Fowlpox Virus Strains Research Articles

Retention of 1.2 kbp of ‘novel’ genomic sequence in two European field isolates and some vaccine strains of Fowlpox virus extends open reading frame fpv241

The emergence of variant fowlpox viruses (FWPVs) and increasing field use of recombinants against avian influenza H5N1 emphasize the need to monitor vaccines and to distinguish them from field strains. Five commercial vaccines, two laboratory viruses and two European field isolates were characterized by PCR and sequencing at 18 loci differing between attenuated FP9 and its pathogenic progenitor. PCR failed to discriminate between the viruses and sequence determination revealed no significant differences at any locus, except for a polymorphic locus encompassed by deletion 24 (9.3 kbp) in FP9. Surprisingly, 'novel' previously unreported sequence (spanning 1.2 kbp) was found in both European field isolates and three of the vaccines. It was absent from the other two vaccines, removed by a 1.2 kbp deletion identical to that surprisingly also observed in the completely sequenced genome of FPV USDA. This locus (H9) adds a potentially useful tool for discriminating between FWPV field isolates and vaccines.

Isolation and Molecular Biological Investigations of Avian Poxviruses from Chickens, a Turkey, and a Pigeon in Croatia

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.

Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies

The FP9 strain of F has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modified V Ankara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens from Plasmodium berghei (circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These "passage-specific" alterations are hypothesized to be involved in determining the immunogenicity of fowlpox virus as a recombinant vaccine vector.

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2–3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression.

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.

In ovo inhibition of fowlpoxvirus replication by a gall extract from Guiera senegalensis

Several field isolates of fowlpoxvirus (FPV) from Burkina Faso, West Africa, were isolated and partly evaluated by molecular analysis. In addition, the in ovo antiviral activity against FPV of a gall extract from Guiera senegalensis was determined. Three viral isolates were obtained from suspected fowlpox cases after passage in embryonating chicken eggs and their poxviral identity confirmed by electron microscopy. All isolates were found to be pathogenic for chicks and all grew well in cell culture. Polymerase chain reaction and sequencing of amplicons revealed sequences identical with those of other FPV strains. The most studied isolate was then employed for use in an antiviral assay. An aqueous acetone extract from the galls of G. senegalensis was found to inhibit both virus-induced pock formation and to reduce viral titre in embryonating chicken eggs. The suggested mechanism of action is the activation of the alternative complement pathway and the inhibition of FPV-induced cholesterogenesis in ovo by constituents of the gall extract. Plusieurs souches de virus de la variole aviaire (FPV) isolées au Burkina Faso, en Afrique de l'Ouest, ont été partiellement étudiées par une analyse moléculaire. En plus, l'activité antivirale, vis-à-vis de la FPV, d'un extrait de galle du Guiera senegalensis a été déterminée in ovo. Trois souches virales ont été obtenues à partir de suspicions de FP après passages sur œufs embryonnés de poule et leur identité poxvirale a été confirmée en microscopie électronique. Toutes les souches se sont révélées pathogènes pour les poussins et toutes ont bien poussé sur cultures cellulaires. La PCR et le séquençage des amplicons ont révélé des séquences identiques à ceux d'autres souches de FVP. La souche la plus étudiée a ensuite été utilisée dans un essai d'activité antivirale. Un extrait acétone aqueux de galle du G. senegalensis s'est révélé inhiber la formation de pustule induite par le virus et réduire le titre du virus sur œufs embryonnés de poule. Le mécanisme suggéré de l'action des constituants de l'extrait de Galle est l'activation de la voie alterne du complément et l'inhibition de la cholestérogenèse induite par le FPV in ovo. Verschiedene Feldisolate des Geflügelpockenvirus (FPV) aus Burkina Faso, Wesrafrika, wurden isoliert und teilweise mit molekularen Untersuchungsmethoden charakterisiert. Außerdem wurde die antivirale Aktivität eines Galleextrakts aus Guiera senegalensis gegen FPV in ovo ermittelt. Drei Virusstämme wurden von FP-Verdachtsfällen nach Anzüchtung in embryonierten Hühnereiern isoliert und durch elektronenmikroskopische Untersuchungen als Pockenviren identifiziert. Alle Isolate waren pathogen für Hühner und vermehrten sich in der Zellkultur gut. Die PCR und die Sequenzierung von Amplifikaten ließ Sequenzen erkennen, die mit denjenigen von anderen FPV identisch waren. Das am besten untersuchte Isolat wurde dann für den Einsatz in einem antiviralen Test verwendet. Ein wässeriges Acetonextract aus der Galle von G. senegalensis war fähig sowohl die virusinduzierte Pockenformation zu hemmen als auch den Virustiter in embyonierten Hühnereiern zu reduzieren. Es wird vermutet, dass die Wirkungsweise auf der Aktivierung einer alternativen Komplementreihenfolge und der Hemmung der FPV-induzierten in ovo-Cholesterogenese durch Bestandteile des Galleextrakts beruht. Varios aislamientos de campo del virus de viruela (FPV) de Burkina Faso, oeste de África, fueron aislados y parcialmente evaluados mediante análisis moleculares. Además, se determinó la actividad antiviral in ovo frente a FPV de extracto de bilis de Guiera senegalensis. Se obtuvieron tres aislados virales de casos sospechosos de FP, tras realizar un pase en huevos embrionados y la identificación viral fue confirmada por microscopia electrónica. Todos los aislamientos resultaron patógenos en pollos y crecieron bien en cultivo celular. La técnica de PCR y la secuenciación de los amplicones revelaron secuencias idénticas con otras cepas de FPV. El aislado más estudiado fue el empleado posteriormente en una prueba antiviral. Un extracto acuoso y con acetona de bilis de G senegalensis resultó inhibir la formación de placas inducidas por el virus y redujo el título viral en huevos de pollo embrionados. El mecanismo de acción sugerido es la activación de la vía alternativa del complemento y la inhibición de la colesterogénesis inducida por FPV in ovo por constituyentes del extracto de bilis.

