Progress 10/01/03 to 09/30/04
Outputs
A poxvirus isolated from an Andean condor was compared biologically antigenically and genetically with fowlpox virus (FPV), the type species of the genus Avipoxvirus. Susceptible chickens inoculated with the condorpox virus (CPV) isolate developed only mild localized lesions and were not protected against subsequent challenge with FPV. In Western blotting, in addition to the presence of cross-reacting antigens, distinct differences in antigenic profiles of CPV and FPV were observed. Reticuloendotheliosis virus (REV) sequences, which are present in the genome of FPV, were absent in the CPV genome. Thus based on the genetic, antigenic and biological differences from FPV, the poxvirus isolated from the condor is a new member of Avipoxvirus genus.
Impacts
A previously unrecognized condorpox virus (CPV) was determined to be biologically, antigenically, and genetically distinct from fowlpox virus (FPV) and thus may represent a new species of the avipoxvirus genus.
Publications
- Tripathy, D.N. and Kim, T-J. (2002) Diagnosis and Characterization of Avianpox Viruses from Wild Birds. American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002
- Kim, T.J., Schnitzlein, W.M. and Tripathy, D.N. (2002) Genetic Approaches Toward Characterization of Avian Poxviruses. Poster presentation at the American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002
- Kim, T-J, Schnitzlein, W.M., McAloose, D., Pessier, A.P. and Tripathy, D.N.. (2003). Characterization of an avianpox virus isolated from an Andean Condor (Vultur gryphus). Veterinary Microbiology 96: 237-246.
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Progress 10/01/02 to 09/30/03
Outputs
Chickens immunized with two avianpox viruses (Hawaiian goose pox and Palila pox) from Hawaiian endangered forest birds developed very mild localized cutaneous lesions while chickens immunized with a field strain of fowlpox virus had severe lesions, which took more than two weeks for regression. Immunization of chickens with these Hawaiian poxvirus isolates did not provide protection against fowlpox virus indicating that these viruses are biologically different from fowlpox virus.
Impacts
Two avianpox virus isolates from endangered Hawaiian forest birds revealed mild pathogenicity for chickens and did not provide protection against fowlpox virus. Thus, they are biologically different from fowlpox virus.
Publications
- Kim, T-J, Schnitzlein, W.M., McAloose, D., Pessier, A.P. and Tripathy, D.N.. (2003). Characterization of an avianpox virus isolated from an Andean Condor (Vultur gryphus). Veterinary Microbiology 96: 237-246.
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Progress 10/01/01 to 09/30/02
Outputs
1.Hawaiian goose poxvirus (HGPV), a new species of the avipoxvirus genus. A poxvirus was isolated from the cutaneous lesions of a Hawaiian goose (Branta sandwicensis) by inoculation of ground suspension of infected lesions on the chorioallantoic membrane of developing chicken embryos. Restriction fragment length polymorphism of HGPV genome showed a different profile from those of fowlpox virus (FPV) and avianpox viruses previously isolated from Hawaiian forest birds (Alalapox virus and Apapanepox virus). Interestingly, nucleotide sequence analysis of a 5.3 kb PstI-HindIII fragment of HGPV DNA corresponding to FPV ORF192-203 revealed that three FPV ORFs (199, 200 and 202) are not present in its genome. The predicted amino acid sequences of the encoded proteins showed a high similarity to condorpox virus (CPV) and a moderate similarity to FPV counterparts. In addition, none of reticuloendotheliosis virus (REV) sequences were amplified in HGPV genome by PCR
amplification. Western blotting using polyclonal antiserum against either FPV or CPV showed similarity in antigenic profile between HGPV and two previous Hawaiian isolates (Alalapox virus and Apapanepox virus). However, HGPV showed differences from those of FPV and CPV. Interestingly, still three avianpox viruses from Hawaiian birds showed distinguishable reaction in approximately 26, 34, 35 and 81 kDa proteins with anti-Alalapox serum. In spite of antigenic similarity of HGPV with previous Hawaiian isolates, it has unique genetic and antigenic characteristics to be considered as a new species of the avipoxvirus genus. 2. Isolation of avianpox viruses from Palila (Loxioides bailleui). Avian poxvirus infection in two Palila (Loxioides bailleui), an endangered Hawaiian forest bird was confirmed using either chicken embryos or avian cell cultures for virus isolation.. In one case the virus was isolated by inoculation of chicken embryos from a SPF flock and poxvirus infection was
confirmed by histopathology. In the second suspected case, the virus was isolated by inoculation of permanent avian cell lines (QT-35, LMH cells). 3. Development of a PCR based diagnostic test for rapid diagnosis of pox infection in wild birds. In order to amplify DNA sequences in suspected samples of avianpox virus from a variety of wild birds, we have designed two degenerate PCR primer sets. The first set represents the conserved region of FPV ORF198 and the conserved region of FPV ORF201. Similarly, the second set of primers represents the conserved region of FPV ORF 201 and the conserved region of FPV ORF203. These primer sets are based on sequences that are conserved in the genomes of the fowlpox virus and condorpox virus. In a comparative study, using these primer sets, poxvirus specific DNA could be amplified from majority of field samples suspected of avianpox virus infection (including scab, eye and toe lesions). Although both primer sets were effective in specific
amplification of poxvirus DNA, the second set provided more consistent results. Thus the second set can be used for rapid and specific diagnosis and identification of avian poxvirus infection.
