Isolation and genetic characterization of swinepox virus from pigs in India

Swinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in India.     

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.     

Differentiation of avian poxvirus strains on the basis of nucleotide sequences of 4b gene fragment

Investigations for detection and differentiation of nine avian poxviruses (APVs) were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis (REA) and further nucleotide sequence analysis. With one primer set, which framed a region within the fowl poxvirus 4b core protein gene, we were able to detect APV-specific DNA from 19 tested strains and isolates belonging to five defined Avipoxvirus species and four previously undefined isolated species. PCR results revealed no recognizable differences in size of amplified fragments among the different APVs. REA of PCR products with MseI and EcoRV allowed us to differentiate most of the tested avipox species. Nucleotide sequence analysis of the amplified fragments showed a nucleotide similarity of 72%-100% among the different species. Phylogenetic analysis documented five distinguishable sequence clusters in accordance with results obtained by REA. PCR in combination with REA and sequencing of the amplified fragments is a rapid and effective diagnostic system, and it is a new approach to refine epidemiologic studies of APV infections.     

Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox

The present report documents the occurrence of a poxvirus infection in commercial meat turkeys. The affected farm had six flocks, with a total of 11,680 birds at different ages; birds from two of these flocks were affected. The clinical picture was characterized by severe epithelial lesions and proliferations on the head and neck regions as reported for the cutaneous form of poxvirus infection. Except for these lesions, no adverse clinical signs or gross pathologic lesions were observed. Only a low number of birds was affected (n = 20) and no increase of mortality could be seen. Bacteriologic investigations from the lesions revealed multiresistant Staphylococcus aureus. Eosinophilic inclusions (Bollinger bodies) in histologic examinations in the cytoplasm of keratinocytes were noticeable. Typical pox virions were demonstrated by electron microscopy, and poxvirus was isolated on the chorioallantoic membrane of specific-pathogen-free chicken eggs. Further identification of the poxvirus species was carried out by PCR and sequencing, revealing an infection with the species fowlpox. Layers in vicinity of the turkey farm that also were affected by fowlpox were considered as potential source of infection. Although it is assumed that avian poxviruses are strongly species specific, the present case report reinforces the changing picture of poxvirus infections in turkeys. Furthermore, it supports the assumption of previous data that fowlpox virus has to be seen as recently emerging pathogen in turkeys.     

Unusual presentations of cowpox infection in cats

Cowpox virus infections are reported typically to cause focal ulcerated, crusted skin lesions, sometimes with mild systemic illness and concurrent oral lesions. Severe systemic illness usually only occurs in young or immunosuppressed individuals. This report describes four cases of cowpox infection in cats which illustrate variations to the usual presentation of the virus. The poxvirus infections were confirmed histopathologically, serologically and by PCR analysis.     

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.     

Comparative sequence analysis of B5R gene of zoonotic buffalo pox virus isolates with other orthopoxviruses

The present paper describes the isolation of buffalo pox virus from scab lesions and its molecular characterization through B5R gene sequencing. During our study, pustular pox lesions were observed on the teats and mammary parenchyma of cattle and buffaloes, and the disease was of significant zoonotic importance since similar lesions were produced on the hands, legs, and face of people in close contact with the affected animals. The collected scab materials were subjected for virus isolation in 9–11-day-old chicken embryos by the chorioallontoic membrane route and in the Vero cell line. The virus was confirmed by a sensitive and rapid diagnostic polymerase chain reaction using the primers that amplify “A type inclusion” gene, and further, B5R gene of the virus was sequenced and compared with the corresponding sequences of other orthopoxviruses. The results showed high sequence homology of our isolates with other orthopoxviruses.     

Poxvirus infection in a cat with presumptive human transmission

The present report describes a case of generalized cowpox virus infection with necrotizing facial dermatitis in a cat and a likely transmission to an animal keeper. The viral aetiology was confirmed by histopathology, immunohistochemistry, PCR, virus isolation, DNA sequencing and electron microscopy. Histopathological examination of the cat's skin revealed a severe, necrotizing dermatitis with ballooning degeneration and hyperplasia of epithelial cells with pathognomonic cytoplasmic eosinophilic inclusion bodies. Additionally, at post-mortem examination, a systemic poxvirus infection was detected affecting pancreas, thymus, lymph node, liver and lung. The human patient's skin biopsy revealed an ulcerative dermatitis with epidermal hyperplasia and ballooning degeneration. Serological investigation displayed a high orthopoxvirus-specific antibody titre in the human patient. Environmental factors increase the natural reservoir host population for cowpox viruses, such as voles, which results in a higher risk of infection for cats and subsequently for humans. Due to this zoonotic potential, a cowpox virus infection must be considered as an aetiological differential in cases of necrotizing dermatitis in cats.     

Reticuloendotheliosis virus integration in the fowl poxvirus genome: not a recent event

Integration of reticuloendotheliosis virus (REV) into the genome of fowl poxvirus (FPV) has been reported recently. With a view to determine whether this event had occurred in the past, we screened by polymerase chain reaction (PCR) for the presence of REV provirus in the DNAs of nine avian poxviruses, some of which had been lyophilized 50 yr ago. For REV, 5' long terminal repeat (LTR) and REV envelope sequences were amplified, whereas for FPV, the major envelope antigen gene and the region flanking REV sequences were amplified. In six of seven FPV strains examined, the specific PCR amplicons were obtained for both REV provirus and FPV sequences. One isolate in which presence of REV 5' LTR and envelope was not detected by PCR, a LTR remnant was detected by Southern hybridization. Interestingly, no REV sequence was detected in either canary poxvirus or pigeon poxvirus genome. These observations indicate that REV integration in the FPV genome is not a recent phenomenon but probably occurred prior to 1949.     

