Genomic characterization of two novel pathogenic avipoxviruses isolated from pacific shearwaters (Ardenna spp.).

BACKGROUND: Over the past 20 years, many marine seabird populations have been gradually declining and the factors driving this ongoing deterioration are not always well understood. Avipoxvirus infections have been found in a wide range of bird species worldwide, however, very little is known about the disease ecology of avian poxviruses in seabirds. Here we present two novel avipoxviruses from pacific shearwaters (Ardenna spp), one from a Flesh-footed Shearwater (A. carneipes) (SWPV-1) and the other from a Wedge-tailed Shearwater (A. pacificus) (SWPV-2). RESULTS: Epidermal pox lesions, liver, and blood samples were examined from A. carneipes and A. pacificus of breeding colonies in eastern Australia. After histopathological confirmation of the disease, PCR screening was conducted for avipoxvirus, circovirus, reticuloendotheliosis virus, and fungal agents. Two samples that were PCR positive for poxvirus were further assessed by next generation sequencing, which yielded complete Shearwaterpox virus (SWPV) genomes from A. pacificus and A. carneipes, both showing the highest degree of similarity with Canarypox virus (98% and 67%, respectively). The novel SWPV-1 complete genome from A. carneipes is missing 43 genes compared to CNPV and contains 4 predicted genes which are not found in any other poxvirus, whilst, SWPV-2 complete genome was deemed to be missing 18 genes compared to CNPV and a further 15 genes significantly fragmented as to probably cause them to be non-functional. CONCLUSION: These are the first avipoxvirus complete genome sequences that infect marine seabirds. In the comparison of SWPV-1 and -2 to existing avipoxvirus sequences, our results indicate that the SWPV complete genome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus genome isolated from avian or other natural host species, and that it likely should be considered a separate species.

Avian Pox in Native Captive Psittacines, Brazil, 2015.

To investigate an outbreak of avian pox in psittacines in a conservation facility, we examined 94 birds of 10 psittacine species, including sick and healthy birds. We found psittacine pox virus in 23 of 27 sick birds and 4 of 67 healthy birds. Further characterization is needed for these isolates.

Avian pox in crimson rosellas (Platycercus elegans) in southern Australia.

Poxviral infection was identified in a crimson rosella presented to the Australian Wildlife Health Centre (Victoria) in 2002, and from a second crimson rosella in 2008. Both cases were characterized by proliferative lesions on non-feathered skin. Routine histopathology identified intra-lesional epidermal changes consistent with those caused by poxvirus. Electron microscopy confirmed the presence of poxvirus in inclusions in the first case, and genetic analysis of DNA extracted from both cases found an identical viral genome that differs from all other known poxviruses. We conclude that this infection in crimson rosellas is caused by a previously unrecognized avian poxvirus endemic to this region of Australia, and with low virulence.

Avipoxviruses: infection biology and their use as vaccine vectors.

Avipoxviruses (APVs) belong to the Chordopoxvirinae subfamily of the Poxviridae family. APVs are distributed worldwide and cause disease in domestic, pet and wild birds of many species. APVs are transmitted by aerosols and biting insects, particularly mosquitoes and arthropods and are usually named after the bird species from which they were originally isolated. The virus species Fowlpox virus (FWPV) causes disease in poultry and associated mortality is usually low, but in flocks under stress (other diseases, high production) mortality can reach up to 50%. APVs are also major players in viral vaccine vector development for diseases in human and veterinary medicine. Abortive infection in mammalian cells (no production of progeny viruses) and their ability to accommodate multiple gene inserts are some of the characteristics that make APVs promising vaccine vectors. Although abortive infection in mammalian cells conceivably represents a major vaccine bio-safety advantage, molecular mechanisms restricting APVs to certain hosts are not yet fully understood. This review summarizes the current knowledge relating to APVs, including classification, morphogenesis, host-virus interactions, diagnostics and disease, and also highlights the use of APVs as recombinant vaccine vectors.

