ancient captures of cressdnavirus Rep genes by avipoxviruses-large dsDNA pathogens of birds and other saurians. As gene transfers must have occurred during virus coinfections, saurian hosts were implied for the cressdnavirus donor lineage. Surprisingly, phylogenetic analysis revealed that donors were not members of the vertebrate-infecting Circoviridae, instead belonging to a previously unclassified family that we name Draupnirviridae. While draupnirviruses still circulate today, we show that those in the genus Krikovirus infected saurian vertebrates at least 114 Mya, leaving endogenous viral elements inside snake, lizard, and turtle genomes throughout the Cretaceous Period. Endogenous krikovirus elements in some insect genomes and frequent detection in mosquitoes imply that spillover to vertebrates was arthropod mediated, while ancestral draupnirviruses likely infected protists before their emergence in animals. A modern krikovirus sampled from an avipoxvirus-induced lesion shows that their interaction with poxviruses is ongoing. Captured Rep genes in poxvirus genomes often have inactivated catalytic motifs, yet near-total presence across the Avipoxvirus genus, and evidence of both expression and purifying selection on them suggests currently unknown functions.

Molecular characterisation of a novel pathogenic avipoxvirus from an Australian little crow (Corvus bennetti) directly from the clinical sample.

Avipoxviruses are thought to be restricted to avian hosts and considered significant pathogens that may impact the conservation of many birds. However, reports of avipoxvirus-like viruses from reptiles suggest that cross-species transmission, within birds and other species, may be possible. The vast majority of avipoxviruses in wild birds remain uncharacterised and their genetic variability is unclear. Here, cutaneous pox lesions were used to recover a novel full-length crowpox virus genome from an Australian little crow (Corvus bennetti), followed by the detection of immature and intracellular mature virions using electron microscopy. The CRPV genome was 328,768 bp in length and contained 403 predicted open-reading frames. While 356 of the ORFs of CRPV genome had the greatest similarity with other avipoxviruses gene products, a further 47 ORFs were novel. Subsequent phylogenetic analyses showed that the CRPV was most closely related to other avipoxviruses isolated from passerine and marine bird species and demonstrated the highest sequence similarity with an albatrosspox virus (84.4%). Considering the sequence similarity observed between CRPV and other avipoxviruses and phylogenetic position, this study concluded that the CRPV to be a distinct available candidate of avipoxviruses.

Detection and molecular characterization of Avipoxvirus in Culex spp. (Culicidae) captured in domestic areas in Rio de Janeiro, Brazil.

Avian pox is a highly contagious poultry disease that causes significant economic losses. Mosquitoes belonging to the genus Culex (Diptera: Culicidae) have a fundamental role in disseminating Avipoxvirus (Poxviridae). This study proposes investigating the presence of Avipoxvirus (APV) DNA in Culex spp. from Rio de Janeiro to determine its frequency and perform a phylogenetic analysis based on the core like the 4b protein (p4b) gene. The detection of APVs was conducted individually on four hundred Culex spp. mosquitoes. A total of 12.23% (47/384) of the Culex spp. were positive in the PCR. Sequencing the p4b gene revealed that this study's sequences displayed 98.8-99% identity with Fowlpoxvirus (FWPW) sequences available in GenBank. In the phylogenetic analysis, these APVs were clustered in the A1 subclade together with FWPW sequences from several countries. The evolutionary distance of the p4b gene was 0.61 ± 0.21% in rural areas and 0.38 ± 0.16% in peri-urban areas. The current investigation is the first study to report the detection of APVs in field-caught mosquitoes. Moreover, a high frequency of APV DNA was observed in Culex spp. captured in domestic areas, where backyard poultry is present. This data demonstrates the importance of implementing control measures for Culex spp. to mitigate the transmission of APVs in backyard poultry in Rio de Janeiro.

Outbreaks of Avipoxvirus Clade E in Vaccinated Broiler Breeders with Exacerbated Beak Injuries and Sex Differences in Severity.

