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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.

3' Untranslated Regions↗

Mapping of a major early/late gene of fowlpox virus.

Identification, cloning and mapping of a major gene expressed during the early and late stages of infection with fowlpox virus is described. The gene is located within a 17.3 kb PstI fragment of the fowlpox virus genome and has an open reading frame of 501 bp. Analysis of the 5'-ends of mRNA transcribed from this gene showed that the start sites of both early and late transcripts map to the sequence TAAAT near the translation start site (ATG). This is the first poxvirus early/late gene described in which both early and late transcription start sites map to same DNA sequence. From northern hybridization analysis it was shown that the early function of this gene gives rise to the most abundant early mRNA coded by 17% of the fowlpox virus genome. The strong early function of this gene promoter will be useful in the construction of recombinant fowlpox viruses.

Base Sequence↗

Gene translocations in poxviruses: the fowlpox virus thymidine kinase gene is flanked by 15 bp direct repeats and occupies the locus which in vaccinia virus is occupied by the ribonucleotide reductase large subunit gene.

By sequencing a fragment of 7351 bp the fowlpox virus thymidine kinase gene has been found to map to a position within the equivalent of the vaccinia HindIII I fragment. The deduced gene arrangement in fowlpox virus is I3, X, TK, I5, I6, I7, I8, G1, indicating that the homologue of the vaccinia I4 gene has been replaced by two genes X and TK. The non-essential TK gene has therefore replaced another non-essential gene, I4 (the ribonucleotide reductase large subunit) in this region. The X/TK insertion in fowlpox virus is precisely flanked by direct repeats of 15 bp suggesting that the translocation event may have involved transposition. The % identities between the fowlpox virus and vaccinia virus proteins ranged between 58.5% and 31.3%.

Amino Acid Sequence↗

A 39,000 Mr immunodominant protein of fowlpox virus contains multiple copies of a 12 amino acid repeat sequence.

The nucleotide sequence of an unusual fowlpox virus gene which maps immediately upstream from the fowlpox virus 4b gene has been determined. The 34,000 Mr protein predicted to be encoded by the gene contains 11 copies of a 12 amino acid serine-rich repeat sequence. The seven amino-terminal copies of the repeat sequence are perfectly conserved but variation exists in the four carboxy-terminal copies. Three peptides were synthesized which contained either one copy of the repeat sequence, two copies of the repeat sequence or a hydrophilic amino-terminal region of the protein. All three peptides when injected with adjuvant into rabbits gave rise to antibodies which reacted strongly on Western blots of purified fowlpox virus proteins with a 39,000 Mr protein. When directly compared in Western blots the antipeptide sera were shown to recognize a protein comigrating with one of the two immunodominant proteins recognized by chicken anti-fowlpox virus sera taken 2 weeks post-infection. The virion protein is removed by treatment with sodium deoxycholate suggesting that it is located at or near the surface of the virus.

Amino Acid Sequence↗

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus.

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5. The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA polymerase offers an alternative to the widely used vaccinia virus vTF7-3, or the recently developed modified vaccinia virus Ankara (MVA) T7 RNA polymerase recombinant, a highly attenuated strain with restricted host-range. Recombinant fowlpox viruses have the advantage that as no infectious virus are produced from mammalian cells they do not have to be used under stringent microbiological safety conditions.

Animals↗

The potential role of fowlpox virus in rational vaccine design.

The design of optimal vaccines requires detailed knowledge of how protective immune responses are generated in vivo under normal circumstances. This approach to vaccine development, where the immune correlates of protection are defined and vaccines are designed to elicit the same response, is called rational vaccine design. Poxviruses are attractive candidates for inclusion in such design strategies owing to their large genome, which allows for the inclusion of multiple heterologous genes, including those encoding antigens, co-stimulatory molecules and cytokines. Fowlpox virus, the prototypical member of the Avipoxvirus genus, is particularly suitable, as it is also incapable of replicating in mammalian cells. The potential of recombinant fowlpox virus as a safe vaccine vector is being evaluated currently in a number of clinical trials for diseases, including HIV, malaria and various types of cancer. Despite their promise, intricate details regarding how fowlpox virus interacts with the host immune system have not been resolved. In this review, the issues surrounding the use of fowlpox virus as a vaccine vector and possible strategies for enhancing its efficacy are discussed.

AIDS Vaccines↗

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.

Animals↗

The safe and effective use of fowlpox virus as a vector for poultry vaccines.

The safety and efficacy of a fowlpox-Newcastle disease vaccine were evaluated by in vitro and in vivo methods. Genetic and phenotypic stability following cell culture and chick passage were demonstrated. The safety characteristics of the recombinant virus equalled or exceeded those of the parent fowlpox virus, as determined by lack of shed and spread to contacts, failure to revert to virulence following passage in chicks and innocuity in other avian species. The fowlpox-Newcastle Disease virus effectively immunized against virulent fowlpox challenge and virulent Newcastle disease challenge (intramuscular or intra-ocular administration). These results indicate that the recombinant FPV/NDV virus is a safe and effective vaccine for poultry.

Animals↗

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.

Animals↗

Phenotypic and kinetic analysis of effective simian-human immunodeficiency virus-specific T cell responses in DNA--and fowlpox virus-vaccinated macaques.

