Isolation and pathogenicity testing of avian reticuloendotheliosis virus from layer chickens in China.

Reticuloendotheliosis virus (REV) can cause runting, immunosuppression, acute reticulum cell neoplasia, and chronic lymphoid tumors in a variety of domestic and wild birds. We diagnosed a case of reticuloendotheliosis with obvious tumors in liver and kidney. We isolated and sequenced the virus and performed pathogenicity testing of the REV strain. Immunohistochemistry and PCR confirmed that the diseased layer chickens were infected with REV. The strain, named BJ1503, was successfully isolated from the case by inoculation of tissue homogenates onto chicken embryo fibroblasts. The length of the proviral REV genome is 8,293 nucleotides. The isolate had 99.7% identity with REV-HA9901 (AY842951.1), which was isolated from Jiangsu, China, in 1999. The chickens infected with REV-BJ1503 had depressed weight gain and lymphoid atrophy. Our findings suggest that REV isolate BJ1503 was phylogenetically close to the earlier strain found in China, with minor variations, and the virus was associated with severe production problems.

Co-Infection with Marek's Disease Virus and Reticuloendotheliosis Virus Increases Illness Severity and Reduces Marek's Disease Vaccine Efficacy.

Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) cause Marek's disease (MD) and reticuloendotheliosis (RE), respectively. Co-infection with MDV and REV is common in chickens, causing serious losses to the poultry industry. However, experimental studies of such co-infection are lacking. In this study, Chinese field strains of MDV (ZW/15) and REV (JLR1501) were used as challenge viruses to evaluate the pathogenicity of co-infection and the influence of MD vaccination in chickens. Compared to the MDV-challenged group, the mortality and tumor rates increased significantly by 20.0% (76.7 to 96.7%) and 26.7% (53.3 to 80.0%), in the co-challenged group, respectively. The protective index of the MD vaccines CVI988 and 814 decreased by 33.3 (80.0 to 47.7) and 13.3 (90.0 to 76.7), respectively. These results indicated that MDV and REV co-infection significantly increased disease severity and reduced the vaccine efficacy. The MDV genome load showed no difference in the feather pulps and spleen, and pathogenicity-related MDV gene expression (meq, pp38, vIL-8, and ICP4) in the spleen significantly increased at some time points in the co-challenged group. Clearly, synergistic pathogenicity occurred between MDV and REV, and the protective efficacy of existing MD vaccines was attenuated by co-infection with Chinese field MDV and REV strains.

Synergistic pathogenic effects of co-infection of subgroup J avian leukosis virus and reticuloendotheliosis virus in broiler chickens.

To study interactions between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) and the effects of co-infection on pathogenicity of these viruses, 1-day-old broiler chicks were infected with ALV-J, REV or both ALV-J and REV. The results indicated that co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection (P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression than those with a single infection. This was manifested by significantly lower bursa of Fabricius and thymus to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus (P < 0.05). Perihepatitis and pericarditis related to severe infection with Escherichia coli were found in many of the dead birds. E. coli was isolated from each case of perihepatitis and pericarditis. The mortality associated with E. coli infection in the co-infection groups was significantly higher than in the other groups (P < 0.05). Among 516 tested E. coli isolates from 58 dead birds, 12 serotypes of the O-antigen were identified in two experiments. Different serotypes of E. coli strains were even isolated from the same organ of the same bird. Diversification of O-serotypes suggested that perihepatitis and pericarditis associated with E. coli infection was the most frequent secondary infection following the immunosuppression induced by ALV-J and REV co-infection. These results suggested that the co-infection of ALV-J and REV caused more serious synergistic pathogenic effects, growth retardation, immunosuppression, and secondary E. coli infection in broiler chickens.

Industrial hygiene assessment of reticuloendotheliosis viruses exposure in the poultry industry.

OBJECTIVES: Reticuloendotheliosis viruses (REV) are a group of retroviruses like avian leukosis/sarcoma viruses (ALSV) that naturally infect and cause cancers in chickens. We recently found that ALSV antibody levels were associated with job tasks in the poultry industry. The objectives of this study are to examine whether a similar association can be found with REV antibody levels and to examine the correlation between REV and ALSV antibody levels. METHODS: Relative risk was estimated comparing REV antibody levels of 45 poultry workers with those of 44 controls. The expected mean antibody level was predicted for the association with employment by a generalized linear model. Correlation coefficient was measured between ALSV and REV antibody levels. RESULTS: REV antibody levels were significantly higher in poultry workers than in control subjects and were associated with gender and employment conditions, especially employment duration. The relative risk was significantly higher for some job categories. A significant correlation was observed between REV and ALSV antibody levels, which was strong among poultry workers, but weak among the control subjects. CONCLUSION: Antibody levels can be validly used to identify certain job tasks associated with high risk of exposure to REV in the workplace, and the practical implication is recommendations for protection at these job tasks. Importantly, in situations where there is exposure to multiple pathogens in the workplace, the analysis of antibody levels of one pathogen may sufficiently represent exposure to the other correlated pathogens. This suggested exposure assessment may hold true for pathogens with a similar route of transmission.