Avipoxvirus affects chickens and wild birds, and it is characterized by lesions on the nonfeathered parts of the body (the cutaneous form), or necrotic lesions in the upper respiratory tract (the diphtheritic form). In poultry farming, avian pox is usually controlled by live attenuated vaccines. However, there have been many reports of outbreaks, even in flocks of vaccinated birds. In the present study, different outbreaks of the emerging clade E avipoxvirus were detected in commercial breeder flocks of chickens vaccinated against fowlpox virus in Southeast Brazil. Clinical manifestations of these outbreaks included a marked prevalence of moderate to severe progressive lesions in the beaks of affected birds, especially in roosters with increased mortality (up to 8.48%). Also, a reduced hatchability (up to 20.77% fewer hatching eggs) was observed in these flocks. Analysis of clinical samples through light and transmission electron microscopy revealed the presence of Bollinger bodies and poxvirus particles in epithelial cells and affecting chondrocytes. PCR, sequencing, and phylogenetic analysis of major core protein (P4b) and DNA polymerase (pol) genes identified this virus as clade E avipoxvirus. We also developed qPCR assays for open reading frames (ORFs) 49, 114, and 159 to detect and quantify this emergent virus. These results show the arrival and initial spread of this pathogen in the poultry industry, which was associated with harmful outbreaks and exacerbated clinical manifestations in vaccinated commercial breeder flocks. This study also highlights the relevance of permanent vigilance and the need to improve sanitary and vaccination programs.

Poxvirus infection in house finches (Haemorhous mexicanus): Genome sequence analysis and patterns of infection in wild birds.

Poxviruses (family: Poxviridae) infect many avian species, causing several disease outcomes, the most common of which are proliferative lesions on the legs, feet, and/or head. Few avian studies of poxvirus to date have combined molecular and ecological analyses to obtain a more comprehensive understanding of the identity and distribution of the disease in a population. Here, we describe patterns of poxvirus infection in an urban population of house finches (Haemorhous mexicanus) in Arizona (USA) and use high-throughput sequencing to determine the genome sequence of the virus. We found that poxvirus prevalence, based on visual identification of pox lesions, was 7.2% (17 infected birds out of a total of 235 sampled) in our population during summer 2021. Disease severity was low; 14 of the 17 infected birds had a single small lesion on the skin overlaying the eye, leg, and ear canal. All but two lesions were found on the feet; one bird had a lesion on the eye and the other in the ear opening. We also investigated possible temporal (i.e., date of capture) and biological correlates (e.g., age, sex, body condition, degree of infection with coccidian endoparasites) of poxvirus infection in urban-caught house finches during this time but found that none of these significantly correlated with poxvirus presence/absence. Two complete poxvirus genomes were determined from two infected birds. These genomes are ∼354,000 bp and share 99.7% similarity with each other, and 82% with a canarypox virus genome, the most closely related avipoxvirus. This novel finchpox virus is the first to be reported in house finches and has a similar genome organization to other avipoxviruses.

Molecular characterization of avipoxviruses circulating in Windhoek district, Namibia 2021.

Samples from eleven birds (chicken, dove and peacock) with symptoms of fowlpox, caused by the avipoxvirus (APV), were collected in seven different areas of the Windhoek district, Namibia between April and October 2021. A fragment of the 4b core protein and the DNA polymerase gene of APV were amplified by PCR from the DNA of the samples and sequenced. Phylogenetic analysis revealed that the viruses present in the chickens all belonged to clade A1 while the viruses in the doves and peacock were from subclade A3.1. This is the first report of subclade A3.1 avipoxvirus in peacock. In addition, all of the samples obtained from chickens were shown by PCR to be positive for the integration of reticuloendotheliosis virus while those from the doves and peacocks were negative. This study is the first characterization of avipoxvirus in Namibia and provides additional information on the presence of avipoxvirus in southern Africa.

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.

Characterization of Avian Pox in a Ruffed Grouse (Bonasa umbellus) from Pennsylvania.