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%).

Beak necrosis in Hungarian partridges (Perdix perdix) associated with beak-bits and avian poxvirus infection.

Proliferative growth, consistent with poxvirus infection, encapsulated plastic beak-bits and covered the dorsal portion of the upper beak and nares of adult male and female captive-raised Hungarian partridges. Three representative birds were submitted to the Wisconsin Veterinary Diagnostic Laboratory for necropsy. Lesions in the necropsied birds extended through the nares, where the plastic bit ends are designed to rest. The lesions also variably extended caudally into the oropharynx and cranially within the beak epithelium, and included palate deformity and beak necrosis. Poxvirus was diagnosed in all of the birds examined based on histopathology, electron microscopy, and polymerase chain reaction amplification and sequencing. This report is the first to describe avian pox lesions associated with the application of beak-bits and the resulting beak and oral pathology.

Unusual pathology of canary poxvirus infection associated with high mortality in young and adult breeder canaries (Serinus canaria).

Mortality in excess of 65% occurred in a flock of 450 canaries (Serinus canaria). Clinical signs in the canaries included severe respiratory distress, loss of feathers and/or scaly skin on the head, neck and back, anorexia, loss of weight and fluffed-up appearance of several days duration before death. Gross pathology in most of the canaries included thickened eye lids and small scab-like nodules on the skin of the head and neck, enlarged thymus, mild to severe consolidation of lungs and exudate in the sinuses and trachea. A few birds also had thickened air sacs and enlarged and pale spleens. Microscopically unusual lesions included severe epithelial proliferation and hypertrophy and mononuclear inflammatory cells containing eosinophilic intracytoplasmic inclusion bodies of poxvirus in the thymus, bursa of Fabricius, spleen, bone marrow, air sac, peritoneum, external and middle ears, and lachrymal gland. Similar inclusion bodies associated with inflammation were also seen in the epidermis, dermis, feather follicles, conjunctivae, sinuses, turbinates, choana, oral mucosa including tongue, oesophagus, larynx, trachea, syrinx and bronchi and parabronchi of lungs. Some of the birds also had concurrent bacterial, mycotic and polyomavirus infections. Poxvirus was isolated from lungs and skin in chicken embryo liver cells and confirmed as avian poxvirus by polymerase chain reaction.

Poxvirus Infection in a Colony of Laboratory Pigeons (Columba livia).

Pigeons (Columba livia) are used in biomedical research for studies of vision, cognition, neuronal pathways, and spatial orientation. Because there are few commercial laboratory sources, research pigeons are typically acquired from local fancier breeders or bred onsite. For acquired pigeons, the health and vaccine status is often unknown. A juvenile pigeon, born onsite and living in an enclosed outdoor loft, presented with small, bleeding, wart-like lesions on the medial aspects of digits 1 and 4. Topical treatment was initiated. Within a week, 4 fledglings were reported for small, dark papular lesions on the face, head, neck, and beak, and shortly thereafter, 2 additional juvenile pigeons developed similar lesions. The fledglings were euthanized, and histologic examination revealed numerous intralesional eosinophilic cytoplasmic viral inclusions (Bollinger bodies) confirming a diagnosis of poxvirus infection, likely pigeon pox. Although usually self-limiting, pigeon pox can cause moderate to severe lesions in fledgling and juvenile birds. Vaccination with a modified live poxvirus labeled for chickens was used to create herd immunity to pigeon poxvirus. Since vaccination of our entire flock and implementation of more stringent health protocols, all lesions have resolved, and no new lesions have been noted.

Molecular characterization of a poxvirus isolated from an American flamingo (Phoeniconais ruber rubber).