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.     

Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.

A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains.     

Poxvirus infection in a great tit (Parus major)

Four great tits (Parus major) with nodular lesions suggestive of poxvirus infections were observed in a garden in Vienna, Austria. One of the birds was submitted for examination. Because of its poor condition, the bird had to be euthanatized and was subsequently necropsied. An avipoxvirus infection was confirmed by histopathology, electron microscopy, and polymerase chain reaction and sequencing. This is the second report of naturally occurring poxvirus infection in great tits and the first of its kind in central Europe.     

Antigenic and genetic characterization of an avian poxvirus isolated from an endangered Hawaiian goose (Branta sandvicensis)

An avian poxvirus from cutaneous lesions in a Hawaiian goose (Branta sandvicensis) was characterized in this study. The virus was isolated by inoculation onto the chorioallantoic membranes (CAMs) of developing chicken embryos. Cytoplasmic inclusion bodies were observed on histopathological examination of CAM lesions. Western blotting analysis using polyclonal antiserum against fowl poxvirus (FWPV) showed differences from FWPV, but a similar antigenic profile between Hawaiian goosepox (HGP) isolate and two previous Hawaiian poxvirus isolates were observed. Still three avian poxviruses from Hawaiian birds showed distinguishable reaction in approximately 27, 34, 35, and 81 kDa proteins when polyclonal antibodies against the Hawaiian poxvirus isolate (Alala/lanakila) were used. Restriction fragment length polymorphisms (RFLP) of DNA of this isolate also showed differences from those of FWPV and previous avianpox isolates from Hawaiian forest birds. While nucleotide sequences of a 5.3-kb PstI-HindIII fragment of the genome of HGP isolate revealed very high homology (99% identities) with Canary poxvirus (CNPV) ORF266-274, and like CNPV, homologs of three FWPV ORFs (199, 200, and 202) including any reticuloendotheliosis virus (REV) sequences are not present in the genome of HGP isolate.     

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.     

Swinepox--skin disease with sporadic occurrence

Swinepox virus infection results in an acute, mild or subclinical course and is characterised by typical poxvirus skin lesions in affected pigs. Additionally, sporadic vertical swinepox virus transmission leads to congenital generalised infection and subsequent abortion or stillbirth. The present report describes the occurrence of epidermal efflorescences in two piglets after intrauterine natural suipoxvirus infection. No clinical abnormalities of the gilt and littermates as well as in other pigs from this herd were present. One of the affected piglets was stillborn and submitted for necropsy, the other animal was alive at birth, but died 3 days later. Histologically, a proliferative to ulcerative dermatitis with epithelial ballooning degeneration and characteristic intracytoplasmatic inclusion bodies was observed. The pathomorphological and histopathological suspected diagnosis of a poxvirus infection was confirmed by electron microscopy. Furthermore, the agent was identified as suipoxvirus by polymerase chain reaction. As demonstrated here, obvious skin lesions in suipoxvirus infection leads to a suspected diagnosis in newborn piglets on macroscopic examination. However, further post mortem examinations, including electron microscopy as well as molecular techniques are essential for the identification of the aetiology and the exclusion of differential diagnoses. Because the disease only affected two pigs there was only a small economic loss. A valid diagnostic plays an important role in advising farmers and for herd health monitoring.     

Detection of Fowl Adenovirus Associated With Hydropericardium Hepatitis Syndrome by a Polymerase Chain Reaction

Hydropericardium hepatitis syndrome HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction PCR) was developed to detect the fowl adenovirus FAV) associated with HHS. The virus from infectedl ivers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.     

An epizootic of avian pox in endemic short-toed larks (Calandrella rufescens) and Berthelot's pipits (Anthus berthelotti) in the Canary Islands, Spain

Between January 2002 and November 2003, 50% (n = 395) of short-toed larks (Calandrella rufescens) and 28% (n = 139) of Berthelot's pipits (Anthus berthelotti) examined on the islands of Fuerteventura and Lanzarote, Canary Islands, had gross lesions compatible with avian pox. However, Spanish sparrows (Passer hispaniolensis, n = 128) and trumpeter finches (Bucanetes githagineus, n = 228), which inhabit the same steppe habitats associated with goat husbandry, did not have poxlike lesions. Histopathology and electron microscopy confirmed poxvirus in the lesions, whereas serology using standard, fowl poxvirus-and pigeon poxvirus-based diagnostic agar gel immunodiffusion techniques was negative, likely because of the limited (74.6% pipit; 74.9% lark) similarity between the viruses in our species and fowlpox virus on which the serologic tests rely. On the basis of polymerase chain reaction analyses, the virus isolated from dried lesions of C. rufescens has 80.5% similarity with the virus isolated from A. berthelotti and 91.3% similarity with canarypox, whereas A. berthelotti poxvirus has only 80% similarity with canarypox. We have two distinct and possibly new avian poxviruses. Both poultry and the wild birds on the farms were heavily infested by fleas, which may have acted as vectors in transmission of poxvirus. Disease prevalence in these Canary Island passerines is higher than that described in song birds in Hawaii that are now threatened, endangered, or extinct. Environmental and biological factors contributing to increased disease susceptibility of these isolated populations must be investigated.     

Study of vertical transmission of fowl adenoviruses

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.     

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.