A free-ranging, adult male ruffed grouse (Bonasa umbellus) was harvested by a hunter during November 2019 in Forest County, PA. The bird was submitted for necropsy due to a skin mass on its left leg. Upon necropsy, two proliferative skin masses were grossly visible, one on the left leg and one on the cere. An additional mass was present on the oropharyngeal mucosa covering the hard palate. These masses were diagnosed as avian pox based on histopathologic and cytologic findings, including marked epithelial hypertrophy, hyperplasia, vacuolar degeneration with eosinophilic stippling, and intracytoplasmic inclusion bodies. An avipoxvirus was detected using PCR and was identified as fowlpox virus through sequencing of the 4b core gene segment. The avipoxvirus from this case showed genetic similarity to isolates from Eastern wild turkeys (Meleagris gallopavo silvestris).

Caracterización de la viruela aviar en un grévol engolado (Bonasa umbellus) en el estado de Pensilvania. Un cazador recolectó un grévol engolado macho adulto silvestre (Bonasa umbellus) durante noviembre del 2019 en el condado de Forest, Pensilvania. El ave fue sometida a necropsia debido a una masa cutánea en su pata izquierda. Durante la necropsia, dos masas cutáneas proliferativas fueron claramente visibles, una en la pierna izquierda y otra en la cera. Había una masa adicional en la mucosa orofaríngea que cubría el paladar duro. Estas masas se diagnosticaron como viruela aviar con base en los hallazgos histopatológicos y citológicos, que incluyeron hipertrofia epitelial marcada, hiperplasia, degeneración vacuolar con punteado eosinofílico y cuerpos de inclusión intracitoplasmáticos. Se detectó un avipoxvirus mediante PCR y se identificó como virus de la viruela aviar mediante la secuenciación del segmento del gene 4b del centro viral. El avipoxvirus de este caso mostró similitud genética con aislamientos de pavos salvajes del este (Meleagris gallopavo silvestris).

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.

Isolation and molecular detection of pigeon pox virus in Assiut and New Valley governorates.

Pigeon Poxvirus (PPV) was detected in eight pigeons suffering from wart like nodular lesions in two Egyptian governorates (Assiut and New Valley) during summer 2018. Different serological and molecular techniques were carried out for isolation and detection of the virus on chorio-allantoic membranes (CAM) of specific-pathogen-free (SPF) embryonated chicken eggs. The characteristic pock lesions were detected on CAMs, whereas PPV was isolated. Electron microscopy revealed enveloped brick shaped Avipoxvirions. The neutralizing antibodies against PPV were detected in six out of eight samples. Serum neutralization test revealed a neutralization index of ≥ 1.6, while ELISA revealed an S/P ratio of ≥ 1.4 in the affected pigeons. Nucleotide sequence of P4b of Pigeon poxvirus isolated from nodule 1 sample (PPVNV1), revealed 100 % nucleotide identity to PPV and only 90 % nucleotide identity with Fowl poxvirus (FPV). P4b locus based SYBR green QPCR produced PPV amplicons of 77.33-77.83 °C melting temperature (Tm). QPCR SYBR green assay successfully differentiated PPV from FPV amplicon which revealed a dissociation curve of Tm =75.85 °C. This is the first report discriminating PPV from FPV based on SYBR green qPCR approach of P4b locus. This isolated local Egyptian strain can be used in vaccine production for optimal vaccination strategy.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Yellow-Eyed Penguin (Megadyptes antipodes).

Emerging viral diseases have become a significant concern due to their potential consequences for animal and environmental health. Over the past few decades, it has become clear that viruses emerging in wildlife may pose a major threat to vulnerable or endangered species. Diphtheritic stomatitis, likely to be caused by an avipoxvirus, has been recognised as a significant cause of mortality for the endangered yellow-eyed penguin (Megadyptes antipodes) in New Zealand. However, the avipoxvirus that infects yellow-eyed penguins has remained uncharacterised. Here, we report the complete genome of a novel avipoxvirus, penguinpox virus 2 (PEPV2), which was derived from a virus isolate obtained from a skin lesion of a yellow-eyed penguin. The PEPV2 genome is 349.8 kbp in length and contains 327 predicted genes; five of these genes were found to be unique, while a further two genes were absent compared to shearwaterpox virus 2 (SWPV2). In comparison with penguinpox virus (PEPV) isolated from an African penguin, there was a lack of conservation within the central region of the genome. Subsequent phylogenetic analyses of the PEPV2 genome positioned it within a distinct subclade comprising the recently isolated avipoxvirus genome sequences from shearwater, canary, and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV2 (96.27%). This is the first reported genome sequence of PEPV2 from a yellow-eyed penguin and will help to track the evolution of avipoxvirus infections in this rare and endangered species.

