Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

Survival of two avian respiratory viruses on porous and nonporous surfaces

The transmission of pathogens from infected to susceptible hosts may occur through contaminated fomites and inanimate objects. This type of transmission depends on the ability of the pathogens to survive in the environment. In this report, we describe the survivability of two avian respiratory viruses, e.g., avian metapneumovirus and avian influenza virus on 12 different porous and nonporous surfaces. The viruses survived on some of the surfaces for up to 6 days postcontamination but not after 9 days. Both viruses survived longer on nonporous surfaces than on porous ones. One of the reasons for poor survival on porous surfaces could be inefficient elution of virus from these surfaces. These results should be helpful in determining how long the premises should be left vacant after an outbreak of these viruses has occurred in poultry houses.     

The role of backyard poultry flocks in the epidemic of highly pathogenic avian influenza virus (H7N7) in the Netherlands in 2003

In recent years, outbreaks of highly pathogenic avian influenza (HPAI) viruses have caused the death of millions of poultry and of more than 200 humans worldwide. A proper understanding of the transmission dynamics and risk factors for epidemic spread of these viruses is key to devising effective control strategies. The aim of this study was to quantify the epidemiological contributions of backyard flocks using data from the H7N7 HPAI epidemic in the Netherlands in 2003. A dataset was constructed in which flocks in the affected area were classified as susceptible (S), infected but not yet infectious (E), infectious (I), and removed (R). The analyses were based on a two-type SEIR epidemic model, with the two types representing commercial poultry farms and backyard poultry flocks. The analyses were aimed at estimation of the susceptibility (g) and infectiousness (f) of backyard flocks relative to commercial farms. The results show that backyard flocks were considerably less susceptible to infection than commercial farms (), while estimates of the relative infectiousness of backyard flocks varied widely (). Our results indicate that, from an epidemiological perspective, backyard flocks played a marginal role in the outbreak of highly pathogenic avian influenza in the Netherlands in 2003.     

Pathogenicity of two recombinant avian leukosis viruses

We have recently described the isolation and molecular characteristics of two recombinant avian leukosis subgroup J viruses (ALV J) with an avian leukosis virus subgroup A envelope (r5701A and r6803A). In the present study, we examined the role of the subgroup A envelope in the pathogenesis of these recombinant viruses. Chickens of line 151(5) x 7(1) were inoculated at 1 day of age with r5701A, r6803A, Rous-associated virus type 1 (RAV-1), or strain ADOL-Hcl of ALV-J. At 2, 4, 10, 18, and 32 wk postinoculation (PI), chickens were tested for avian leukosis virus (ALV)-induced viremia, shedding, and neutralizing antibodies. All except one chicken inoculated with the recombinant viruses (98%) developed neutralizing antibodies by 10 wk PI compared with only 16% and 46% of the ADOL-Hcl and RAV-1-inoculated birds, respectively. ALV-induced tumors and mortality in the two groups inoculated with recombinant viruses were different. The incidence of tumors in groups inoculated with r5701A or RAV-1 was 100% compared with only 9% in the groups inoculated with r6803A or ADOL-Hcl. The data suggest that differences in pathogenicity between the two recombinant viruses might be due to differences in the sequence of the 3' untranslated region (presence or absence of the E element), and, therefore, not only the envelope but also other elements of the viral genome play an important role in the pathogenesis of ALV.     

Conventional and future diagnostics for avian influenza

The significant and continued transboundary spread of Asian avian influenza H5N1 since 2003, paired with documented transmission from avian species to humans and other mammals, has focused global attention on avian influenza virus detection and diagnostic strategies. While the historic and conventional laboratory methods used for isolation and identification of the virus and for detection of specific antibodies continued to be widely applied, new and emerging technologies are rapidly being adapted to support avian influenza virus surveillance and diagnosis worldwide. Molecular tools in particular are advancing toward lab-on-chip and fully integrated technologies that are capable of same day detection, pathotyping, and phylogenetic characterization of influenza A viruses obtained from clinical specimens. The future of avian influenza diagnostics, rather than moving toward a single approach, is wisely adopting a strategy that takes advantage of the range of conventional and advancing technologies to be used in "fit-for-purpose" testing.     

