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%).

Molecular characterisation of a novel pathogenic avipoxvirus from the Australian magpie (Gymnorhina tibicen).

Avipoxviruses are significant pathogens infecting a wide range of wild and domestic bird species globally. Here, we describe a novel genome sequence of magpiepox virus (MPPV) isolated from an Australian magpie. In the present study, histopathologically confirmed cutaneous pox lesions were used for transmission electron microscopic analysis, which demonstrated brick-shaped virions with regular spaced thread-like ridges, indicative of likely infectious particles. Subsequent analysis of the recovered MPPV genome positioned phylogenetically to a distinct sub-clade with the recently isolated avipoxvirus genome sequences from shearwater and canary bird species, and demonstrates a high degree of sequence similarity with CNPV (96.14%) and SWPV-2 (95.87%). The novel MPPV complete genome is missing 19 genes with a further 41 genes being truncated/fragmented compared to SWPV-2 and contains nine predicted unique genes. This is the first avipoxvirus complete genome sequence that infects Australian magpie.

Emergence of a novel avian pox disease in British tit species.

Avian pox is a viral disease with a wide host range. In Great Britain, avian pox in birds of the Paridae family was first diagnosed in a great tit (Parus major) from south-east England in 2006. An increasing number of avian pox incidents in Paridae have been reported each year since, indicative of an emergent infection. Here, we utilise a database of opportunistic reports of garden bird mortality and morbidity to analyse spatial and temporal patterns of suspected avian pox throughout Great Britain, 2006-2010. Reports of affected Paridae (211 incidents) outnumbered reports in non-Paridae (91 incidents). The majority (90%) of Paridae incidents involved great tits. Paridae pox incidents were more likely to involve multiple individuals (77.3%) than were incidents in non-Paridae hosts (31.9%). Unlike the small wart-like lesions usually seen in non-Paridae with avian pox in Great Britain, lesions in Paridae were frequently large, often with an ulcerated surface and caseous core. Spatial analyses revealed strong clustering of suspected avian pox incidents involving Paridae hosts, but only weak, inconsistent clustering of incidents involving non-Paridae hosts. There was no spatial association between Paridae and non-Paridae incidents. We documented significant spatial spread of Paridae pox from an origin in south-east England; no spatial spread was evident for non-Paridae pox. For both host clades, there was an annual peak of reports in August/September. Sequencing of the avian poxvirus 4b core protein produced an identical viral sequence from each of 20 great tits tested from Great Britain. This sequence was identical to that from great tits from central Europe and Scandinavia. In contrast, sequence variation was evident amongst virus tested from 17 non-Paridae hosts of 5 species. Our findings show Paridae pox to be an emerging infectious disease in wild birds in Great Britain, apparently originating from viral incursion from central Europe or Scandinavia.

Evaluation of tip-enhanced Raman spectroscopy for characterizing different virus strains.

Tip-enhanced Raman spectroscopy (TERS) is a highly sensitive spectroscopic technique which combines the advantages of optical spectroscopy with the requirements needed for the characterization of biological nano-structures. In this study, TERS was used to investigate the applicability of this spectroscopic technique for the detection of different virus strains like avipoxvirus and adeno-associated virus. TERS spectra obtained from different particles of the same virus strain show variations in relative peak intensities and positions of most spectral features observed. These spectral variations were higher for the larger avipoxvirus particles (∅≈350 nm) than for the smaller adeno-associated virus particles (∅≈26 nm).

Follow-up of the development of femoral degeneration lesions in broilers / Seguimiento del desarrollo de lesiones por degeneración en pollos de engorde

A study was carried out in the experimental facilities of FMVZ/UNESP-Botucatu, with the aim of following-up the development and the incidence of femoral degeneration (FD). A total of 305 one-day-old male broilers were housed in six pens of 5m² each. Histological analyses of femur head collected when broilers were 0, 7, 14, 21, 28, 35, and 42 days of age were carried out. At 42 days of age, 30 birds were taken to the experimental processing plant of FMVZ for leg gross examination. Ten legs per FD score where selected, and histologically analyzed to determine the most probable age at the beginning of the lesions, and to standardize femoral degeneration lesion scores. The histological results showed that cell architecture started to disorganize at 21 days of age in the resting and proliferation zones, and that angiogenesis increased, invading the joint cartilage, The gross lesion indexes due to femoral degeneration were 22.5 percent, 42.5%, and 65% at 28, 35, and 42 days of age, respectively.