Molecular characterisation of a novel pathogenic avipoxvirus from an Australian passerine bird, mudlark (Grallina cyanoleuca).

Avipoxviruses have been recognised as significant pathogens in the conservation of numerous bird species. However, the vast majority of the avipoxviruses that infect wild birds remain uncharacterised. Here, we characterise a novel avipoxvirus, mudlarkpox virus (MLPV) isolated from an Australian passerine bird, mudlark (Grallina cyanoleuca). In this study, tissues with histopathologically confirmed lesions consistent with avian pox were used for transmission electron microscopy, and showed characteristic ovoid to brick-shaped virions, indicative of infectious particles. The MLPV genome was >342.7 Kbp in length and contained six predicted novel genes and a further six genes were missing compared to shearwaterpox virus-2 (SWPV-2). Subsequent phylogenetic analyses of the MLPV genome positioned the virus within a distinct subclade also containing recently characterised avipoxvirus genomes from shearwater, canary and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV-2 (94.92%).

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

BACKGROUND: Avian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on obtaining a tissue biopsy, which poses significant risks to birds and challenges in the field. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. Our aim was to address whether pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds. METHODOLOGY AND PRINCIPAL FINDINGS: Pox-like lesion tissue, pectoral muscle, feathers, toenail clippings, blood, and swabs (both pox-like lesion tissue and non pox-like lesion tissue) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types obtained from 27 individuals. These results were compared to those of conventional PCR and comparisons were also made among sample types, utilizing lesion tissue samples as the gold standard. CONCLUSIONS AND SIGNIFICANCE: Hummingbird avian pox can be diagnosed without relying on tissue biopsies. We identify that feather samples, of which contour feathers yielded the best results, can be used for diagnosing infected birds, thus reducing sampling risk. In sum, the real-time PCR assay detected viral DNA in various integumentary system sample types and will be useful in future studies of hummingbird disease ecology.

Avipoxvirus infection in two captive Japanese cormorants (Phalacrocorax capillatus).

Cormorant fishing is a traditional Japanese fishing method using captive Japanese cormorants (Phalacrocorax capillatus). Between June and July 2017, an avian pox outbreak was reported in captive cormorant populations throughout several distant cities in Japan. We examined the lesions obtained from two such affected cormorants, which were raised in distant cities. The affected cormorants were grossly characterized by the development of cutaneous nodules around the base of the beak. Histopathologically, these nodules consisted of marked epidermal hyperplasia with ballooning degeneration of spinous cells and eosinophilic intracytoplasmic inclusions (Bollinger bodies). The lesions displayed 4b core protein (P4b) of Avipoxvirus (APV) and DNA polymerase genes, which were detected by PCR. Moreover, the nucleotide sequences detected from both cormorants were found to be identical. No identical sequence was found in any international database. These findings suggest that both examined cormorants were infected with an identical APV, which has never been previously reported. According to the phylogenetic analysis, the detected sequences were observed to cluster in subclade A3, which consists mainly of the sequences detected from several marine birds, including other cormorant species. This observation suggests that the viruses might be maintained in Japanese cormorants in nature.

Molecular Phylogeny of an Avipoxvirus Isolated from Red-Flanked Blue Robin in China.

