Reticuloendotheliosis Virus Provirus Research Articles

Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria: Detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus.

Cutaneous fowlpox is a disease of chickens and turkeys caused by the fowlpox virus (FWPV), characterized by the development of proliferative lesions and scabs on unfeathered areas. FWPVs regularly carry an integrated, active copy of the reticuloendotheliosis virus (REV), and it has been hypothesized that such FWPVs are more problematic in the field. Extensive outbreaks are usually observed in tropical and sub-tropical climates, where biting insects are more difficult to control. Here, we report an epidemic of 65 cutaneous fowlpox cases in Austria in layer chickens (91% of the cases) and broiler breeders and turkeys, all of them unvaccinated against the disease, from October 2018 to February 2020. The field data revealed appearance in flocks of different sizes ranging from less than 5000 birds up to more than 20,000 animals, with the majority raised indoors in a barn system. The clinical presentation was characterized by typical epithelial lesions on the head of the affected birds, with an average decrease of 6% in egg production and an average weekly mortality of 1.2% being observed in the flocks. A real-time multiplex polymerase chain reaction (PCR) confirmed the presence of FWPV-REV DNA, not only in the lesions but also in the environmental dust from the poultry houses. The integration of the REV provirus into the FWPV genome was confirmed by PCR, and revealed different FWPV genome populations carrying either the REV long terminal repeats (LTRs) or the full-length REV genome, reiterating the instability of the inserted REV. Two selected samples were fully sequenced by next generation sequencing (NGS), and the whole genome phylogenetic analysis revealed a regional clustering of the FWPV genomes. The extensive nature of these outbreaks in host populations naïve for the virus is a remarkable feature of the present report, highlighting new challenges associated with FWPV infections that need to be considered.

Atypical Manifestation of Cutaneous Fowlpox in Broiler Chickens Associated with High Condemnation at a Processing Plant

The present case is an unusual report of cutaneous fowlpox with an atypical appearance and incidence in broilers. Gross skin lesions were noticed in 41-day-old commercial broilers during the veterinary inspection at a processing plant in the north of Iran. The skin lesions were only observed on feathered skin areas of the broilers and remained unnoticed until slaughter. Round, nodular or coalescent, elongated, reddish-brown proliferative lesions were mainly located on the back, thighs, and proximal areas of the neck of broilers. Nonfeathered skin, including the wattle, comb, eyelids, and legs, were not affected. This condition incurred high losses due to a 5.3% condemnation and trimming of carcasses. Cutaneous lesions were sampled for histopathology and molecular virology for further investigations. Histopathology revealed multifocal necrotic dermatitis with epidermal eosinophilic cytoplasmic inclusion bodies in the skin lesions. Molecular investigations confirmed the presence of fowlpox virus (FWPV) in the proliferative lesions, with further investigations identifying two FWPV genome populations, one carrying a portion of the reticuloendotheliosis virus (REV) and the other a nearly complete REV provirus. Furthermore, the 4b core protein gene-based molecular analysis clustered the field virus into clade A of the genus Avipoxvirus.

First report on molecular characterization and phylogenetic analysis of Reticuloendotheliosis virus in Sudan.

The reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The infection can result in immunosuppression, runting syndrome, high mortality, acute reticular cell neoplasia, or T- and/or B-cell lymphoma. One PCR positive chicken spleen sample obtained in a previous study in addition to one Marek's disease and three fowl pox (FP) vaccine samples were investigated in this study. A PCR assay was performed to detect the presence of REV provirus DNA in these samples. The results indicated the contamination of fowl pox virus and Marek's disease vaccines with REV. In addition, detection of integration of REV inside the genome of fowl pox vaccine was confirmed using primers corresponding to the FPV DNA regions flanking the REV integration site. Alignments of two sequences, one from the spleen tissue and the other from contaminated FP vaccine with REV, with other REV (env) gene sequences obtained from GenBank indicated their high similarity. Furthermore, phylogenetic analysis indicated that the partial part of (env) gene of our two isolates was closely related to variants from India, USA, Taiwan, and China. These results confirmed the contamination of commercial fowl pox and Marek's disease vaccines used in Sudan with REV. Phylogenetic analysis indicated that the partial part of (env) gene sequences from Sudan was closely related to variants from India, USA, Taiwan, and China.

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.

Full Genome Sequences of Two Reticuloendotheliosis Viruses Contaminating Commercial Vaccines

Reticuloendotheliosis virus (REV) fragments are a common contaminant in some commercial vaccines such as fowl poxvirus (FPV) and Marek's disease virus. However, only those strains integrating or containing a near-intact REV provirus are more likely to cause problems in the field. We confirm here, by PCR assays and animal experiments, that vaccines against FPV and herpes virus of turkeys were contaminated with full genome sequences of REV. Further, we determined the complete proviral sequence of two REV isolates from contaminated vaccines. Two REV isolates (REV-99 and REV-06) present in the vaccines were both replication competent, and their proviral genome was 8286 nucleotides in length with two identical long terminal repeats (LTR). The complete genome in these two REV isolates shared 99.8% identity to APC-566 and fowl poxvirus REV proviral inserts (FPV-REV). REV-99 and REV-06 LTR showed over 99% identity to chicken syncytial virus (CSV), but an identity of only 75.8% and 78.0%, respectively, to SNV. Alignments with other available REV gag, pol, and env sequences revealed high similarity at the nucleotide level. The results further indicated that the prototype CSV may be the most-important REV contaminant in the commercial vaccines, and distinct genotypes of REVs may cocirculate in chicken flocks of China at the present time.

