Genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes.

Fowlpox virus (FWPV) is currently used as a vector to express foreign genes of various poultry and mammalian pathogens. However, due to limited information available about the primary structure of FWPV promoters required for an optimal transcriptional efficiency, the full potential of FWPV as an expression vector has not been completely realized. To dissect such transcriptional regulatory elements at the molecular level, we selected two FWPV promoters dictating contrasting levels of expression of acidic-type inclusion body protein gene (P190) and A15L vaccinia virus homolog of FWPV (P180) for site-directed mutagenesis studies. The transcriptional activity of mutated promoters was analyzed based on their ability to transcribe a reporter gene, lacZ, and translation of the resultant mRNA into functional protein. Replacement of the spacer sequences of P180 with those of P190 resulted in a five-fold increase in mRNA and a 17.6-fold increase in protein over those with its parental promoter, P180. Similarly, replacement of a thymidine after the start codon with guanosine resulted in a 2.3-fold increase in lacZ mRNA and a seven-fold increase in protein. Combining these substitutions in P180SG produced a maximum increase in mRNA and protein of 6.7- and 29.9-fold, respectively, over concentrations with its parental P180 promoter. The promoter activity of P180SG was comparable to that of the strongest natural promoter, P190. The amount of protein per transcript generated by the mutated promoters of P180 increased to at least three times that with the parental P180. In contrast, similar replacements in P190 resulted in a 40-50% reduction in mRNA and protein in all the mutated promoters. We discuss the significance of spacer sequence and the purine after the start codon in the context of a high level of expression.

A consideration of previously uncharacterized fowl poxvirus unidirectional and bidirectional late promoters for inclusion in homologous recombinant vaccines.

Because of the limited analysis of fowl poxvirus (FPV) promoters, expression of foreign proteins by recombinant FPV has usually been directed by heterologous vaccinia virus or synthetic poxvirus promoters. Thus, the impact of completely homologous recombinant virus vaccines has yet to be realized by the poultry industry. In an effort to increase the availability of such transcriptional regulatory elements, the modulation of gene expression by six previously uncharacterized FPV late promoters was examined. To simplify this comparison, each promoter region was separately coupled to the same reporter gene (lacZ) in individual plasmid constructs, and their activities in transfected, virus-infected cells were monitored. In each of the four selected unidirectional transcriptional regulatory elements as well as a 30-base pair representative of the bidirectional promoter region, the predicted temporal specificity of expressing at late stages of virus replicative cycle was verified. Stable lacZ gene transcripts arising from each plasmid varied less than threefold in quantity, whereas the amounts of beta-galactosidase product ranged within a 130-fold interval. Only the promoter that naturally regulates expression of the A type inclusion body protein gene directed production of beta-galactosidase at a level comparable with that associated with the strong vaccinia virus P11 promoter. Because one of the remaining unidirectional transcriptional regulatory elements, P174, was only 2.4-fold less efficient, both of these promoters, P174 and P190, should be satisfactory for directing the expression of poultry pathogen genes inserted into the genomes of FPV recombinant vaccines.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop.

As a result of the first-round cluster analysis, a panel of 16 novel monoclonal antibodies (mAbs) was assigned for detailed analysis to the CD45 subgroup of the Third International Swine CD Workshop. The specificity of the mAbs was initially determined by examining their reactivity with Chinese hamster ovary (CHO) cells engineered to express individual isoforms of porcine CD45. These analyses indicated that seven of the mAbs (PG77A, PG96A, PG167A, PGB78A, 3C/9, MIL13, NHT 101) recognized the portion of the CD45 molecule encoded by the A exon (CD45RA), while one (MIL15) was specific for that portion encoded by the C exon (CD45RC). In each case, the designation was supported by the demonstration that the molecular weight(s) of the recognized antigen(s) in porcine mononuclear cells, as determined by immunoprecipitation, corresponded to the predicted size(s) according to their specificity. As expected, a similar correlation was obtained for five standard mAbs whose specificity for either common or restricted epitopes of porcine CD45 had been established in previous workshops. Screening of the remaining 174 mAbs that comprised this workshop but were excluded from the CD45 subgroup by cluster analysis failed to detect any additional ones reactive with the porcine CD45-expressing cells.

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.

Fowlpox virus encodes a novel DNA repair enzyme, CPD-photolyase, that restores infectivity of UV light-damaged virus.