Se realizó un estudio en las instalaciones experimentales de FMVZ/UNESP-Botucatu, con el objetivo de seguir el desarrollo y la incidencia de degeneración femoral (DF) en pollos. Se utilizaron 305 polluelos de un día, machos, distribuidos en seis corrales de 5m² cada uno. Se analizaron cortes histológicos de cabezas de fémur recolectadas a los 0, 7, 14, 21, 28, 35 y 42 días de edad. A los 40 días de edad, se llevaron 30 aves al Matadero Experimental de FMVZ, para análisis macroscópico de las piernas. Se escogieron 10 muslos por escore de DF, y se analizaron histológicamente para determinar la edad más probable del inicio de la lesión y estandarizar los escores de lesión por degeneración femoral. Los resultados histológicos indicaron que a los 21 días ocurre el inicio de la desorganización celular en la zona de reposo y de proliferación, además del aumento de la angiogénesis, invadiendo el cartílago articular. Microscópicamente, el índice de lesión por degeneración femoral fue del 22.5 por ciento, 42.5% y 65% a los 28, 35 y 42 días de edad, respectivamente.

Avipoxvirus in blackcaps (Sylvia atricapilla).

From July to September 2005, 1075 wild birds of 37 species were mist-netted at a location in the north-eastern part of the Czech Republic. The birds were examined for the presence of avipoxvirus lesions. This was demonstrated by electron microscopy in skin lesions in nine of 244 blackcaps (Sylvia atricapilla) examined (4% prevalence). Blackcaps skin bioptates were processed using the ultrathin section method. In skin bioptates, avipoxviruses were demonstrated in intracytoplasmic inclusions where, in addition to mature viruses, lipids and filamentous structures concentrated into large circular formations were found. The so-called additional inclusions were also found. These did not contain any virus components, and they served as the precursor of A-type intracytoplasmic inclusions. Blackcap avipoxvirus was isolated by passage on the chorioallantoic membrane of 9-day-old chicken embryos. The virus was successfully adapted after 11 passages (each passage lasted 5 to 7 days), at which time a marked changes in the form of tiny nodules 2 to 3 mm in diameter were observed on the chorioallantoic membrane. Further identification of field isolates and of the cultured virus was carried out using polymerase chain reaction and sequencing. Sequences were compared with consensus sequences of both canarypoxviruses and fowlpoxviruses. Our sequence was found to be 98.8% identical to the canarypox consensus sequence, but only 63% identical to the fowlpox consensus sequence. Our avipoxvirus sequence was proven to be significantly more closely related to canarypoxviruses than to fowlpoxviruses also by phylogenetic analysis.

Avipoxvirus infection in a collection of captive stone curlews (Burhinus oedicnemus).

A natural outbreak of avipoxvirus occurred in recently purchased stone curlews (Burhinus oedicnemus) at a breeding farm and subsequently spread to other stone curlews residing at the farm. The initial outbreak was characterized by mild vesicular skin lesions on the legs, which then developed crusts and bled. The overall morbidity rate was 100%, but none of the birds died, and all recovered without complication. Four gallinaceous species, also kept on the farm, did not develop lesions. Avipoxvirus was identified from the skin lesions by virus isolation, electron microscopy, and monoclonal antibody testing, as well as by polymerase chain reaction testing. Eight months after this outbreak, 7 male stone curlews developed large, round, crusty lesions on their legs. Although poxvirus virions were identified in the lesions, results of virus isolation were negative. These lesions possibly were the result of a recrudescence of the original infection in male birds that were stressed because they were housed together during the breeding season. This is the first clinical description of an avipoxvirus infection in stone curlews.

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.

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.

Budding of fowlpox and pigeonpox viruses at the surface of infected cells.