Phylogenetic analysis of avian poxviruses among free-ranging birds of Virginia

Polymerase chain reaction was used to amplify a portion of the avian poxvirus core 4b gene of infected free-ranging birds that presented at the Wildlife Center of Virginia during the 2003 and early 2004 years. The species of bird infected were a great blue heron (Ardea herodias), two American crows (Corvus brachyrhyncos), two American robins (Turdus migratorius), two mourning doves (Zenaida macroura), a red-tailed hawk (Buteo jamaicensis), a blue-gray gnatcatcher (Polioptila caerulea), a northern mockingbird (Mimus polyglottos), a house finch (Carpodacus mexicanus), and a northern cardinal (Cardinalis cardinalis). Phylogenetic analysis was performed using the consensus sequences determined for each avian case in Virginia in combination with avian poxvirus core 4b gene sequence from isolates previously described in Europe and that of vaccinia virus. Alignment of DNA sequences identified areas of point mutations and, in the case of a single mourning dove, the incorporation of a triplet of nucleotides. Maximum-likelihood analysis grouped the 2003-2004 Virginia avian poxviruses into a clade distinct from those reported in European free-ranging birds, with the exception of a single case in a mourning dove that clustered within one European clade. The cladogram that resulted from our analysis of the European isolates is in agreement with those previously published. This study identified a distinct clade of avian poxvirus unique from four clades previously described and associated with epornitics in free-ranging birds, where the core 4b gene DNA sequence has been the basis of comparison.     

Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005

This article reports the genetic and pathogenic characteristics of 34 isolates of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Genetic analyses showed that a unique lineage of H6N1 viruses has been established in domestic chickens in Taiwan since 1997, and this lineage of viruses differs from the H6N1 viruses circulating in Hong Kong and Southeastern China. Pathogenicity tests showed that all Taiwanese H6N1 viruses were of low pathogenicity but might lead to economic loss when associated with other diseases. Hemagglutination inhibition tests showed that antigenic drift has occurred in Taiwanese H6N1 viruses, and sequence comparison has identified a total of five possible antigenic sites on the hemagglutinin molecule of the H6N1 viruses. Some Taiwanese H6N 1 viruses could replicate in mice without preadaptation, indicating that these viruses have the potential to cause cross-species infection into mammals.     

Evolution of avian influenza viruses

Although influenza viruses can infect a wide variety of birds and mammals, the natural host of the virus is wild waterfowl, shorebirds, and gulls. When other species of animals, including chickens, turkeys, swine, horses, and humans, are infected with influenza viruses, they are considered aberrant hosts. The distinction between the normal and aberrant host is important when describing virus evolution in the different host groups. The evolutionary rate of influenza virus in the natural host reservoirs is believed to be slow, while in mammals the rate is much higher. The higher rate of evolution in mammals is thought to be a result of selective pressure on the virus to adapt to an aberrant host species. Chickens and turkey influenza virus isolates have previously and incorrectly been lumped together with wild waterfowl, gull, and shorebird influenza viruses when determining rates of evolutionary change. To determine mutational and evolutionary rates of a virus in any host species, two primary assumptions must be met: first, all isolates included in the analysis must have descended from a single introduction of the virus, and second, the outbreak must continue long enough to determine a trend. For poultry, three recent outbreaks of avian influenza meet these criteria, and the sequences of the hemagglutinin and nonstructural genes were compared. Sequences from all three outbreaks were compared to an avian influenza virus consensus sequence, which at the amino acid level is highly conserved for all the internal viral proteins. The consensus sequence also provides a common point of origin to compare all influenza viruses. The evolutionary rates determined for all three outbreaks were similar to what is observed in mammals, providing strong evidence of adaptation of influenza to the new host species, chickens and turkeys.     

Expression of avian influenza virus hemagglutinin by recombinant fowlpox virus

A vaccine strain of fowlpox virus (FPV) was genetically engineered to produce avian influenza virus hemagglutinin (HA). This was accomplished by inserting a cDNA copy of the avian influenza virus HA gene, which was regulated by a vaccinia virus promoter, into the FPV thymidine kinase (TK) gene. Two types of recombinant viruses, differing only in the orientation of the HA gene relative to an adjacent foreign gene (lacZ), were created. Following preliminary identification of FPV recombinants based on the generation of beta-galactosidase (lacZ gene product), correct insertion of the HA gene into the genomes of these viruses was verified by hybridization studies. Susceptible chickens vaccinated with these FPV recombinants produced specific hemagglutination-inhibiting antibodies against the HA antigen. In view of this immune response, these viruses may serve as vaccines against avian influenza virus. In this regard, they appeared to be less virulent than the parental virus.     