An avian poxvirus from the beak scab of an American flamingo (Phoeniconais ruber rubber) was isolated by inoculation on the chorioallantoic membrane (CAM) of specific-pathogen-free (SPF) chicken embryos. The virus produced multifocal areas of epithelial hyperplasia along with foci of inflammation in the CAM, and rare cells contained small eosinophilic intracytoplasmic bodies. Chickens inoculated with the isolated virus in the feather follicle of the leg did not develop significant lesions. Nucleotide sequence comparison of a PCR-amplified 4.5 kb HindIII fragment of the genome of flamingo poxvirus (FlPV) revealed very high homology (99.7%) with condor poxvirus (CPV), followed by approximately 92% similarity with canary poxvirus (CNPV) and Hawaiian goose poxvirus (HGPV), but less similarity (approximately 69%) to fowl poxvirus (FPV), the type species of the genus Avipoxvirus of family Poxviridae. As in the cases with CPV, CNPV, and HGPV, genetic analysis of FlPV revealed an absence of three corresponding FPV open reading frames (ORF199, 200, and 202) and an absence of any reticuloendotheliosis virus (REV) sequences in this region. There are only nine nucleotide substitutions observed between FlPV and CPV in the 4.5 kb fragment; those were clustered in the ORF201 region, which in FPV genome is a site for integration of REV sequences. Phylogenetic analysis of the predicted amino acid sequences of the ORF201-coded hypothetical protein demonstrated FlPV to be more closely related to CPV, as well as to CNPV and HGPV, than to FPV.

Evaluation of pathogenicity of avian poxvirus isolates from endangered Hawaiian wild birds in chickens.

Pathogenicity of two avian poxviruses isolated from endangered Hawaiian wild birds, the Hawaiian Goose and the Palila, was compared with fowl poxvirus in chickens. Immune responses were measured by ELISA pre- and postimmunization with Hawaiian poxviruses and after challenge with fowl poxvirus. Both isolates from Hawaiian birds developed only a localized lesion of short duration at the site of inoculation in specific-pathogen-free chickens and did not provide protection against subsequent challenge with virulent fowl poxvirus. On the other hand, birds inoculated with virulent fowl poxvirus developed severe lesions. In contrast to high antibody response in chickens immunized with fowl poxvirus, birds immunized with either of the two Hawaiian isolates developed low to moderate antibody responses against viral antigens. The level of immune responses, however, increased in birds of all groups following subsequent challenge.

Interactive effects of turkeypox virus and Plasmodium hermani on Turkey poults.

Two experiments were conducted to examine the interactive effects of two disease agents of wild turkeys (Meleagris gallopavo), turkeypox virus and the malarial organism, Plasmodium hermani, on the health of turkey poults. Groups of domestic broad-breasted white turkey poults of 1 and 10 wk of age were infected with either turkeypox virus, P. hermani, both turkeypox virus and P. hermani, or were maintained as uninfected controls. The strains of turkeypox virus and P. hermani had been isolated from wild turkeys in southern Florida (USA). The goals of these experiments were two-fold and included both an examination of age differences in response to infections, and an examination of the effects of dual versus singular infections with the two agents. Both singular and concomitant infections of turkeypox virus and P. hermani were more detrimental to poults infected at 1 wk of age than to those infected at 10 wk, based on mortality, weight gain, and parasitemia. Dual infections of turkeypox virus and P. hermani were found to be slightly more harmful to 1-wk-old poults than were singular infections. No such interactive effects were noted in the poults infected at 10 wk of age.

Genetic and antigenic characterization of a poxvirus isolate from ostriches.

Avian poxvirus was isolated from nodules on the heads and conjunctiva of two 3-to-4-wk-old ostrich chicks. The ostriches from which poxvirus was isolated had been placed on premises where turkeys that had shown evidence of poxvirus infection had been raised earlier. Microscopically, the nodules from the ostriches were composed of proliferating and hypertrophic epithelial cells that formed large fronds. Most of the hypertrophic epithelial cells contained large eosinophilic intracytoplasmic inclusion bodies characteristic of poxvirus. Characterization of the avian poxvirus isolated from the cutaneous lesions in ostriches was based on western blotting of virus antigen, restriction fragment length polymorphism of genomic DNA, pathogenesis, and cross-protection studies in chickens. Antigenic and genetic studies did not reveal any significant difference between the poxvirus isolated from ostriches (PVO) and fowl poxvirus (FPV). Further, susceptible chickens immunized with the PVO were protected when challenged with a virulent strain of FPV. Thus, the poxvirus isolated from ostriches had similar antigenic, genetic, and biological properties to FPV.