Chick embryo fibroblasts and chorioallantoic membranes infected with fowlpox virus (FWPV) or pigeonpox virus (PPV) were examined by transmission and scanning electron microscopy. Immature virus particles were observed in finely granular areas, i.e. virus factories, of the cytoplasm. These particles had various forms depending on their stages of development. Many tubular structures were also seen in these regions. Mature virus particles with ellipsoidal or brick-shaped forms enclosing electron-dense cores were detected throughout the cytoplasm. Notably, there was a high frequency of virus budding at the cell surface, but only occasional virus wrapping in the cytoplasm. Another remarkable feature of the infected cells was accumulation of many virions just beneath the plasma membrane, indicating that this phenomenon is closely related to virus budding. Based on the observed frequency of budding, this mechanism seems to be the predominant way for FWPV and PPV to exit the cell.

Avian pox in sanderlings from Florida.

Asian pox was diagnosed in three sanderlings (Calidris alba) on Sanibel Island, Florida (USA) in February 1997. All three cases had large tumor-like lesions which contributed significantly to their mortality. Poxvirus infection was confirmed by cytology, histopathology, and electron microscopy. This is the first report of poxvirus infection in sanderlings.

Efficient human immunodeficiency virus ( HIV)-1 Gag-Env pseudovirion formation elicited from mammalian cells by a canarypox HIV vaccine candidate.

Canarypox viruses undergo abortive replication in mammalian cells. Despite this restriction on replication in mammalian cells, significant immune responses have been shown in animals and in humans receiving recombinant canarypox vaccine vectors expressing heterologous immunogens. A recombinant canarypox vaccine candidate (vCP205), which expresses human immunodeficiency virus (HIV)-1 Gag, Env, and protease proteins, is presently under investigation in phase I and phase II human trials in the United States and elsewhere. In this study, the ability of vCP205 to elicit HIV Gag-Env pseudovirion formation in avian and mammalian cells was investigated. Gag-Env pseudovirions were produced from both avian and mammalian cell lines infected by this vaccine vector. A subset of mammalian cells was identified in which pseudovirion production and release was very efficient, surpassing the production from infected avian cells. The production of Gag-Env pseudovirions by canarypox HIV vaccine vectors may have important implications for future HIV vaccine design.

Avian pox in eastern screech owls and barred owls from Florida.

Avian pox was diagnosed in two eastern screech owls (Otus asio) and two barred owls (Strix, varia) living in different regions of Florida (USA) between November 1994 and October 1995. Avian poxvirus infection was confirmed by the presence of eosinophilic intracytoplasmic epidermal inclusions (Bollinger bodies) on light microscopy of tissue from all four owls. Additionally, typical poxvirus particles were demonstrated by electron microscopy of a lesion from one of the eastern screech owls. These are the first published case reports of avian pox in eastern screech owls and barred owls.

An epornitic of avian pox in houbara bustards (Chlamydotis undulata macqueenii).

An epornitic of avian pox occurred in a flock of 123 houbara bustards (Chlamydotis undulata macqueenii) received at the Sulman Falcon Hospital in the State of Bahrain in February 1993. Birds displayed conjunctivitis, excessive lacrimation and papilloma-like growths forming amorphous clusters on the third eyelid and on the conjunctiva. Examination of eyelid samples under transmission electron microscopy revealed pox virus particles displaying the classical morphology of vaccinia-avipox virions. Typical pox lesions were also detected 5 days post infection (p.i.) on chorioallantoic membrane (CAM). The virus titre on CAM was 10(7) focus-forming units (FFU)/ml. In tissue culture, only a slight cytopathogenic effect (CPE) was detected 5 days p.i. the virus titre on cell cultures was 10(4.5)FFU/ml. The virus infection in cell culture appeared to be abortive and no CPE was seen after three passages in secondary chicken embryo fibroblasts. No neutralization of the cell-grown virus was detected on serological studies using antisera directed against fowl, pigeon, canary and sparrowpox viruses. The discussion is framed around the different cultural properties of the houbara bustard pox virus isolate and its relationship to other known viral strains.