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.

Detection of specific reticuloendotheliosis virus sequence and protein from REV-integrated fowlpox virus strains

The detection is described of reticuloendotheliosis virus (REV) protein in tissue culture of chicken embryonated cells (CEFs) infected with field isolates of fowl poxvirus (FPV). By the polymerase chain reaction (PCR), five out of the six field isolates, but two out of the seven vaccine strains of FPV, were found to have had a 291 bp repeat sequence of REV-LTR integrated in their genomic DNA. An immunofluorescence (IF) method was employed using a monoclonal antibody (MAb) known to specify strain common envelope proteins for REV and allowed to detect the presence of a specific REV protein. The IF results indicate the localization of REV proteins in boundaries defined precisely within cells infected with these field strains of FPV carrying REV (FPV-REV). Furthermore, by immunoblotting (IB) using a chemiluminescent detection kit, the REV protein reacted specifically with the MAb and had a relative molecular mass (RMM) of 62 kDa. The data have the potential to advance substantially the current understanding of the integrated REV in FPV strains; and the identification of a unique protein associated with variant forms of FPV will also offer great potential for identification of novel vaccine candidates for use in poultry against variant forms of FPV.

Molecular characterization of reticuloendotheliosis virus insertions in the genome of field and vaccine strains of fowl poxvirus.

Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent "variant" FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5' long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3' LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3' LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5' LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3' LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5' LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5' LTR sequences, positive PCR results with primers that amplify the 5' LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3' LTR PCR.

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.

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.

Vaccination against Fowlpox virus via drinking water.

The oral vaccination against Fowlpox was investigated via drinking water containing the F132-c strain of Fowlpox virus to be effective even though the vaccine virus-titer was 10(4) TCID (50)/dose each time. When the virus-titer of the F132-c strain was 10(4-5 )TCID(50)/dose per single drinking water vaccination, 90% or more of chickens were not protected, however, they were protected when vaccinated twice via drinking water. A weak immune response occurred by a slight infection after the first vaccination, and due to memory cells, a booster could work well after the second vaccination. These results suggest the possibility of reducing both the amount of virus required for a vaccine via drinking water and the labor cost in the field.

Reticuloendotheliosis Virus Integration in the Fowl Poxvirus Genome: Not a Recent Event

Integration of reticuloendotheliosis virus (REV) into the genome of fowl poxvirus (FPV) has been reported recently. With a view to determine whether this event had occurred in the past, we screened by polymerase chain reaction (PCR) for the presence of REV provirus in the DNAs of nine avian poxviruses, some of which had been lyophilized 50 yr ago. For REV, 5' long terminal repeat (LTR) and REV envelope sequences were amplified, whereas for FPV, the major envelope antigen gene and the region flanking REV sequences were amplified. In six of seven FPV strains examined, the specific PCR amplicons were obtained for both REV provirus and FPV sequences. One isolate in which presence of REV 5' LTR and envelope was not detected by PCR, a LTR remnant was detected by Southern hybridization. Interestingly, no REV sequence was detected in either canary poxvirus or pigeon poxvirus genome. These observations indicate that REV integration in the FPV genome is not a recent phenomenon but probably occurred prior to 1949.

