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PubMed2006

Construction and immunogenicity of a recombinant fowlpox virus containing the capsid and 3C protease coding regions of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is an important pathogen with worldwide economic consequences. Consequently, an important goal is the development of a vaccine that can provide rapid protection while overcoming the potential risk associated with the production of conventional inactivated vaccines. An important secondary feature of the vaccine would be the ability to distinguish vaccinated from infected animals. A recombinant fowlpox virus (vUTAL3CP1) containing FMDV capsid polypeptide and 3C coding regions of O/NY00 was constructed and evaluated for its ability to induce humoral and cellular responses in mice and guinea pigs. In addition, the ability to protect guinea pigs against homologous virus challenge was examined. Mice and guinea pigs were given booster vaccinations twice and once, respectively, and guinea pigs were challenged 20 days after the booster vaccination. Control groups included animals inoculated with commercial vaccine, fowlpox virus or phosphate-buffered saline (PBS). All animals vaccinated with vUTAL3CP1 developed specific anti-FMDV antibody and neutralizing antibody, as well as T lymphocyte proliferation response and CTL cytotoxic activity. Three of four guinea pigs vaccinated with vUTAL3CP1 were completely protected from viral challenge. The results demonstrated the potential of a fowlpox virus-based recombinant FMD vaccine.

3C Viral Proteases↗

PubMed2006

Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18.

cDNAs of the HA genes of subtype H5N1 AIV were fused to form a single open reading frame, designated H5HA-H7HA. The H5HA-H7HA cDNA and chicken Interleukin-18 (IL18) were inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce pUTAL-H5-H7-IL18. cDNA of H5N1 AIV HA was inserted into the FPV expression vector pUTA2 to create the recombinant expression plasmid pUTA2-H5. Plasmids were then co-transected into CEF cells. The two recombinant fowlpox viruses (rFPV) were produced by three cycles with the BrdU and verified by RT-PCR, IFA and Western blotting. One-day-old specific pathogen free (SPF) chickens and 7-day-old commercial Leghorn egg-laying chickens were inoculated with 10(6) PFU recombinant or parental fowlpox vaccine viruses by wing-web puncture. Hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were assessed after 1-3 weeks post-immunization. We found that all rFPV-vaccinated groups produced HI-specific antibodies, and the level of cellular immunity induced by the rFPV-H5-H7-IL18 strain was significantly higher than that induced by rFPV-H5HA. At 3 weeks post-inoculation, immunized SPF and Leghorn chickens were challenged with H5N1 HP AIV. The rFPV-H5-H7-IL18 vaccine strains were able to induce complete (10/10) protection, while the rFPV-H5HA vaccine strain induced (9/10) protection. Cloacal swabbing samples were collected from immunized leghorn chickens during the first week post-challenge; no shedding was found in the rFPV-H5-H7-IL18 vaccinated group. The rFPV-H5-H7-IL18 vaccinated group displayed significantly increased weight gain relative to the rFPV-H5HA group. This study reports a significant step in the further development of new AIV vaccines.

Animals↗

PubMed2002

Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy.

Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination. Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA. Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome. Disruption of the F11L coding sequence by integration of an expression cassette for the Escherichia coli lacZ and guanine phosphoribosyltransferase marker genes resulted in the isolation of replication competent knockout viruses. Growth of F11L-knockout viruses in primary chicken embryo fibroblasts was unimpaired in comparison to wild type-virus. To test the generation of vector viruses, an insertion plasmid was constructed that contains F11L-specific sequences for homologous recombination, the E. coli lacZ and gpt genes as transient selectable marker, and the vaccinia virus early/late promoter P7.5 for transcriptional control of target gene expression. The coding sequence of the melanoma-associated antigen tyrosinase was chosen as model recombinant gene. Isolation of tyrosinase-recombinant viruses, which produced stably the insert, demonstrated the usefulness of the F11L-insertion site for the generation of fowlpox vectors. Rapid isolation of those recombinants was achieved by using a double selective system and linearising the vector plasmid before transfection.

Animals↗

PubMed2003

Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes.

Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (NI subtype)genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.

Animals↗

PubMed1992

Comparison of the locations of homologous fowlpox and vaccinia virus genes reveals major genome reorganization.

We have derived a restriction enzyme map for the fowlpox virus FP9 strain. Sites for BamHI, PvuII, PstI and NcoI have been mapped mainly by Southern blotting. The size of the genome derived from the restriction maps (254 kb) corresponds to the figure of 260 +/- 8 kb determined from analysis of genomic DNA by pulsed-field electrophoresis. The map can be compared with a previously published map for a different strain of fowlpox virus using the PstI digest which is common to both studies. Some 65 kb of fowlpox virus sequence, in 11 blocks, as well as individual M13 clones have been aligned with the map. Where those blocks correspond with blocks of homologous genes in vaccinia virus, it is possible to compare the genomic locations for those genes in the two viruses. This comparison reveals that, whereas there are blocks of sequence within which genes exist in the same relative position in the two viruses, the genomic location of those sequence blocks differs widely between the two viruses.

Deoxyribonuclease BamHI↗

PubMed1982

Effects of fowlpox virus infection on lipid metabolism in cultured chicken embryo cells.

Lipid biosynthesis was measured in cultured chicken embryo cells after infection with fowlpox virus. Between 24 and 72 h postinfection, fowlpox virus-infected cells incorporated less [14C]acetate and 3H2O into fatty acids and sterols than did mock-infected cells, demonstrating a virus-dependent inhibition of general lipid metabolism. Two specific effects of fowlpox virus infection were an accumulation of C-4 alkylated sterol intermediates and inhibition of monounsaturated fatty acid biosynthesis.

Animals↗

PubMed1990

Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.

A cDNA copy of the RNA encoding the fusion (F) protein of Newcastle disease virus (NDV) strain Texas, a velogenic strain of NDV, was obtained and the sequence was determined. The 1,792-base-pair sequence encodes a protein of 553 amino acids which has essential features previously established for the F protein of virulent NDV strains. These include the presence of three strongly hydrophobic regions and pairs of dibasic amino acids in the pentapeptide Arg-Arg-Gln-Arg-Arg preceding the putative cleavage site. When inserted into a fowlpox virus vector, a glycosylated protein was expressed and presented on the surface of infected chicken embryo fibroblast cells. The F protein expressed by the recombinant fowlpox virus was cleaved into two polypeptides. When inoculated into susceptible birds by a variety of routes, an immunological response was induced. Ocular or oral administration of the recombinant fowlpox virus gave partial protection, whereas both intramuscular and wing-web routes of inoculation gave complete protection after a single inoculation.

Amino Acid Sequence↗

PubMed1998

The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic.

The immunodominant, 39,000-molecular weight core protein (39K protein) of fowlpox virus (FP9 strain), equivalent to the vaccinia virus A4L gene product, contains highly charged domains at each end of the protein and multiple copies of a 12-amino-acid serine-rich repeat sequence in the middle of the protein. Similar repeats were also detected in other fowlpox virus strains, suggesting that they might confer a selective advantage to the virus. The molloscum contagiosum virus homolog (MC107L) also contains repeats, unlike the vaccinia virus protein. The number of repeats in the fowlpox virus protein does not seem to be crucial, since some strains have a different number of repeats, as shown by the difference in the size of the protein in these strains. The repeat region could be deleted, indicating that it is not essential for replication in vitro. It was not possible to delete the entire 39K protein, indicating that it was essential (transcriptional control signals for the flanking genes were left intact). The repeat region is partly responsible for the immunodominance of the protein, but the C-terminal part of the protein also contains highly antigenic linear epitopes. A role for the 39K protein in immune system modulation is discussed.

Amino Acid Sequence↗

PubMed2006

Development and use of fowlpox vectored vaccines for avian influenza.