Avipox sp. in a colony of gray-crowned rosy finches (Leucosticte tephrocotis).

Members of a wild-caught colony of 16 gray-crowned rosy finches (Leucosticte tephrocotis) were presented with dermal and mucosal lesions, anorexia, emaciation, lethargy, and sudden death. Lesions included dermatitis, conjunctivitis, and glossitis. Skin scrapings from and bacterial culture of dermal lesions yielded Staphylococcus aureus and Candida albicans. Necropsy and histologic examination revealed characteristic epidermal and mucosal pox lesions, with the presence of characteristic Bollinger body intracellular inclusions. Electron microscopy (EM) provided confirmation of pox virus infection. This epornitic resulted in the death or euthanasia of 12 birds (75% morbidity and associated mortality) and was brought to conclusion through culling of affected birds. The source of infection remains unknown, although multiple modes of introduction exist. Similar epornitics may be prevented through indoor, species-specific housing, and quarantine. Vaccination and antiparasitic treatment may reduce the risk of disease spread.

Characterization of avipoxviruses from wild birds in Norway.

Avipoxviruses from different geographic regions of the world have been characterized to study their genetic and biological properties, but so far, no such work has been performed on Norwegian isolates. Lesions suggestive of avian pox, found on a Norwegian wild sparrow (Passer domesticus) and wood pigeon (Palumbus palumbus), were obtained in 1972 and 1996, respectively. Histologically, these lesions were demonstrated to be characteristic of poxvirus infections and the poxvirus was observed using an electron microscope. The resulting viruses were propagated in chicken embryo fibroblast cells. Restriction fragment length polymorphism of genomes from 2 Norwegian isolates and fowl pox vaccine strain, generated by BamHI, revealed a high degree of heterogeneity among the isolates. The profiles of avipoxviruses isolated from wild birds were clearly distinct from each other and also to the fowl poxvirus strain. Furthermore, chickens experimentally infected with pigeon poxvirus had higher antibody titers and extensive lesions compared to other isolates. This may suggest that pigeon poxvirus is more virulent than the other isolates.

Characterization of poxviruses from forest birds in Hawaii.

Two strains of avian pox viruses were isolated from cutaneous lesions in Hawaiian crows (Corvus hawaiiensis) examined in 1994 and a third from a biopsy obtained in 1992 from an infected bird of the Apapane species (Himatione sanguinea) by inoculation of the chorioallantoic membranes (CAM) of developing chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies characteristic of pox virus infection. The pathogenicity of these three viruses in domestic chickens was mild as evidenced by the development of relatively minor lesions of short duration at the sites of inoculation. Their virulence in this host was similar to that of a fowlpox virus (FPV) vaccine strain and contrasted greatly with the ability of two field strains of FPV to produce extensive proliferative lesions. One of the Hawaiian crow pox virus isolates as well as the one originating from the Apapane species could be propagated in two secondary avian cell lines, QT-35 and LMH. A comparison of the restriction fragment length polymorphisms (RFLP) of the genomes of the two cell line-adapted viruses, generated by EcoRI digestion, revealed a limited degree of similarity. Moreover, neither profile was comparable to those of the two field isolates of FPV, which were almost indistinguishable from each other. Thus, based on the genetic distinctness of the two Hawaiian bird viruses, they appear to represent different strains of avipoxvirus.

