Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

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.

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.

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences.

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.

The impact of vaccines and the future of genetically modified poxvirus vaccines for poultry.

The regular use of live or killed vaccines against infectious agents has remarkably improved the efficiency of poultry production. In some cases eradication of disease has been possible when the pathogen is antigenically stable and confined to a certain geographical area. In other instances monovalent or polyvalent live or killed vaccines have been effective in reducing mortality and morbidity. Many conventional vaccines are developed by trial and error and basic information about their genetic make-up is not known. While the poultry industry has benefited from the regular use of conventional vaccines, there is need for a new generation of effective vaccines that require minimal handling of birds during administration. Using molecular techniques, it is possible to identify the genes associated with virulence and protection. In genetically engineered vaccines, genes that encode protective antigens can be expressed in bacterial or viral vectors. In this regard, avianpox virus vectors appear to be promising for the generation of polyvalent vaccines expressing antigens from multiple pathogens.

Characterization of an avianpox virus isolated from an Andean condor (Vultur gryphus).

A novel pox virus, condorpox virus (CPV) isolated from the spleen of an Andean condor (Vultur gryphus) by inoculation of chorioallantoic membranes (CAM) of specific pathogen free (SPF) chicken embryos was compared biologically, antigenically and genetically with fowlpox virus (FPV), the type species of the genus Avipoxvirus. Susceptible chickens inoculated with CPV developed only mild localized lesions but were not protected against subsequent challenge with FPV. Based on Western blotting, in addition to the presence of cross-reacting antigens, distinct differences in antigenic profiles of CPV and FPV were observed. Sequence analysis of a 4.5 kb HindIII fragment of CPV genomic DNA revealed the presence of eight co-linear genes corresponding to FPV open reading frame (ORF)193-198, 201 and 203. Interestingly, reticuloendotheliosis virus (REV) sequences present in the genome of all FPV were absent in CPV. Although, the results of a phylogenic analysis suggested that CPV is a member of the genus Avipoxvirus, its unique antigenic, biologic and genetic characteristics distinguish it from FPV to be considered as a new member of this genus.

Fowlpox virus infection causes a lymphoproliferative response in chickens.

While attenuated fowlpox virus (FPV) strains are widely used for vaccination of chickens and turkeys for prevention of fowlpox, recombinant FPV expressing various foreign genes have been evaluated for their ability to offer protection against various diseases in poultry as well as mammals. Little is known regarding the cell-mediated immune responses to FPV infection. In this study, immune response in chickens infected with a virulent and a vaccine strain of FPV were compared by a lymphoproliferation assay. Interestingly, a lymphoproliferative response was seen during 2-4 weeks post-infection irrespective of the FPV strain used in this study. Analyses of the buffy coat cultures with (35)S-methionine pulse labeling revealed an elevated protein of approximately 48-50 kDa in the culture supernatants. Furthermore, those supernatants could stimulate naive, non-adherent cells of the buffy coat cultures, in a dose dependant manner, suggestive of stimulatory cytokines. FPV, a complex virus presumably stimulates a variety of cytokines in vivo causing a proliferative cellular response. Knowledge of those cytokines or a better understanding of the proliferative responses is pivotal in evaluation of FPV vaccines and in the design of FPV-based recombinant vaccines.

Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability.

Nine field strains of fowlpox virus (FPV) isolated during a 24-year span from geographically diverse outbreaks of fowlpox in the United States were screened for the presence of reticuloendotheliosis virus (REV) sequences in their genomes by PCR. Each isolate appeared to be heterogeneous in that either a nearly intact provirus or just a 248- or 508-nucleotide fusion of portions of the integrated REV 5' and 3' long terminal repeats (LTRs) was exclusively present at the same genomic site. In contrast, four fowlpox vaccines of FPV origin and three originating from pigeonpox virus were genetically homogeneous in having retained only the 248-bp LTR fusion, whereas two other FPV-based vaccines had only the larger one. These remnants of integrated REV presumably arose during homologous recombination at one of the two regions common to both LTRs or during retroviral excision from the FPV genome. Loss of the provirus appeared to be a natural event because the tripartite population could be detected in a field sample (tracheal lesion). Moreover, the provirus was also readily deleted during propagation of FPV in cultured cells, as evidenced by the detection of truncated LTRs after one passage of a plaque-purified FPV recombinant having a "genetically marked" provirus. However, the deletion mutants did not appear to have a substantial replicative advantage in vitro because even after 55 serial passages the original recombinant FPV was still prevalent. As to the in vivo environment, retention of the REV provirus may confer some benefit to FPV for infection of poultry previously vaccinated against fowlpox.

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.

Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus.

The spike 1 (S1) surface glycoprotein of infectious bronchitis virus (IBV) is the major inducer of the generation of virus neutralizing antibodies, and the administration of purified S1 has been shown to elicit a protective immune response against virulent virus challenge. On the basis of these observations, recombinant fowl poxvirus (rFPV) containing a cDNA copy of the S1 gene of IBV Mass 41 (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 in vito by indirect immunofluorescence staining and western blot analyses. Later, in vivo expression was demonstrated by the detection of IBV-specific serum immunoglobulin G and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histologic changes of the tracheas in virulent IBV Mass 41-challenged chickens previously receiving rFPV-S1 as compared with parental fowl poxvirus (FPV)-vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent virulent FPV challenge. As to a preferred method of immunization, wing web administration appeared to be superior to the subcutaneous route because a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowl pox and infectious bronchitis.

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.