The avian influenza (AI) vaccine designated TROVAC-AIV H5 (TROVAC-H5) contains a live recombinant fowlpox rec. (FP) recombinant (recFP), expressing the hemagglutinin (HA) gene of an AI H5 subtype isolate. This recombinant vaccine was granted a license in the United States for emergency use in 1998 and full registration in Mexico, Guatemala, and El Salvador where over 2 billion doses have been administered. One injection of TROVAC-H5 protects chickens against AI-induced mortality and morbidity for at least 20 weeks, and significantly decreases shedding after challenge with a wide panel of H5-subtype AI strains, regardless of neuraminidase subtype. Recently, excellent protection was demonstrated against 2003 and 2004 Asian highly pathogenic H5N1 isolates. Whereas TROVAC-H5 AI H5 efficacy was not inhibited by anti-AI or anti-fowlpox maternal antibodies (passive immunity), protection to AI was significantly decreased in chickens previously vaccinated or infected with FP (active immunity). Advantages of the TROVAC-H5 vaccine over inactivated AI vaccines are: (a) single administration at 1 day of age and early onset (1 week) of protection, (b) easy monitoring of AI infection in vaccinated flocks with agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) used as tests to differentiate infected from vaccinated animals (DIVA tests), and (c) no residue problem due to adjuvant. These features make TROVAC-H5 an ideal AI vaccine for routine administration of day-of-age chicks in hatcheries. RecFP expressing HA from three lineages of H7 subtype (Eurasian, American, and Australian) were also tested for efficacy against a highly pathogenic avian influenza (HPAI) Eurasian HPAI H7N1. Only the recFP expressing the Eurasian H7 gene provided sufficient protection indicating that the breadth of protection induced by recFP is apparently restricted for H7 isolates. The fowlpox vector technology can also be used for the production of an emergency vaccine: once the HA sequence of an emerging AI virus is known, recFP can be rapidly generated. TROVAC-H5 has recently been shown to be immunogenic in cats and could therefore also be considered for use in mammals.

Animals↗

PubMed2004

[Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice].

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

AIDS Vaccines↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Occurrence of atypical fowlpox in poultry farms in Kenya.

Atypical fowlpox occurred in several poultry farms in Kenya. On two occasions layers had their eyes closed and egg production dropped. Fowlpox virus was isolated from lesions on the inner surfaces of the closed eyelids. Other chickens had lesions covered by yellow caseous necrotic material in the mouth, around the epiglottis, and in the trachea and choanae. Typical proliferative cutaneous lesions were observed in birds of all ages in other flocks examined. Fowlpox virus was recovered from both cutaneous and diphtheritic lesions. The infected chorioallantoic membranes had focal hyperplastic lesions containing pink-staining intracytoplasmic inclusion bodies in most cells. Transmission studies showed that the virus was highly virulent to susceptible chickens.

Animals↗

PubMed1996

Fowlpox virus structural protein immunogens and characterization of single band polypeptide.

A fowlpox virus isolate obtained from an outbreak of disease in a vaccinated poultry flock was propagated in chicken embryo fibroblast cell culture. Analysis of purified virus polypeptide on 7.5-15% gradient polyacrylamide gel revealed 45 structural polypeptides after Coomassie blue staining. The mol.wt. of polypeptides ranged between 225.53 and 10.50 kDa with total mol.wt. of 2650 kDa. Variable numbers of immunogenic virion polypeptides were detected in immunoblot with fowlpox virus infected chicken sera collected at different time intervals. A total of 29 polypeptides reacted with sera collected at 1st week post-infection and the number gradually declined to 27, 26, 20, 17 and 15 when reacted with 2nd, 3rd, 4th, 5th and 6th week post-infection sera, respectively. Reaction with fowlpox virus hyperimmune sera revealed 35 immunogenic polypeptides. A number of major and minor immunogens were detected. Antisera against seven major single band polypeptides including one double band polypeptide showed very low reactivity both in ELISA and serum neutralization test. Involvement of multigenic components in virus neutralization is indicated.

Animals↗

PubMed1969

Accelerated plaque formation by fowlpox virus in the presence of chymotrypsin.