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.

Pathogenicity and transmission of reticuloendotheliosis virus isolated from endangered prairie chickens.

The pathogenicity and transmission of a field isolate of reticuloendotheliosis virus (REV) was studied using an experimental model in Japanese quail. Oncogenicity was also evaluated after inoculations in chickens and turkeys. The original REV (designated APC-566) was isolated from Attwater's prairie chickens (Tympanuchus cupido attwateri), an endangered wild avian species of the southern United States. The transmissibility of the REV isolate was studied in young naive Japanese quail in contact with experimentally infected quail. Vertical transmission was not detected by virus isolation and indirect immunofluorescence. Seroconversion was detected in few contact quails, suggesting horizontal transmission. The APC-566 isolate induced tumors beginning at 6 wk of age in quails infected as embryos. Most of the tumors detected in Japanese quail were lymphosarcomas, and 81% of these neoplasias contained CD3+ cells by immunoperoxidase. REV APC-566 was also oncogenic in chickens and turkeys infected at 1 day of age, with tumors appearing as early as 58 days after infection in chickens and at 13 wk of age in turkeys. This study was conducted in part as an attempt to understand the potential for pathogenicity and transmission of REV isolated from endangered avian species.

Experimental infection of Attwater's/greater prairie chicken hybrids with the reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), a common pathogen of poultry, has been associated with runting and neoplasia in an endangered subspecies of grouse, the Attwater's prairie chicken. The pathogenesis of REV infection was examined in experimentally infected prairie chickens. Three groups of four Attwater's/greater prairie chicken hybrids were infected intravenously with varying doses (tissue culture infective dose [TCID50], 200, 1000, and 5000) of a prairie chicken-isolated REV. A fourth group of four birds was not infected. Blood was collected prior to infection, and at various times up to 37 wk following infection. Peripheral blood mononuclear cells were examined for integrated proviral DNA by a single-amplification polymerase chain reaction (PCR) and nested PCR of a region within the pol gene. The nested PCR identified REV proviral DNA in all REV-inoculated birds by 2 wk postinfection and confirmed chronic infection throughout the study. With the exception of a bird that died from bacterial pneumonia 8 wk postinfection, neoplasia, resembling that seen in naturally occurring infections, was observed in all birds, even those receiving as little as 200 TCID50 of virus.

Avian reticuloendotheliosis virus strain A and spleen necrosis virus do not infect human cells.

Spleen necrosis virus (SNV) and Reticuloendotheliosis virus strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769-4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301-310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.

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.

Structural genes, not the LTRs, are the primary determinants of reticuloendotheliosis virus A-induced runting and bursal atrophy.

Reticuloendotheliosis virus strain A (REV-A) and chicken syncytial virus (CSV), two replication competent avian retroviruses, differ in the extent to which they induce a runting syndrome that includes anemia, lymphoid organ atrophy, and reduced body size. We have isolated an infectious clone of CSV, the less pathogenic of the two viruses, and compared it to REV-A. Partial DNA sequence analysis suggests that it differs from REV-A by no more than 1 to 2% at the nucleotide level. Analysis of viral interference indicates that these two viruses use the same cell receptor for infection of both fibroblasts and hematopoietic cells, DNA sequence of the CSV and REV-A long terminal repeats (LTRs) reveals that these structures differ principally by two small insertions (5 and 19 bp) present in the U3 region of REV-A. The larger of these may encode enhancer sequences that have been reported to influence transcription rates in vitro. Measurement of steady-state levels of viral RNA in infected cells, however, as well as circulating virus in infected chicks indicates that the different pathogenic responses elicited by these two viruses are not due to large differences in viral transcription or replication. Chimeric viruses were constructed in which the LTRs from one virus were used to express the structural genes of the second virus. Infection of 1-day-old chicks by parental virus as well as the reciprocal chimeric constructs demonstrated that the ability to induce both runting and bursal atrophy segregated with the structural genes of REV-A. Infection of birds with additional chimeric viruses in which the env genes of REV-A and CSV were exchanged indicated that the pathogenic response resulting from REV-A infection was due to at least two regions of the viral genome encoding structural genes.