Outbreak of cutaneous form of avian poxvirus disease in previously pox-vaccinated commercial turkeys / Surto de bouba aviária na forma cutânea em perus comerciais previamente vacinados

This study describes an outbreak of avian poxvirus disease in previously pox-vaccinated turkeys in Brazil. The turkeys had suggestive gross lesions of cutaneous avian poxvirus in the skin of the head and cervical area without changes in the flock mortality rates. In the slaughterhouse, 30 carcasses were removed from the slaughter line to collect tissue from cutaneous lesions for histological analyses and characterization of the virus. The virus was identified by conventional polymerase chain reaction (PCR) and subsequent gene sequencing. Acanthosis, hyperkeratosis, and hydropic degeneration were seen on skin histopathology. Eosinophilic intracytoplasmic inclusion bodies (Bollinger) on keratinocytes were observed in 46.6% of the samples. Avian poxvirus DNA was detected on PCR in 83.3% of the total samples. PCR associated with histopathology had 93.3% of positivity for avian poxvirus. In the phylogenetic study, samples show 100% matching suggesting that the outbreak occurred by a single viral strain and was different from those strains affecting other wild birds such as canaries and sparrows. A single mutation (Adenine for Guanine) was detected in our study's strain and in the strains of turkey, chickens, and vaccine strains published in GenBank. Also, when the sequence strain of the present study and sequences from GenBank of canarypox and sparrowpox strains were aligned, a Thymine was found replacing the Adenine or Guanine. The in ovo vaccination method as single-use in turkeys of this study apparently did not provide adequate protection against avianpox disease, but additional vaccination administered by wing-web when turkeys were 45-60 days old in the new flocks controlled the disease. In the subsequent year, new cases of this disease were not found. It was not possible to confirm the source of the virus strain, but infection with a field strain derived from chickens is one possibility, considering the poultry farm population in the area and biosecurity aspects. For wide characterization of avipoxvirus and differentiation among strains, the complete sequence of the viral genome is required.(AU)

Este estudo descreve um surto de bouba aviária em perus previamente vacinados contra poxvirus aviário no Brasil. Os perus apresentaram lesões macroscópicas, sugestivas de bouba aviaria cutânea, na pele da cabeça e região cervical sem alteração nas taxas de mortalidade do lote. No abatedouro, 30 carcaças foram retiradas da linha de abate para coleta de dois fragmentos de pele com lesões para análise histológica e caracterização do vírus. A identificação do vírus foi realizada por PCR convencional e posterior sequenciamento. No exame histopatológico das lesões de pele, houve acantose, hiperqueratose e degeneração hidrópica. Corpúsculos de inclusão intracitoplasmáticos eosinofílicos (Bollinger) foram encontrados em 46,6% das amostras. A técnica de PCR detectou o DNA do vírus da bouba aviária em 83,3% do total de amostras. PCR associado com a histopatologia resultou em 93,3% de positividade para o vírus da bouba aviária. No estudo filogenético, as sequências resultaram em 100% de identidade, sugerindo que o surto ocorreu por uma única estirpe de vírus diferenciada das outras estirpes que acometem canários e pardais. Uma única mutação (Adenina para Guanina) foi detectada nas estirpes deste estudo e nas sequências de perus, galinhas e estirpes vacinais publicadas no GenBank. Além disso, quando a sequência da estirpe do presente estudo e as sequências das estirpes de canarypox e sparrowpox foram comparadas, a Timina foi encontrada em substituição a Adenina ou Guanina. A vacinação in ovo em dose única utilizada nos perus deste estudo aparentemente não forneceu proteção adequada contra a doença causada pelo poxvirus aviário. Entretanto, a revacinação na membrana da asa em perus com 45-60 dias de idade dos novos lotes controlou a doença. No ano subsequente, novos casos desta doença não foram registrados. Não foi possível confirmar a origem da estirpe viral, mas estirpes de campo oriundas de galinhas seria uma possibilidade, considerando a população na área e os aspectos de biosseguridade. Para caracterização ampla do avipoxvirus e diferenciação entre as estirpes, a sequência completa do genoma viral é requerida.(AU)

Evaluation of the pathogenicity of avipoxvirus strains isolated from wild birds in New Zealand and the efficacy of a fowlpox vaccine in passerines.

