Preliminary molecular characterization of a fowl poxvirus isolate in Grenada.

Two 1-mo-old local breed chickens, with gross lesions in the skin of the head region suspected to be fowl poxvirus infection, were submitted to the Diagnostic Laboratory of the School of Veterinary Medicine, Grenada, West Indies. Cutaneous lesions were collected from these birds for virus isolation, histopathologic diagnosis, and molecular analysis. Fowl poxvirus infection was confirmed by virus isolation in chicken embryo and by histopathology. Molecular characterization of the fowl poxvirus was conducted by PCR amplification of selected genomic fragments and by nucleotide sequencing. Integration of reticuloendotheliosis virus fragments into the fowl poxvirus genome was confirmed by PCR and DNA sequencing. This is the first report from the Caribbean region on the preliminary molecular characterization of a fowl poxvirus isolate.

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.

The DNA repair enzyme, CPD-photolyase restores the infectivity of UV-damaged fowlpox virus isolated from infected scabs of chickens.

Fowlpox virus (FWPV), an important pathogen of poultry, replicates very efficiently in the featherless areas of skin, and persists in dried and desiccated scabs for prolonged periods. Although the molecular mechanisms underlying the stability of the virus are not completely known, we recently identified the presence of a virus-encoded novel DNA repair enzyme, CPD-photolyase, in FWPV. This enzyme repairs the ultraviolet (UV)-induced pyrimidine dimers, converting them to monomers using photons from white light as a renewable source of energy. In this study, we examined the role of photolyase in the pathogenesis of fowlpox. A comparison of pathogenesis of fowlpox in chickens infected with parental FWPV with that in chickens infected with photolyase-deficient FWPV (Phr(-) FWPV) found no significant differences in terms of replication of virus or formation of secondary lesions. When the virions isolated from infected scabs were exposed to UV light, UV-damaged parental FWPV, unlike Phr(-) FWPV, were rescued through the CPD-photolyase-mediated photoreactivation pathway by at least 48%. However, the mutant virus triggered host's immune response and conferred complete protection against subsequent challenge with virus similar to that conferred by the parental virus. Since the mutant virus is less stable than the parental virus in the infected scabs but is as immunogenic, Phr(-) FWPV might be less persistent in the environment. Furthermore, this particular genetic locus can also be used to insert foreign genes for the development of FWPV recombinant vaccines.

Genomic and antigenic characterization of avipoxviruses.

A permanent cell line of avian origin, QT-35, was used for the propagation of avipoxvirus isolates, including juncopox, pigeon pox, and field and vaccine strains of fowlpox viruses. The genomes of these avipoxvirus isolates were compared by restriction enzyme analysis using BamHI and HindIII endonuclease digestion and subsequent agarose gel electrophoresis. The genetic profiles of the virus strains were very similar, with a high proportion of comigrating fragments, although most strains could still be distinguished; therefore, these avipoxviruses appear to be closely related. Similar results were obtained when the immunogenic proteins of 6 fowlpox virus strains were examined by immunoblotting. Although the majority of the antigens were common, the strains could be differentiated by unique proteins of differing electrophoretic mobilities.

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.

Re-emerging fowlpox: evaluation of isolates from vaccinated flocks.

Vaccines of fowlpox or pigeonpox virus origin have been routinely used for more than half a century to prevent fowlpox in commercial poultry in areas where the disease is endemic. However, in recent years, outbreaks of fowlpox have occurred in previously vaccinated flocks. One possible explanation for this problem is the emergence of variant strains of fowlpox virus (FPV). A second, not mutually exclusive, postulate is that the novel FPV exhibit enhanced virulence due to the integration of avian reticuloendotheliosis virus (REV) into their genomes. To determine if immunological variance and/or the acquisition of REV nucleotide sequences could be responsible for the ineffectiveness of current vaccines, the ability of two commercial vaccine viruses and four, recently isolated, field strains to protect chickens against challenge with one of the more virulent field viruses was evaluated. Adequate protection was provided by the vaccines and two of the four field isolates. Interestingly, the two isolates that were not protective, as well as the challenge strain, failed to elicit a strong humoral antibody response. As to possible REV participation, an antibody response to this virus was only found in those chickens receiving one of the ''protective'' field strains, despite the presence of REV coding sequences in all four field viruses. While REV long terminal repeats of variable lengths were detected in the genomes of all FPV strains used in this study, only the DNAs of the field strains appeared to have intact REV provirus. This retention of foreign DNA may enhance the pathogenesis of FPV, although other factors may be involved.

Characterization of monoclonal antibodies against fowl poxvirus.

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.

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.

Taxonomy of pasteurella anatipestifer. 2. Cellular fatty-acid profile by gas chromatography.

Methylated cellular fatty acids of representative strains of Pasteurella spp., Moraxella spp., and P. anatipestifer were subjected to gas chromatography in an attempt to further support the independence of P. anatipestifer from both Pasteurella and Moraxella. All Pasteurella spp. and Moraxella spp. revealed group characteristics specific for each genus that could be easily differentiated from the unique profile of P. anatipestifer. All P. anatipestifer strains tested showed similar fatty-acid profiles in gas chromatography, regardless of host of origin.

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.

Pathogenesis of fowlpox in laying hens.

Egg production dropped after hens were inoculated with fowlpox virus originally isolated from a natural mild infection. The drop started from the 2nd week and continued to the 5th week after inoculation. All birds developed focal lesions at the site of inoculation, and some developed secondary lesions. The drop in individual birds was related to the severity of lesions. Birds challenged with fowlpox virus fifteen months after vaccination with pigeon pox vaccine were susceptible.

A smear technique for staining elementary bodies of fowlpox.

Red-stained elementary bodies of fowlpox virus were detected in smears from lesions of infected chorioallantoic membrane and from skin of infected birds stained by the Giménez method.

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.

Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter.

A vaccinia virus promoter was evaluated for regulation of a foreign gene in fowlpox virus by a transient expression assay. Fowlpox virus-infected quail cells, transfected with plasmid DNA containing chloramphenicol acetyltransferase (CAT) gene ligated to a vaccinia virus promoter, expressed CAT activity. No CAT activity was detected either in uninfected cells or fowlpox virus-infected cells. These results indicated that a heterologous vaccinia virus promoter can regulate expression of a foreign gene in fowlpox virus.

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.

Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis.

DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.

Studies of Pasteurella anatipestifer: an approach to its classification.

Moraxella spp., Pasteurella spp., and strains of P. anatipestifer were tested for biochemical reactions, growth temperature, viability, antibacterial sensitivity, and DNA base composition. P. anatipestifer was viable for shorter periods at 37 C, showed high resistance to polymyxin B and kanamycin, and had lower base composition than reported for Moraxella and Pasteurella spp. Because of these conditions, P. anatipestifer should be excluded from the genera Pasteurella and Moraxella.

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.

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.

Natural pox and herpes as a dual virus infection in chickens.

A natural dual viral infection was confirmed in chickens of University of Illinois Poultry Farm by histopathologic, immunologic, and electron-microscopic examination of formalin-fixed tracheal tissue. Histopathologic examination of the tracheal mucosa revealed eosinophilic cytoplasmic inclusions characteristic of fowlpox and intranuclear inclusions suggestive of Marek's disease. Treatment of the formalin-fixed tracheal tissue with peroxidase and fluorescent-labeled antibody against Marek's disease virus revealed specificity of the reaction. Electron microscopy showed viral particles of two morphologic forms, i.e., pox and herpes. A single cell having dual infection with herpes virus in the nucleus and pox virus in the cytoplasm was also encountered on electron-microscopic examination.