We first report avipoxvirus (APV) infection and an isolate named APV/03/2016 from a red-flanked blue robin (Tarsiger cyanurus) captured at Songhua Lake Scenic Area in Jilin City (Jilin Province, China) on March 24, 2016. The partial sequence of the 4b core protein gene and DNA polymerase gene of APV/03/2016 suggests that the virus belongs to the subclade B1 cluster of clade B (canarypox virus). The BLAST results showed the highest similarity of the two genes with the Pacific shearwater-isolated strain SWPV-2 (KX857215), canarypox virus strain D98-11133 (GQ487567), canarypox virus strain ATCC VR-111 (AY318871), avipoxvirus Mississippi isolate P89 (KC018048), and avipoxvirus Wisconsin isolate P92 (KC018051). The results indicate that APV/03/2016 is a canarypox-like virus. These findings demonstrate the continuous emergence of new APV hosts such as red-flanked blue robins and suggest that monitoring of APV circulation and evolution should be strengthened for T. cyanurus conservation.

Filogenia molecular de un Avipoxvirus aislado de ruiseñor coliazul en China. Se reporta por primera vez la infección por poxvirus aviar (APV) y un aislamiento denominado APV/03/2016 obtenido de un ruiseñor coliazul (Tarsiger cyanurus) capturado en el área escénica del Lago Songhua en la ciudad de Jilin (provincia de Jilin, China) el 24 de marzo de 2016. La secuencia parcial del gene de la proteína central 4b y el gene de la polimerasa de ADN del virus APV/03/2016 sugiere que el virus pertenece al subclado B1 del clado B (virus de la viruela del canario). Los resultados de la búsqueda mediante BLAST mostraron la mayor similitud de los dos genes con la cepa aislada de aves pelágicas del Pacífico (KX857215), virus de la viruela del canario cepa de virus D98-11133 (GQ487567), cepa de virus de la viruela del canario ATCC VR-111 (AY318871), aislamiento de avipoxvirus de Mississippi P89 (KC018048), y el aislamiento P89 de avipoxvirus en Wisconsin (KC018051). Los resultados indican que el virus APV/03/2016 es un virus similar al de la viruela del canario. Estos hallazgos demuestran la aparición continua de nuevos hospedadores de poxvirus aviares como el ruiseñor coliazul y sugieren que el monitoreo de la circulación y evolución de poxvirus aviares debería fortalecerse para la conservación del T. cyanurus.

WHOLE GENOME SEQUENCING OF AN AVIPOXVIRUS ASSOCIATED WITH INFECTIONS IN A GROUP OF AVIARY-HOUSED SNOW BUNTINGS (PLECTROPHENAX NIVALIS).

Avipoxvirus infections have been reported in both free-ranging and domestic birds worldwide. Fowlpox and canarypox viruses belong to the genus Avipoxvirus among the virus family Poxviridae. They cause cutaneous lesions with proliferative growths on the unfeathered parts of the skin and/or diphtheritic lesions generally associated with necrosis in the upper respiratory and digestive tracts. In this study, a poxvirus has been identified in wild-caught snow buntings (Plectrophenax nivalis) housed in an outdoor aviary in the region of Rimouski, Quebec. During the falls and winters of 2015 and 2016, eight snow buntings affected by this infection were examined. Macroscopic and microscopic lesions observed were characteristic of an avipoxvirus infection. Electron microscopy imaging of an ultrathin section of the histopathological lesions of two birds confirmed the presence of the poxvirus. Afterward, the presence of the poxvirus was confirmed in three birds by a specific polymerase chain reaction assay that amplified a segment of the gene encoding the fowlpox virus 4b core protein. A 576-nucleotide amplicon was obtained from one of them and sequenced. The analyses revealed a 99% homology to other previously described avipoxviruses. Using high-throughput sequencing, almost the entire viral genome of this avipoxvirus was revealed and found to possess a 359,853-nucleotide sequence in length. Bioinformatic analyses revealed that the virus was genetically related to canarypox virus. To our knowledge, this is the first confirmed case and full description of a poxviral infection in this species. This episode suggests a high susceptibility of this northern species of passerine to avipoxviruses circulating in southeastern Canada during the summer months. Even if the source of the viral infections remains undetermined, transmission by local biological vectors is suspected. Management of poxviral infections in snow buntings housed outdoors in southeastern Canada could rely on the control of biting insects.