Avipoxvirus (APV) infection is a highly contagious disease of birds and has been reported in more than 200 bird species, affecting both domesticated and free-ranging birds around the world. In New Zealand, at least three different strains of Avipoxvirus (APV) have been identified in a range of bird species.The pathogenicityof two APV strains isolated from wild birds in New Zealand, representing subclade A1 and subclade B1 were compared in zebra finches (Taeniopygia guttata). The efficacy of fowlpox vaccine at preventing clinical disease in passerines was also evaluated. Twenty-five zebra finches were divided into five groups (I-IV and a control group). Birds from Groups II and IV were vaccinated using fowl poxvirus vaccine prior to challenge. Subsequently two groups (I and II) were inoculated with a silvereye isolate (A1) and the other two groups (Group III and IV) were inoculated with a blackbird isolate (B1). Both inocula were previously propagated in chicken fibroblast cell culture. Birds in the control group were inoculated with sterile PBS. Skin thickness at the inoculation sites was measured and the development of additional skin lesions was monitored. Antibody development was measured by ELISA pre- and post virus inoculation. Both APV strains caused either swelling or hyperplasia at the inoculation site of non-vaccinated birds (4/5 in Group I and 5/5 in Group III). The swelling was milder and no foot lesions were observed in vaccinated birds before or after challenge with the silvereye or blackbird APV strains. These findings indicated that the fowlpox vaccine provided safe and appropriate protection for zebra finches exposed to the two wild APV strains and suggest that the vaccine has the potential to be used where APV threatens the captive management or translocation of endangered passerines.

Ecology and conservation biology of avian malaria.

Avian malaria is a worldwide mosquito-borne disease caused by Plasmodium parasites. These parasites occur in many avian species but primarily affect passerine birds that have not evolved with the parasite. Host pathogenicity, fitness, and population impacts are poorly understood. In contrast to continental species, introduced avian malaria poses a substantial threat to naive birds on Hawaii, the Galapagos, and other archipelagoes. In Hawaii, transmission is maintained by susceptible native birds, competence and abundance of mosquitoes, and a disease reservoir of chronically infected native birds. Although vector habitat and avian communities determine the geographic distribution of disease, climate drives transmission patterns ranging from continuous high infection in warm lowland forests, seasonal infection in midelevation forests, and disease-free refugia in cool high-elevation forests. Global warming is expected to increase the occurrence, distribution, and intensity of avian malaria across this elevational gradient and threaten high-elevation refugia, which is the key to survival of many susceptible Hawaiian birds. Increased temperatures may have already increased global avian malaria prevalence and contributed to an emergence of disease in New Zealand.

Disease and the dynamics of extinction.

Invading infectious diseases can, in theory, lead to the extinction of host populations, particularly if reservoir species are present or if disease transmission is frequency-dependent. The number of historic or prehistoric extinctions that can unequivocally be attributed to infectious disease is relatively small, but gathering firm evidence in retrospect is extremely difficult. Amphibian chytridiomycosis and Tasmanian devil facial tumour disease (DFTD) are two very different infectious diseases that are currently threatening to cause extinctions in Australia. These provide an unusual opportunity to investigate the processes of disease-induced extinction and possible management strategies. Both diseases are apparently recent in origin. Tasmanian DFTD is entirely host-specific but potentially able to cause extinction because transmission depends weakly, if at all, on host density. Amphibian chytridiomycosis has a broad host range but is highly pathogenic only to some populations of some species. At present, both diseases can only be managed by attempting to isolate individuals or populations from disease. Management options to accelerate the process of evolution of host resistance or tolerance are being investigated in both cases. Anthropogenic changes including movement of diseases and hosts, habitat destruction and fragmentation and climate change are likely to increase emerging disease threats to biodiversity and it is critical to further develop strategies to manage these threats.