Characterization of avian reovirus strain-specific polymorphisms

Avian reoviruses have been associated with several pathologic conditions, but correlative relationships between genotypes and specific diseases have not been demonstrated. Six avian reoviruses (883, 176, 81-5, S1133, FC, and TX) were selected for this study, and a comparative study of the pathogenic properties of the viruses in chickens, following peroral and footpad inoculation, was carried out, along with a comparison of the electrophoretic mobility of viral genomic segments and viral proteins encoded by the gene segments. The pathogenic properties of the viruses were shown to be diverse, with three distinct pathotypes being defined: Pathotype I (883) caused only a syndrome that we have termed "transient digestive system disorder" (TDSD); Pathotype II (FC, TX, and S1133) caused only "viral arthritis syndrome" (VAS), whereas Pathotype III (176 and 81-5) caused both TDSD and VAS. Likewise, the genomes of the viruses were shown to be extremely polymorphic, with a maximum of five segments co-migrating between any two strains. Considerable variation in the electrophoretic mobility of the encoded proteins also was demonstrated with pronounced variation in the molecular size of the sigma 4 protein, the purported viral attachment protein, being evident. These results show that the genomes of avian reoviruses were extremely polymorphic, preventing correlation between genotypes and pathotypes. But these studies have provided us with the genetic elements needed to characterize the gene functions involved in viral pathogenesis.     

Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.     

Antigenic cross-reactivity among avian pneumoviruses of subgroups A,B, and C at the matrix but not nucleocapsid proteins

Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.     

Reflections on the pathogenesis of diseases caused by the acute avian leukosis/sarcoma viruses with special reference to avian erythroblastosis

The various diseases that follow experimental infection with the acute and non-acute avian oncoviruses are discussed with special reference to the pathogenesis of avian erythroblastosis. One view, based onin vitro studies, sees erythroblastosis as the product of a failure in the differentiation of virus-infected stem cells to mature erythrocytes, as a result of cell transformation. The results of somein vivo studies, however, point to a resemblance of the disease to a haemolytic anaemia, where cellular death is an important component. It seems probable that the disease is the result of transformation of cells of the erythroblastic series followed by the death of many of these cells due to influences that have not yet been determined. Determination of the causes of this cellular death may prove to be as important for our understanding of the problem of leukaemia as the work that has already been accomplished in explaining the causes of cell transformation. It is also suggested that the tendency ofgs amino acid sequences of the avian leukosis viruses and mouse leukaemia viruses to form fusion proteins with a variety of proto-oncogenes may be part of a wider phenomenon, and that these sequences may fuse with other proteins, altering their properties. More work is required on the possibility that there is an undiscovered immunological component in the progression of the L/S diseases.Abbreviations AGID agar gel immunodiffusion - AEV avian erythroblastosis virus - AMV avian myeloblastosis virus - ATPase adenosine triphosphatase - CEF chicken embryo fibroblasts - EGF epidermal growth factor - ELISA enzyme-linked immunosorbent assay - gs group specific antigen - gag viral gene expressing the group specific antigen - L/S leukosis/sarcoma - LLV lymphoid leukosis virus - PEBLEs proerythroblast-like elements - REV reticuloendotheliosis virus - RAV-1 etc. Rousassociated virus isolates     

Molecular characteristics and pathogenicity of an avian leukosis virus isolated from avian neurofibrosarcoma

Peripheral nerve sheath tumors (PNSTs) are rare in chickens and their etiology remains to be elucidated. In this study, a naturally occurring PNST in a Japanese native fowl (Gallus gallus domesticus) was pathologically examined and the strain of avian leukosis virus (ALV) isolated from the neoplasm was characterized by molecular biological analysis. The fowl presented with a firm subcutaneous mass in the neck. The mass, connected to the adjacent spinal cord (C9-14), was microscopically composed of highly cellular tissue of spindle cells arranged in interlacing bundles, streams, and palisading patterns with Verocay bodies and less cellular tissue with abundant collagen. Immunohistochemically, neoplastic cells were divided into two types: perineurial cells positive for vimentin, glucose transporter 1 (GLUT1), and claudin1; and Schwann cells positive for vimentin, occasionally positive for S-100 alpha/beta but negative for GLUT1. Based on these findings, a diagnosis of neurofibrosarcoma was made. The complete nucleotide sequence of an ALV strain, CTS_5371, isolated from the neoplasm was determined and phylogenetic analysis indicated that the strain was a novel recombinant virus from avian leukosis/sarcoma viruses previously reported. Additionally, experimental infection revealed that CTS_5371 induced the proliferation of Schwann cells and perineurial cells. These results suggest that this ALV strain has the ability to induce PNSTs in chickens.     

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.