Although T cell immunity is important in the control of HIV-1 infection, the characteristics of effective HIV-specific T cell responses are unclear. We previously observed protection from virulent SHIV challenges in macaques administered priming with DNA vaccines and boosting with recombinant fowlpox viruses expressing shared SIV Gag antigens. We therefore performed a detailed kinetic and phenotypic study of the T cell immunity induced by these vaccines prior to and following SHIV challenge utilizing intracellular cytokine staining. Pigtail macaques vaccinated intramuscularly with DNA/recombinant fowlpox virus exhibited a coordinated induction of first Gag-specific CD4 T cell responses and then a week later Gag-specific CD8 T cell responses following the fowlpox virus boost. Overall, the magnitude and timing of the peak CD8 T cell responses following challenge was significantly associated with reductions in SHIV viremia following pathogenic challenge. After pathogenic lentiviral challenge, virus-specific effector memory T cells derived from animals controlling SHIV infection recognized a broad array of epitopes, expressed multiple effector cytokines and rapidly recognized virus-exposed cells ex vivo. These results shed light on some of the requirements for T cells in the control of pathogenic lentiviral infections.

Animals↗

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.

Animals↗

Antitumor activity of the intratumoral injection of fowlpox vectors expressing a triad of costimulatory molecules and granulocyte/macrophage colony stimulating factor in mesothelioma.

Deficiency in costimulatory molecule expression has been implicated in the ability of tumors to escape immune effectors. The activity of the intratumoral administration of recombinant fowlpox vectors expressing a triad of costimulatory molecules (rF-TRICOM) was evaluated in the asbestos-induced AB12 and AC29 mouse models of mesothelioma. Mesothelioma cell infected with rF-TRICOM expressed high levels of the costimulatory molecules. Prolongation of survival was observed in mice receiving rF-TRICOM in AB12 and AC29 intraperitoneal models. Complete tumor regressions were observed in mice receiving intratumoral rF-TRICOM in the AB12 subcutaneous tumor model. Tumor regressions were associated with the development of serum IgG reactivities to mesothelioma-associated determinants and specific systemic cytolytic activity, and responding mice were capable of rejecting tumors upon re-challenge. Antitumor activity was also observed in mice with established AB12 tumor vaccinated with irradiated rF-TRICOM-infected AB12 cells. The antitumor activity of intratumoral rF-TRICOM was superior to that of the intratumoral injection of a fowlpox vector expressing granulocyte-macrophage colony stimulating factor (rF-GM-CSF). AB12 and AC29 tumors were found to produce GM-CSF and to have substantial macrophage infiltration. Production of GM-CSF decreased in vivo in tumors injected with rF-TRICOM. rF-TRICOM and wild-type fowlpox inhibited the growth of AB12 and AC29 cells in vitro; less inhibition was observed with rF-GM-CSF. These results indicate that the intratumoral injection of rF-TRICOM has significant activity in mouse models of mesothelioma and can elicit a systemic antitumor immune response. The results also suggest potential limitations to the intratumoral administration of cytokines, such as GM-CSF, in mesothelioma.

Animals↗

Infectious bursal disease virus structural protein VP2 expressed by a fowlpox virus recombinant confers protection against disease in chickens.

Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Amino Acid Sequence↗

Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindIII J fragment despite divergent location of the thymidine kinase gene.

DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.

Amino Acid Sequence↗

Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutinin-neuraminidase gene.

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) strain Texas was generated. Immunoprecipitation with chicken anti-NDV serum confirmed authentic expression of the HN protein. Protection of chickens from infection with NDV was observed when birds were immunized with the recombinant HN fowlpox virus by the intramuscular route after one or two inoculations. Vaccination by the ocular route with a mixture of fowlpox recombinants expressing the fusion and HN proteins did not show added protection over that seen with the individual viruses.

Animals↗

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV.

The hemagglutinin-neuraminidase (HN) gene from the Beaudette C strain of Newcastle disease virus (NDV) has been expressed in a recombinant fowlpox virus vector. The HN gene, under the control of the vaccinia p7.5 promoter, was inserted into a nonessential gene in the terminal inverted repeats of fowlpox virus. Expression was demonstrated in tissue culture, a protein of the correct size for fully glycosylated HN protein being recognized by an HN-specific monoclonal antibody on Western blots. When the recombinant fowlpox virus was inoculated into chickens by intravenous or wing-web routes, antibody which recognizes HN from purified NDV virions was produced. Protective immunity to NDV was generated in the chickens; at the highest dose of vaccine 100% of the chickens tested were protected against challenge with a virulent strain of NDV.

Animals↗

Protective immunity against avian influenza induced by a fowlpox virus recombinant.

Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.

Animals↗

Protection against turkey rhinotracheitis pneumovirus (TRTV) induced by a fowlpox virus recombinant expressing the TRTV fusion glycoprotein (F).

A recombinant fowlpox virus was produced which expressed the fusion protein (F) of turkey rhinotracheitis virus (TRTV), a pneumovirus. Turkey poults were vaccinated twice, at an interval of 2 weeks, intramuscularly and by wing web on each occasion, with the recombinant or a control fowlpox virus. Two weeks after the second vaccination the poults were challenged superconjunctivally and intranasally with virulent TRTV. A partially protective immune response was achieved; turkeys vaccinated with the F recombinant showed milder clinical signs and 1000-fold less challenge virus was recovered from the nose and trachea compared with turkeys that had been vaccinated with control fowlpox virus. Expression of the F protein induced antibodies which were detectable both by an ELISA and a virus neutralization test. These results show that the immune responses to the F protein play a major role in protection against TRTV and indicate that recombinant viruses expressing the TRTV F protein have potential as vaccines against TRT.

Animals↗