Impacts
1. Genetically different avianpox viruses infect Hawaiian endangered forest birds. In this regard, Hawaiian goose poxvirus as a new species of the avipoxvirus genus has been identified 2. PCR can be used effectively in diagnosis of poxvirus infection in wild birds.
Publications
- Tripathy, D.N. and Kim, T-J. (2002) Diagnosis and Characterization of Avianpox Viruses from Wild Birds American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002
- Kim, T.J., Schnitzlein, W.M. and Tripathy, D.N. (2002) Genetic Approaches Toward Characterization of Avian Poxviruses. Poster presentation at the American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002
- Kim, T.J. and Tripathy (2002). Genetic characterization of Poxvirus isolated from an endangered Hawaiian forest bird. Poster presentation, XIV International Poxvirus and Iridovirus Workshop, Lake Placid, NY, Sept. 20-25, P70, p 164.
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Progress 10/01/00 to 09/30/01
Outputs
A virus was isolated from the spleen of a condor and determined to be a pox virus based on electron microscopic examination. Further studies revealed this pathogen to be biologically, antigenically, and genetically distinct from fowlpox virus. This virus could be grown in the chorioallantoic membranes of chicken embryos, but could not be propagated in either of two avian cell lines that typically support the replication of fowlpox virus. Immunization of chickens with this novel virus did not protect them against a subsequent challenge with virulent fowlpox virus. Further immunogenic differences were apparent due to the inability to detect many cross-reacting antigens in these two pox viruses by Western blot analyses. The gene organization within a 4.5 kbp Hind III fragment of the novel virus's DNA was similar to that fowlpox virus genomic region containing open reading frames (ORFs) 193-203. For instance, each gene conserved in both viruses was transcribed in the same
direction and the encoded proteins exhibited 72.5 to 94.6 % homology at the predicted amino acid level. However, homologues of fowlpox virus ORFs 199, 200 and 202 were not present in the corresponding locations in the genome of the novel virus. Based on these observations, the novel virus has been designated condorpox virus and is considered to represent a new species of the avipoxvirus genus. This proposed taxonomic inclusion has been forwarded to the International Committee on Taxonomy of Poxviruses for consideration.
Impacts
(N/A)
Publications
- Kim, T-J., Pessier, A.P. and Tripathy, D.N. (2001). Condor poxvirus with biological differences from fowlpox virus. (2001). Poster Presentation at the AAAP/AVMA Scientific Program, Boston, MA. P. 54.
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Progress 10/01/99 to 09/30/00
Outputs
A strain of pox virus was isolated from the spleen of a condor. Pathogenicity of this condorpox virus for specific pathogen free (SPF) chickens was compared with that of a fowlpox virus strain. While all birds immunized with fowlpox virus were protected following challenge with fowlpox virus, birds immunized with condorpox virus were not protected against fowlpox virus challenge. The observed lack of cross-protection indicates that condorpox virus is biologically different from fowlpox virus.
Impacts
(N/A)
Publications
- Tripathy, D. N., Schnitzlein, W. M., Morris, P. J., Janssen, D. L., Zuba, J. K., Messy, G. and Atkinson, C. T. (2000) Characterization of poxviruses from forest birds in Hawaii. J. Wildlife Dis. 36:225-230
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Progress 10/01/98 to 09/30/99
Outputs
A comparison of the transcriptional efficiencies of natural avipoxvirus and orthopoxvirus promoters in the presence of fowlpox virus. Currently, two vaccinia virus promoters, P7.5 and P11, associated with the expression of an early-late 7.5 or late 11 kDa protein, respectively, are commonly used in the construction of pox virus recombinants. Since it is possible that homologous promoters may function more efficiently, we have attempted to identify novel fowlpox virus (FPV) transcriptional regulatory elements based on their proximity to virus genes. Ones of particular interest would be those that are active late during the virus infectious cycle, since these have been shown to be the strongest. Earlier we reported identification of a putative promoter regulating a 37kDa protein of unknown function in the genome of Hawaiian crow poxvirus. Expression efficiency of this promoter was compared with two FPV promoters and the two heterologous vaccinia virus promoters.