Fowlpox virus (FPV), a pathogen of poultry, can persist in desiccated scabs shed from infected hosts. Although the mechanisms which ensure virus survival are unknown, it is likely that some type of remedial action against environmentally induced damage is required. In this regard, we have identified an open reading frame (ORF) coding for a putative class II cyclobutane pyrimidine dimer (CPD)-photolyase in the genome of FPV. This enzyme repairs the UV light-induced formation of CPDs in DNA by using blue light as an energy source and thus could enhance the viability of FPV during its exposure to sunlight. Based on transcriptional analyses, the photolyase gene was found to be expressed late during the FPV replicative cycle. That the resultant protein retained DNA repair activity was demonstrated by the ability of the corresponding FPV ORF to complement functionally a photolyase-deficient Escherichia coli strain. Interestingly, insertional inactivation of the FPV photolyase gene did not impair the replication of such a genetically altered virus in cultured cells. However, greater sensitivity of this mutant than of the parental virus to UV light irradiation was evident when both were subsequently photoreactivated in the absence of host participation. Therefore, FPV appears to incorporate its photolyase into mature virions where the enzyme can promote their survival in the environment. Although expression of a homologous protein has been predicted for some chordopoxviruses, this report is the first to demonstrate that a poxvirus can utilize light to repair damage to its genome.

Identification of the pseudorabies virus promoter required for latency-associated transcript gene expression in the natural host.

Expression of the latency-associated transcript (LAT) gene is a hallmark of alphaherpesvirus latency, and yet its control and function remain an enigma. Resolution of this problem will require verification and subsequent elimination or disabling of elements regulating LAT gene transcription so that the influence of the resultant RNA can be evaluated. Toward this end, we generated a novel pseudorabies virus (PrV) recombinant in which a 282-bp region containing the LAP1 (first latency-active promoter) consensus sequence was replaced by a reporter cassette. Despite this substitution, replication of the recombinant was comparable to that of the parental and rescuant viruses both in cultured mammalian cells and in the natural host, swine. Furthermore, production of the LAT gene-associated 2.0- and 8.0-kb RNAs during an in vitro lytic infection of cultured neuronal cells was unaffected. However, the otherwise constitutively produced and processed 8.4-kb LAT was not detected in porcine trigeminal ganglia latently infected with this novel recombinant, although the viral genome was shown to be present. Therefore, LAP1 is apparently the basal promoter for PrV LAT gene expression during viral latency but is not required for such activity during an in vitro lytic infection of neuronal cells. More importantly, the ability of PrV to persist in a latent state in the absence of LAT suggests that other factors are responsible for this event in the natural host.

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.

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop.

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.

Determination of the specificity of CD45 and CD45R monoclonal antibodies through the use of transfected hamster cells producing individual porcine CD45 isoforms.

The exclusive presence of the tyrosine phosphatase, CD45, on the surface of hematopoietic cells coupled with the differential expression of its various isoforms has enabled the selection of lymphocyte subsets based on their reactivity with monoclonal antibodies (mAbs) specific for one or more CD45 species. As a prelude to defining the specificity of anti-porcine CD45 mAbs for this purpose, Chinese hamster ovary cells were transfected with constructs containing cDNAs encoding the extracellular and transmembrane domains of four pig CD45 isoforms. Cells expressing only one of the three predominant types (CD45RO, CD45RC, and CD45RAC) or the minor species (CD45RA) of porcine CD45 on their surface were sorted based on positive reactivity with the CD45 mAb K252.1E4. Initially, these CD45+ cells were used as a source of antigen when determining the specificities of nine mAbs, which had been identified during the First and Second International Swine CD Workshops as being reactive with porcine CD45. Later, cloned cell lines were established and phenotypically verified for the production of the correct CD45 transcript by RT-PCR. Binding of two more mAbs (74-9-3 and 10-14-1) in addition to the original mAb panel to these cell lines was assessed by using a cell ELISA in lieu of one-color flow cytometry. Despite differences in detection methodology, identical mAb binding results were obtained. As anticipated, CD45 mAbs K252.1E4, MAC 323, and 74-9-3 which recognize an epitope(s) in the common portion of porcine CD45, reacted with cells expressing any one of the four isoforms but not with the parental CHO cells. In contrast, none of the restricted (CD45R) mAbs bound to cells producing the CD45RO isoform which lacks any of the alternate extracellular regions. However, three of these mABs (6E3/7, FG2F9 and STH267) did react specifically with the CD45RA and CD45RAC isoforms, indicating their specificity for an epitope(s) encoded by the CD45 A exon. The other four CD45R mAbs (MAC326, 3a56, MIL5, and -a2) recognized the CD45RC isoform. Interestingly, only CD45R mAb MAC326 also bound to cells expressing the CD45RAC isoform, suggesting that the epitope(s) recognized by the other three may have arisen due to the juncture of the invariant 5' leader sequence with the CD45C exon. The eleventh mAb (10-14-1) was unique in that it did not react with any of the expressed CD45 isoforms. This inability coupled with the previously demonstrated recognition of a 240 kDa protein suggests that it may be specific for CD45RABC. Overall, this panel of CD45R mAbs should prove useful for obtaining functionally distinct subpopulations of B and T lymphocytes.