Serologic Response Against Fowl Poxvirus and Reticuloendotheliosis Virus After Experimental and Natural Infections of Chickens with Fowl Poxvirus

Two infection studies in chickens were done to investigate the humoral immune response against fowl poxvirus (FPV) and reticuloendotheliosis virus (REV) after intradermal infection with different passages of a field isolate and with the vaccine strain HP B. The field isolate in a low passage carried the near-full-length REV provirus and induced antibodies to REV, but not to FPV. The vaccine strain carried only remnants of the long terminal repeat and induced antibodies against FPV, but not against REV. The field isolate lost the provirus after 36 passages in vitro, and it induced few antibodies against FPV and no antibodies against REV. Intravenous challenge with the low passage field isolate caused some antibody development against FPV in the birds that had previously been infected with the field isolate, but it caused no antibodies against REV in the previously vaccinated birds. REV proviral DNA was found in peripheral blood mononuclear cells of most birds that had been infected with the low passage field isolate. However, FPV DNA was found only once. The findings showed that the integrated REV provirus had an effect on the pathogenesis of fowlpox and that the tested vaccine strain is effective against FPV strains carrying REV provirus. Investigation of sera from FPV diseased flocks and flocks vaccinated against FPV showed a similar proportion of sera with antibodies against FPV. Sera from all diseased flocks but only from two of 10 vaccinated flocks had antibodies against REV. This indicated that the integrated REV provirus is common in FPV field strains.

Quantitative PCR as a Tool to Determine the Reticuloendotheliosis Virus–Proviral Load of Fowl Poxvirus

Sequences of the reticuloendotheliosis virus (REV), an avian retrovirus, are integrated into the genome of fowl poxvirus (FPV). We developed and evaluated a quantitative multiplex real-time PCR (multiplex qPCR) assay to determine the REV-proviral load of FPV strains. Amplification efficiencies were 98.7% for the amplification of the FPV DNA and 88.7% for the amplification of REV-proviral DNA. The ratio between FPV DNA and REV-proviral DNA was calculated from the PCR efficiencies and the threshold cycle deviation of the unknown samples vs. a standard. The intraassay variation was determined by investigating triplets of different dilutions of the standard. The coefficient of variation between the threshold cycles was below 0.05 in all tested dilutions. The ratios of the triplet had a coefficient of variation of 0.201. Generally, the method overestimated the relative amount of REV-proviral DNA. Skin lesions from fowlpox outbreaks were investigated with the multiplex qPCR. The FPV:REV ratio was between 1:0.803 and 1:1.411 in samples with sufficient DNA to allow a conclusion. In addition, the investigation of cell culture material of several passages of a FPV field isolate showed a complete loss of the REV provirus after 36 passages. The loss rate of the REV provirus was approximately 50% per passage. In conclusion, we established the multiplex qPCR assay as a convenient and reliable method to determine the REV-proviral load of FPV. The first results we obtained with it show that it is of value for further investigations about the significance of the integration of the REV provirus into the genome of FPV.

Integrated reticuloendotheliosis virus genomic sequences in fowlpox virus.

Abstract Fowlpox is a common viral disease in domestic poultry, with a worldwide distribution. The negative impacts of the disease are reduction in egg production and retarded growth in younger birds. Vaccination with live vaccines has long been used for the control of the disease in the poultry industry and economic losses have been minimized. However, in recent years, this disease re-emerged as a new threat to poultry production, particularly in vaccinated flocks. Reticuloendotheliosis virus (REV) sequences were detected in the genomes of the fowlpox virus (FPV) field isolates causing the disease outbreaks and subsequently in archival virus isolates. It is interesting that this integration event occurred over 50 years ago. It has been found that most fowlpox field viruses contain REV provirus, while vaccine strains (except FPV S strain, an obsolete Australian vaccine strain) contain only remnants of REV long terminal repeats (LTRs). The integrated REV provirus appears to be infectious, as antibody responses to REV have been detected in infected birds. The presence of REV provirus in FPV field isolates has resulted in enhanced pathogenicity of the virus. In addition, clinical signs associated with REV, including runting disease syndrome and immunosuppression, have been demonstrated in young birds. The retention of the REV provirus may have resulted in modification of host gene (FPV genome) activities such as gene activation or inactivation, giving rise to alteration of the viral phenotype. This may have contributed to antigenic variation, leading to inadequate protection in vaccinated flocks.

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.

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.

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.

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.

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.