Chymotrypsin enhanced fowlpox virus plaque formation in chick embryo cell cultures. A simplified plaque assay for fowlpox virus is described. Plaques were produced in 3 days when chymotrypsin was included in a serum-free fluid overlay. Plaques were also produced in 5 to 6 days under an agar overlay when a medium containing fetal calf serum was employed. Kinetics of plaque formation were also studied, and it was shown that fowlpox virus plaque diameters grow at a linear rate.

Agar↗

PubMed1966

Physicochemical properties of fowlpox virus deoxyribonucleic acid and its anomalous infectious behavior.

Randall, Charles C. (University of Mississippi, Jackson), Lanelle G. Gafford, Richard L. Soehner, and James M. Hyde. Physicochemical properties of fowlpox virus deoxyribonucleic acid and its anomalous infectious behavior. J. Bacteriol. 91:95-100. 1966.-Deoxyribonucleic acid (DNA) was extracted from fowlpox virus-infected tissue, purified inclusions, and purified virus by five variations of detergent and phenol methods. Phenol methods gave a poor yield, whereas detergent techniques extracted up to 78% of the DNA. The buoyant density was 1.695 g/ml, and the melting temperature in 7.2 m NaClO(4) was 39 C, both approximately equivalent to a guanine plus cytosine content of 35 moles per cent. Further proof of the double-stranded nature of the DNA was shown by the characteristic behavior toward deoxyribonuclease, formaldehyde, and heat. Infectious DNA was obtained by the various methods described, but this manifestation of biological activity was capricious and for unknown reasons was often not evident. The infectivity could not be related quantitatively to the amount of DNA employed. Furthermore, the infectious nature of fowlpox virus DNA was demonstrable only when the route of infection was the chorioallantoic membrane. In contrast, whole virus infected both membrane and chick skin with equal efficiency.

Biological Assay↗

PubMed1994

Influence of dose and route of inoculation on responses of chickens to recombinant fowlpox virus vaccines.

The influence of dose and route of inoculation on responses of chickens to vaccination with recombinant fowlpox viruses (rFPVs) expressing an influenza haemagglutinin (HA) (FPV-HA) and the infectious bursal disease virus (IBDV) VP2 antigen (FPV-VP2) has been evaluated. Antibody responses to influenza and fowlpox virus were generated following vaccination via the wing web by subcutaneous inoculation or skin scarification. Intranasal and conjunctival inoculation failed to induce antibodies to FPV or influenza. Following direct intratracheal inoculation antibodies developed to influenza but not FPV. Dose response studies with the FPV-HA and FPV-VP2 recombinants showed that good responses to FPV and the vaccine antigen could be generated over a wide (10000 fold) dose range following wing web inoculation. The responses generated by the FPV-VP2 recombinant over this vaccine dose range protected against IBDV infection of the bursae following challenge with the Australian IBDV 002/73 isolate. These data suggest that effective application of rFPVs for poultry vaccination may be restricted to wing web and parenteral routes of inoculation.

Animals↗

PubMed1993

Recombinant fowlpox virus vaccines for poultry.

The intensive poultry industries rely heavily upon the use of vaccines for disease control. Viral vector based vaccines offer new avenues for the development of vaccines for effective disease control in poultry. Techniques developed for the construction of recombinant vaccinia viruses have been readily adapted to the construction of recombinant viruses based on fowlpox virus (rFPV). The ability to insert several genes into the large genome of fowlpox may enable the development of multivalent vaccines and vaccines incorporating immune response modifiers such as lymphokines. Newcastle disease, avian influenza, infectious bursal disease and Marek's disease antigens expressed by rFPV have been shown to be effective vaccines in poultry. None appear, however, to provide a substantial improvement in vaccine efficacy. Recombinant FPV will be a valuable adjunct to conventional vaccines currently in widespread use. Whether rFPV or other vector based vaccines can circumvent the problems of vaccination in the presence of high maternally derived antibodies is yet to be resolved. The observation that avipoxvirus recombinants may be suitable for the vaccination of non-avian species provides an added dimension to vaccines based on FPV or other avipoxviruses. Recombinant FPV will find a useful role in poultry disease control when used in conjunction with conventional vaccines.

Animals↗

PubMed2003

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.

Animals↗