Promoter constructs were fusions to the lacZ gene using natural restriction sites located within the open reading frames. Recognition of the transcriptional constructs was examined by comparing the transient expression levels of B-galactosidase (lacZ gene product) in virus-infected cells. For this purpose, purified plasmids containing the various promoters were separately transfected into FPV-infected QT-35 (Japanese quail liver cell line) monolayers and the specific activity of the bacterial enzyme in the cell lysates was determined. Differences in the efficiencies of FPV promoters were observed and promoter P37 appeared to direct expression of the lacZ gene at approximately 50% of the level of P11 vaccinia and a FPV promoter. These observations indicate that the P37 promoter from a distantly related avipoxvirus should be suitable for generating poxvirus recombinants.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/97 to 09/30/98
Outputs
Previously, the presence of a presumably novel open reading frame (ORF) of 975 nucleotides in the DNA derived from a Hawaiian crow (Corvus hawaiiensis) pox virus (HCPV) was reported. Both BLAST and FASTA searches of the Swiss Protein Data Base failed to detect any significant matches to the encoded polypeptide. The only pox virus protein match was to the small subunit of the mRNA capping enzyme conserved in orthopox and swinepox viruses. However, the promoter of this mammalian pox virus gene is an early one, unlike the predictably late one having the characteristic TAAAT(G) motif at the start of the HCPV gene. Moreover, the pox virus early transcription terminator sequence, TTTTTNT, is centrally located within this gene which should preclude its expression during the onset of the infectious cycle. Thus, currently, this HCPV gene must be relegated to the status of unknown function. To determine whether this ORF is conserved in avianpox viruses, hybridization of the
HCVP gene to the genome of FPV was attempted. The unknown HCPV gene seems to be conserved among avian pox viruses, since it hybridizes with the genomes of FPV and a pox virus originating in an Apapane species of Hawaiian bird (Himatione sanguinea), albeit under conditions of low and high stringency, respectively. In contrast, the reverse situation, in regards to the degree of stringency required for successful hybridization, was observed when the thymidine kinase gene of FPV was used as a probe. Thus, the two HCVP are probably not closely related to FPV as evidenced by the apparent low homology of two conserved genes. To test the utility of the putative promoter which regulates transcription of the novel HCVP gene, the ninth codon of the Escherichia coli lacZ gene was fused in frame to the 46th codon of the novel HCVP ORF. The resulting plasmid was transfected into both HCVP- and FPV-infected chicken liver cells and the cell lysates were assayed for the presence of galactosidase. The
levels of galactosidase activity by the putative HCVP promoter in HCVP- and FPV-infected chicken liver cells were 77 and 36% respectively of that obtained using a similar construct in which expression was directed by the vaccinia virus strong late P11 promoter. Thus, this transcriptional regulatory element may be of value in producing recombinant avian poxviruses when a homologous promoter is desired.
Impacts
(N/A)
Publications
- Tripathy, D.N., Schnitzlein, W.M. , Morris, P.J., Janssen, D.L., Zuba, J.K., Masey, G., Atkinson, C.T. 1998. Characterization of poxviruses from Hawaiian forest birds. Poster presented at the XIIth Int. Pox Symposium, Renaissance Grand Beach Resort, St. Thomas, p. 29.
- Tripathy, D.N., Schnitzlein, W.M. 1998. Identification of a novel open reading frame in the genome of an avianpox virus isolated from a Hawaiian crow. Poster presented at the XIIth Int. Pox Symposium, Renaissance Grand Beach Resort, St. Thomas, p. 31.
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Progress 10/01/96 to 09/30/97
Outputs
Earlier we reported that avianpox viruses infecting Hawaiian crows were genetically distinct from fowlpox virus. Recently, the nucleotide sequence of a 1.13 kb HindIII-XbaI fragment from the genome of a Hawaiian crow pox isolate (Alala/Lanakila) was determined. An intact open reading frame (ORF) of 975 nucleotides encoding for a polypeptide of predicted molecular weight of 37,144 daltons was found to be present within the 1.13 kb fragment of the Hawaiian crow pox virus genome. The predicted polypeptide lacks significant homology with any known pox virus proteins. The pox virus late promoter signature sequence, TAAAT, comprises part of the initiation codon of this gene and is preceded by the expected A/T rich region. Thus, this novel protein is probably produced late in the viral infection. This study indicates that a novel gene which is transcriptionally regulated by a late promoter is present in the genome of a Hawaiian crow pox virus.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/95 to 09/30/96
Outputs
A comparison of the propagation of avian pox viruses isolated from either an Hawaiian crow (Corvus hawaiiensis) or Apapane species (Himatione sanguinea) revealed better growth in the LMH cell line than in AT-35 cells. However, regardless of the host, the respective restriction fragment length polymorphisms (RFLPs) resulting from either EcoRI or HindIII digestion of their genomes were distinct from each other as well as from those produced with the fowlpox virus genome. Therefore, these new isolates appear to be discrete strains which are not related to fowlpox virus. This uniqueness has been further demonstrated by a lack of cross-hybridization between the Hawaiian pox virus genomes and the fowlpox virus thymidine kinase gene except under conditions of low stringency.
Impacts
(N/A)
Publications
- Tripathy, D.N., Schnitzlein, W.M., Morris, P., Zuba, J. 1996. Characterization of avian pox viruses from Hawaiian forest birds. Abst. 68th Northeastern Conference of Avian Diseases, p13.
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