An atypical clinicopathological manifestation of fowlpox virus associated with reticuloendotheliosis virus in commercial laying hen flocks in Brazil.

Fowlpox (FP) is a common epitheliotropic disease in chickens that is usually controlled by live attenuated vaccines. However, there have been some reports of outbreaks of FP in recent years, even in vaccinated flocks, presenting as atypical lesions and feathering abnormalities in chickens. These findings can be associated with fowlpox virus (FPV) with the reticuloendotheliosis virus (REV) integrated into its genome. In the present study, outbreaks of atypical FP were explored in vaccinated commercial laying hen flocks to determine the nature of the causative agent by histopathologic and molecular approaches. FPV and REV were detected and classified into subclade A1 of the genus Avipoxvirus and subtype 3 of REV (REV3), respectively. Additionally, heterogeneous populations of FPV with partial (containing only a remnant long terminal repeat-LTR) or total (all functional genes) integration of REV were identified by heterologous PCRs and detected considering reference integration sites. These results indicate the mechanism of chimeric genome FPV-REV associated with outbreaks and atypical clinicopathological manifestations in commercial laying hens for the first time in Brazil and in South America. In addition, this study demonstrates the emergence of REV integrated in the FPV genome in Brazilian chicken flocks.

Construction and characterization of novel fowlpox virus shuttle vectors.

Viral vectors are important vehicles in vaccine research. Avipoxviruses including fowlpox virus (FPV) play major roles in viral vaccine vector development for the prevention and therapy of human and other veterinary diseases due to their immunomodulatory effects and safety profile. Recently, we analyzed the genomic and proteomic backgrounds of the Chinese FPV282E4 strain. Based on analysis of the whole genome of FPV282E4, the FPV150 and FPV193 loci were chosen as insertion sites for foreign genes, and two shuttle vectors with a triple-gene expression cassette were designed and constructed. Homologous recombination between the FPV virus genome and sequences within the shuttle plasmids in infected cells was confirmed. The recombinants were obtained through several rounds of plaque purification using enhanced green fluorescent protein as a reporter and evaluated for the correct expression of foreign genes in vitro using RT-PCR, real-time PCR and Western blotting. Morphogenesis and growth kinetics were assayed via transmission electron microscopy and viral titering, respectively. Results showed that recombinant viruses were generated and correctly expressed foreign genes in CEF, BHK-21 and 293T cells. At least three different exogenous genes could be expressed simultaneously and stably over multiple passages. Additionally, the FPV150 mutation, FPV193 deletion and insertion of foreign genes did not affect the morphogenesis, replication and proliferation of recombinant viruses in cells. Our study contributes to the improvement of FPV vectors for multivalent vaccines.

Making an avipoxvirus encoding a tumor-associated antigen and a costimulatory molecule.

Fowlpox virus (FPV) is a double-stranded DNA virus with a history of use as a live attenuated vaccine in commercial poultry production systems. FPV is also highly amenable to genetic engineering, with a large cloning capacity and many nonessential sites available for integration, meaning that in recombinant form, several transgenes can be expressed simultaneously. Recombinant FPV has proven an effective prophylactic vaccine vector for other diseases of birds, as well as other animal species (Brun et al., Vaccine 26:6508-6528, 2008). These vectors do not integrate into the host genome nor do they undergo productive replication in mammalian cells; thus they have a proven and impeccable safety profile and have been progressed as prophylactic and therapeutic vaccine vectors for use in humans (Beukema et al., Expert Rev Vaccines 5:565-577, 2006; Lousberg et al., Expert Rev Vaccines 10:1435-1449, 2011). Furthermore, repeated immunization with FPV does not blunt subsequent vaccine responses, presumably because it is replication-defective, and thus larger doses can be routinely administered (Brun et al., Vaccine 26:6508-6528, 2008). This strengthens the case for FPV as a viable platform vaccine vector, as it means it can be used repeatedly in an individual to achieve different immunological outcomes. Here we describe in detail the construction of a recombinant variant of FPV expressing the prostate tumor-associated antigen prostatic acid phosphatase (PAP) in conjunction with the immunostimulatory cytokine, interleukin-2 (IL-2), which, if undertaken under the appropriate regulatory conditions and with approvals in place, would theoretically be amenable to clinical trial applications.

Results of a randomized phase I gene therapy clinical trial of nononcolytic fowlpox viruses encoding T cell costimulatory molecules.

Oncolytic viruses have shown promise as gene delivery vehicles in the treatment of cancer; however, their efficacy may be inhibited by the induction of anti-viral antibody titers. Fowlpox virus is a nonreplicating and nononcolytic vector that has been associated with lesser humoral but greater cell-mediated immunity in animal tumor models. To test whether fowlpox virus gene therapy is safe and can elicit immune responses in patients with cancer, we conducted a randomized phase I clinical trial of two recombinant fowlpox viruses encoding human B7.1 or a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3; TRICOM). Twelve patients (10 with melanoma and 2 with colon adenocarcinoma) enrolled in the trial and were randomized to rF-B7.1 or rF-TRICOM administered in a dose escalation manner (~3.7×10(7) or ~3.7×10(8) plaque-forming units) by intralesional injection every 4 weeks. The therapy was well tolerated, with only four patients experiencing grade 1 fever or injection site pain, and there were no serious adverse events. All patients developed anti-viral antibody titers after vector delivery, and posttreatment anti-carcinoembryonic antigen antibody titers were detected in the two patients with colon cancer. All patients developed CD8(+) T cell responses against fowlpox virus, but few responses against defined tumor-associated antigens were observed. This is the first clinical trial of direct (intratumoral) gene therapy with a nononcolytic fowlpox virus. Treatment was well tolerated in patients with metastatic cancer; all subjects exhibited anti-viral antibody responses, but limited tumor-specific T cell responses were detected. Nononcolytic fowlpox viruses are safe and induce limited T cell responses in patients with cancer. Further development may include prime-boost strategies using oncolytic viruses for initial priming.

An outbreak of lymphomas in a layer chicken flock previously infected with fowlpox virus containing integrated reticuloendotheliosis virus.

Visceral lymphomas occurred in a 236-day-old layer flock previously diagnosed with reticuloendotheliosis virus (REV)-integrated fowlpox virus (FPV) infection at the age of 77 days. Common pathologic lesions were multiple neoplastic nodules of homogeneous lymphocytes in the livers and spleens of all submitted chickens. All neoplastic tissues were positive for the REV envelope (env) gene by PCR. In a retrospective molecular study of FPV-infected 77-day-old chickens from the same flock, we identified nearly full-length REV provirus integrated into the genome of FPV as well as the REV env gene in trachea samples, whereas only the REV LTR region was present in the FPV strain used to vaccinate this flock. The 622-bp REV env gene nucleotide sequence derived from the trachea and neoplastic tissues was identical. Commercial ELISA of serum samples revealed that all chickens aged between 17 and 263 days in this flock were positive for REV but not for avian leukosis virus. Taken together, the evidence suggests that the visceral lymphomas were caused by a REV-integrated FPV field strain. FPV infections of commercial chickens should be followed up by careful monitoring for manifestations of REV infection, including lymphomas and immune depression, considering the ease with which the REV provirus appears to be able to integrate into the FPV genome.

Gene expression profiling in rMd5- and rMd5deltameq-infected chickens.

Marek's disease (MD) is a lymphoproliferative disorder of domestic chickens caused by a highly contagious and oncogenic alpha-herpesvirus, Marek's disease virus (MDV). MD is characterized by bursal-thymic atrophy and rapid onset of T-cell lymphomas that infiltrate lymphoid tissues, visceral organs, and peripheral nerves with severe clinical signs that include transient paralysis, anemia, weight loss, and neurologic disorders. Using overlapping cosmids- and BAC-cloned MDV, it has been shown that MDV-encoded vIL-8, pp38, vTR, vLIP, RLORF4, and meq are among the many essential genes that play critical roles in viral pathogenesis. Of all the genes investigated so far, only meq has been shown to be consistently expressed in all MDV-derived tumors and lymphoblastoid cell lines. Meq is a basic leucine-zipper protein that shares homology with the jun/fos family of transcriptional factors. There are two copies of meq gene within the MDV genome that are only present in the serotype-1 strains. It has been shown conclusively that deletion of meq results in loss of transformation of T cells in chickens, with no effect on the early cytolytic phase of infection in lymphoid organs, which is essential for induction of innate and adaptive immunity. The goal of this study was to investigate 1) the effect of the meq oncogene on the expression pattern of select chicken immune and nonimmune-related genes, and 2) its potential role in MDV-induced apoptosis. We used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profiling of a panel of chicken genes in rMd5- and rMd5deltameq-infected chickens at 5, 14, 21, and 35 days postinfection (dpi). Although the transcriptional activities of several immune-related genes, including IL-6, IL-10, cMGF, GM-CSF, iNOS, IFNbeta, and INFgamma, were higher in rMd5deltameq-infected chickens at 5 dpi when compared to the rMd5-infected birds, the differences in expression levels of the tested genes between the two viral constructs were not significant. In addition, a reduction in the transcriptional activity of Bdcl2 in recombinant fowlpox virus (rFPV)+meq-infected chicken embryonic fibroblasts suggested that meq alone did not impede FPV-induced apoptosis. The likely suppressive nature and anti-inflammatory function of the meq oncogene and its possible role in virus-induced cell death is discussed.

An efficient method for generating poxvirus recombinants in the absence of selection.

The use of selectable markers (ecogpt) and selection pressures to aid in detection of poxvirus (Vaccinia, VV) recombinants has been implicated in the unintended introduction of second site mutations. We have reinvestigated the use of the helper virus system described by Scheiflinger et al. and adapted by Yao and Evans which produces recombinants at a high frequency in the absence of any selection, at a rate of 6­100%. Our system uses fowlpox virus (FPV) as the infectious helper virus which in infected cells provides the enzymatic apparatus for transcription and replication of a purified, transfected VV genome and for recombination with a second transfected PCR generated DNA fragment. To optimize the system, a PCR DNA fragment was generated that contained poxvirus promoter driven gfp and lacZ genes inserted within the coding sequences of the viral thymidine kinase gene. This PCR fragment was co-transfected together with VV genomic DNA. Recombinant VV was identified by plaquing the mixture on cells non-permissive for FPV and selection of green fluorescent or LacZ positive recombinant vaccinia plaques. The system was optimized using FPV permissive cells (CEF) and non-permissive cells (A549, CV-1) for both the initial infection/transfection and the subsequent selection. Up to 70% of the progeny vaccinia virus contained the gfp/LacZ insertion. In order to test for the presence of FPV/VV intertypic recombinants or other unintended mutations, recombinant wtVV (RwtVV) was regenerated from the gfp/LacZ viruses and evaluated by RFLP analysis and pathogenesis in animals. While all RwtVVs were viable in cell culture, in many of the RwtVV isolates, RFLP differences were noted and while some recombinant viruses exhibited wild type behavior in mice, a wide range of virulence indicative of unintended changes suggests that mutants created by "rescue" systems require careful analysis particularly before use for in vivo studies employing animal models.

Canarypox and fowlpox viruses as recombinant vaccine vectors: a biological and immunological comparison.

Canarypox and fowlpox viruses represent alternative vaccine vectors due to their natural host-range restriction to avian species. Although they cannot replicate in mammals, they correctly express transgenes in human cells and elicit a complete immune response in vaccinated subjects. Several studies have evaluated their genomic differences and protective efficacy in preclinical trials, but detailed information is not available for their transgene expression, cytokine modulation and abortive replication in mammals. This study demonstrates that the heterologous HIV gag/pol and env genes are more efficiently expressed by fowlpox in non-immune and immune cells. The production of retrovirus-like particles, the longer transgene expression, and a balanced cytokine induction may confer to fowlpox-based recombinants the ability to elicit a better immune response.

Canarypox and fowlpox viruses as recombinant vaccine vectors: an ultrastructural comparative analysis.

Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak.

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121.

The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

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.

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.

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.

Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells.

The ability to manipulate the vaccinia virus (VAC) genome, as a plasmid in bacteria, would greatly facilitate genetic studies and provide a powerful alternative method of making recombinant viruses. VAC, like other poxviruses, has a linear, double-stranded DNA genome with covalently closed hairpin ends that are resolved from transient head-to-head and tail-to-tail concatemers during replication in the cytoplasm of infected cells. Our strategy to construct a nearly 200,000-bp VAC-bacterial artificial chromosome (BAC) was based on circularization of head-to-tail concatemers of VAC DNA. Cells were infected with a recombinant VAC containing inserted sequences for plasmid replication and maintenance in Escherichia coli; DNA concatemer resolution was inhibited leading to formation and accumulation of head-to-tail concatemers, in addition to the usual head-to-head and tail-to-tail forms; the concatemers were circularized by homologous or Cre-loxP-mediated recombination; and E. coli were transformed with DNA from the infected cell lysates. Stable plasmids containing the entire VAC genome, with an intact concatemer junction sequence, were identified. Rescue of infectious VAC was consistently achieved by transfecting the VAC-BAC plasmids into mammalian cells that were infected with a helper nonreplicating fowlpox virus.

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.

Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.

Expression of target genes by coinfection with replication-deficient viral vectors.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5' untranslated region (UTR), a luciferase gene and the 3' UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was obtained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and AdexCAT7 was established for high level of expression of a target gene in various cells.

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.

Fowlpox virus host range restriction: gene expression, DNA replication, and morphogenesis in nonpermissive mammalian cells.

Fowlpox virus (FPV), type species of the Avipoxvirus genus, causes a slow-spreading pox disease of chickens. Following infection of mammalian cells there is no evidence of productive replication of FPV although cytopathic effects are induced and FPV recombinants have been shown to express foreign genes from vaccinia virus early/late promoters. Here we report results of a study to investigate the expression of FPV genes, the replication of FPV genomic DNA, and any ultrastructural changes in mammalian cells infected by wild-type virus, undertaken as a first step in elucidating the nature of the block (or blocks) to productive replication of FPV in mammalian cells. Early and late gene expression as well as genomic DNA replication was observed in fibroblast-like cell lines of monkey and human origin. Furthermore, viral morphogenesis was observed in monkey cells, with the production mainly of immature particles though smaller numbers of apparently mature virus particles were observed.