Re-emerging fowlpox: Evaluation of isolates from vaccinated flocks

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

Characterization of poxviruses from forest birds in Hawaii.

Two strains of avian pox viruses were isolated from cutaneous lesions in Hawaiian crows (Corvus hawaiiensis) examined in 1994 and a third from a biopsy obtained in 1992 from an infected bird of the Apapane species (Himatione sanguinea) by inoculation of the chorioallantoic membranes (CAM) of developing chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies characteristic of pox virus infection. The pathogenicity of these three viruses in domestic chickens was mild as evidenced by the development of relatively minor lesions of short duration at the sites of inoculation. Their virulence in this host was similar to that of a fowlpox virus (FPV) vaccine strain and contrasted greatly with the ability of two field strains of FPV to produce extensive proliferative lesions. One of the Hawaiian crow pox virus isolates as well as the one originating from the Apapane species could be propagated in two secondary avian cell lines, QT-35 and LMH. A comparison of the restriction fragment length polymorphisms (RFLP) of the genomes of the two cell line-adapted viruses, generated by EcoRI digestion, revealed a limited degree of similarity. Moreover, neither profile was comparable to those of the two field isolates of FPV, which were almost indistinguishable from each other. Thus, based on the genetic distinctness of the two Hawaiian bird viruses, they appear to represent different strains of avipoxvirus.

Sequence analysis of a 7.3 kbBam H I genomic fragment of fowlpox virus strain 282E4

A 7.3 kb BamH I genomic fragment from fowlpox virus (FPV) strain 282E4 has been sequenced. After comparison with the FPV sequences collected in Genbank, and the entire genomic sequence of vaccinia virus (VV) strain Compenhagen, a ll.2 kb BamH I fragment from FPV strain Munich HP-438 has been found homologous to the fragment. Every predicted open reading frame (ORF) within the fragment has been compared with the ORFs of VV strain Copenhagen at the amino acid level. The signal peptide and transmembrane region of each peptide encoded by each corresponding ORF predicted in the fragment have been studied as well.

Fowlpox virus encodes nonessential homologs of cellular alpha-SNAP, PC-1, and an orphan human homolog of a secreted nematode protein.

The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematode Caenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.

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.

Field and Vaccine Strains of Fowlpox Virus Carry Integrated Sequences from the Avian Retrovirus, Reticuloendotheliosis Virus

For baculoviruses and herpesviruses, integration of transposons or retroviruses into the virus genome has been documented. We report here that field and vaccine strains of fowlpox virus (FPV) carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus (REV). Using PCR and hybridization analysis we observed that vaccine and field strains of FPV carry REV sequences integrated into a previously uncharacterized region of the right 1/3 of the FPV genome. Long-range PCR, hybridization, and nucleotide sequence determination demonstrated that one vaccine strain (FPV S) and recently isolated field strains carry a near-full-length REV provirus. For another vaccine strain (FPV M) a rearranged remnant of the LTR was found at the same insertion site. By Western blotting and reverse transcriptase assays we were unable to demonstrate free REV in supernatants of FPV S cultures. The near-full-length REV provirus integrated into the FPV genome is infectious since FPV S DNA gave rise to REV upon transfection into chicken embryo fibroblasts. Upon infection of chickens with FPV S, all chickens developed high-titered antibodies to REV, and REV was isolated from the blood of half of the inoculated chickens. Our observations add to the list of targets for retrovirus integration into DNA virus genomes. The integration of a near-full-length, and apparently infectious, REV provirus into FPV provides additional transmission routes for the retrovirus by way of the infectious cycle of FPV, including the possibility of mechanical transmission by biting insects since FPV is believed to be transmitted by this route. For large DNA viruses, including the poxviruses, retrovirus integration with attendant possibilities of gene transduction may be an important mechanism for virus evolution, including the acquisition of cellular genes with the potential to modify virus virulence and pathogenicity.