Replication of infectious laryngotracheitis virus in a quail cell line, QT-35.

The relative resistance of the quail fibroblastic cell line QT-35 to infection by infectious laryngotracheitis virus (ILTV) was circumvented by the continual passaging of infected cells in the presence of uninfected monolayers. Such virus-containing cells were used to extend the infection to an otherwise refractory quail liver cell line IQ1A, as well as to a normally permissive chicken liver cell line LMH, with nearly equal efficiency. In contrast, extracellular virus that had been derived from either QT-35 or IQ1A cells could not readily infect either quail cell line, although LMH cells were still susceptible. Therefore, the block to ILTV replication in quail cells is probably at the adsorption/penetration stage.

Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus.

Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.

Propagation of infectious laryngotracheitis virus in an avian liver cell line.

The susceptibility of three avian cell lines (IQ1A, LMH, and QT-35) to infection by three strains of infectious laryngotracheitis virus (ILTV) was assessed both visually and by hybridization using an ILTV glycoprotein B gene probe. In the chicken liver tumor cell line (LMH), cytopathogenicity was observed at the second passage, and plaque formation was observed at the third passage. The identity of the infectious agent was verified to be ILTV by restriction endonuclease analysis of the virus genome and subsequent Southern hybridization. In contrast to LMH cells, which were a suitable host for ILTV, the quail cell line (IQ1A) was refractory to infection by this virus. Moreover, although LMH cell-adapted ILTV could initially replicate to a limited extent in the other quail cell line (QT-35), this ability was not sustained upon continual passaging.

Effect of route of administration on the efficacy of a recombinant fowlpox virus against H5N2 avian influenza.

A recombinant fowlpox vaccine virus containing the H5 hemagglutinin gene of avian influenza virus was administered to susceptible chickens via wing-web puncture, eye drop, instillation into the nares, and drinking water. Even though there was a negligible hemagglutination-inhibition (HI) serologic response, all 10 chickens vaccinated by wing-web puncture remained without obvious signs of disease and survived challenge with a highly pathogenic strain of H5N2 avian influenza virus. All unvaccinated chickens and those vaccinated by nasal and drinking-water routes died following challenge. Eight of 10 chickens vaccinated with the recombinant by eyedrop died. All vaccinates were negative on the agar gel precipitin (AGP) test, and only one chicken had a positive HI titer before challenge. All chickens that survived challenge had high levels of HI antibody and were positive on the AGP test, indicating that they were infected by the challenge virus.

Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus.

A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujesky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.

Identification and nucleotide sequence of the thymidine kinase gene of swinepox virus.

Using degenerative oligonucleotide probes, representing two different conserved regions of poxvirus and mammalian thymidine kinase (TK) genes, the swinepox virus (SPV) TK gene was mapped to a 1.7-kb BamHI-HindIII fragment of the viral genome. Nucleotide sequencing of this DNA piece revealed that the SPV TK gene was encoded by an open reading frame (ORF) of 177 codons. Immediately downstream of the TK gene was a second ORF with homologues at the same location in both capripoxvirus and leporipoxvirus genomes. A similar gene had translocated to near the left hand terminus of the vaccinia virus (orthopoxvirus) genome. Flanking the two SPV genes were ORFs whose counterparts in other poxvirus genera are located at the same relative positions. SPV appeared to be most closely related to capripoxvirus, based on the organization of the four genes and on the percentage of identical amino acid residues of the respective encoded proteins.

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.

Genetic and antigenic differences between fowlpox and quailpox viruses.

The genomes of a fowlpox and quailpox virus isolate were compared by restriction enzyme analysis using BamHI, EcoRI, and HindIII endonucleases. The genetic profiles of the two virus species were very distinct with fragments lacking similar electrophoretic mobilities. In contrast, the patterns of three quailpox virus isolates were very similar with a high proportion of co-migrating fragments. When the immunogenic proteins of two quailpox, three fowlpox, a juncopox, and a pigeonpox virus isolate were examined by immunoblotting, common as well as unique antigens were detected. The greatest disparity was between quailpox virus and the other three species which are genomically conserved. Therefore, on the basis of genetic as well as immunological analysis, quailpox virus is a distinct species of the genus Avipoxvirus.

A rapid method for identifying the thymidine kinase genes of avipoxviruses.

The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK- mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes.