New pathogenetic characters of reticuloendotheliosis virus isolated from Chinese partridge in specific-pathogen-free chickens.

Avian reticuloendotheliosis virus (REV) infection can induce a runting syndrome, immunosuppression, acute reticulum cell neoplasia and lymphomas in a variety of domestic and wild birds. To evaluate the pathogenicity and oncogenicity of REV-JX0927 that isolated from Chinese partridge, experimental inoculated day-old specific-pathogen-free (SPF) White Leghorn chickens were examined at regular intervals. The examination procedures included hematology, serology and histopathology; also including immunohistochemistry and apoptosis assay. Body weight, relative immune organs weight and apoptosis assay results revealed that the immunosuppression of infected birds is associated with apoptosis of lymphocytes in lymphoid tissues, especially in thymus induced by REV-JX0927. Hematology and apoptosis assay results showed that the 7th week of post-infection is a critical time point for lymphocytes to be transformed into tumor cells. Histopathology evidences demonstrated that REV-JX0927 induced reticuloendotheliosis at early stage (1 week), and lymphosarcomas at middle stage (after 7 weeks). In addition, squamous-cell carcinoma, adenocarcinoma and aneurysm were found in infected birds. Arteritis was associated with concentration of serum protein and fat. REV antigen expression was observed in infected birds through the experimental period. REV has high tropism for proventriculus, kidney, liver, lymphoid tissues, pancreas, lymphosarcoma cells and blood vessels. Data from this study showed that several new pathogenitic characters caused by REV-JX0927 were observed. It indicated that REV-JX0927 is a multipotential oncogenic retrovirus.

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.

Semen-Derived Exosomes Mediate Immune Escape and Transmission of Reticuloendotheliosis Virus.

Reticuloendotheliosis virus (REV) causes immune-suppression disease in poultry, leading to a significant economic burden worldwide. Recent evidence demonstrated that the REV can enter the semen and then induce artificial insemination, but how the virus gets into semen was little known. Accumulating studies indicated that exosomes serve as vehicles for virus transmission, but the role of exosomes in viral shedding through the semen remains unclear. In this study, exosomes purified from the REV-positive semen were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and viral proteins, which could also establish productive infections both in vivo and in vitro and escape from the REV-specific neutralizing antibodies. More importantly, compared with the infection caused by free virions, the exosome is more efficient for the virus to ensure effective infection and replication, which can also help the REV compromise the efficacy of the host immune response. In summary, this study demonstrated that semen-derived exosomes can medicate the transmission and immune escape of REV, implicating a novel mechanism for REV entering the semen and leading to vertical transmission.

Detection of reticuloendotheliosis virus as a contaminant of fowl pox vaccines.

This study was designed to detect reticuloendotheliosis virus (REV) as a contaminant in fowl pox vaccines. A total of 30 fowl pox vaccine samples were examined for the presence of REV using both in vitro and in vivo methods. In in vitro testing, the fowl pox vaccine samples were inoculated into chicken embryo fibroblast cultures prepared from specific-pathogen-free embryonated chicken eggs, and the cultures were examined using PCR to detect REV. In in vivo testing, each fowl pox vaccine sample was inoculated into 5-d-old specific-pathogen-free chicks, which were kept under observation for up to 12 wk postinoculation; serum samples were collected at 15, 30, and 45 d postinoculation for the detection of REV-specific antibodies using ELISA. Tissue samples were collected at 8 and 12 wk postinoculation for histopathological examination. Of the tested vaccines, only one imported vaccine sample tested positive for REV using PCR. Serum samples collected from chicks infected with the PCR-positive vaccine batch also tested positive for REV-specific antibodies using ELISA. Histopathological examination of the liver, spleen, and bursa of Fabricius demonstrated the presence of tumor cells in these organs, confirming the results obtained using PCR and ELISA, and indicating that the sample was contaminated with REV. These data clearly indicate that the screening of all commercial poultry vaccines for viruses is an important factor in assuring the biosafety of animal vaccines.

Protective efficacy of vaccination against highly pathogenic avian influenza is dramatically suppressed by early infection of chickens with reticuloendotheliosis virus.

The present study was conducted to understand epidemiological factors that depress the protective efficacy of vaccination against highly pathogenic avian influenza (HPAI). Specific pathogen free chickens were infected at different ages with immunosuppressive reticuloendotheliosis virus (REV), then vaccinated with inactivated vaccine H5N2 and challenged with HPAI H5N1. Compared with control birds, early REV infection significantly inhibited antibody response to H5N2 vaccine and dramatically decreased protective efficacy. Immunosuppressive effects lasted for 4 months, and high mortality occurred in chickens receiving three vaccinations during 3 months prior to challenge with HPAI H5N1. Epidemiological studies indicated that REV infection and vertical transmission is common in chickens in some areas of southern China. We conclude that vaccination programmes against HPAI will not be fully effective if other immunosuppressive viral infections, such as REV, exist in chicken flocks.

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.

Serotherapy of avian reticuloendotheliosis virus-induced tumors.

Immune serum, obtained from animals that had survived sublethal challenge with RE virus, was very effective in achieving tumor cures in chickens infected with this virus. The therapeutic effect could also be obtained by the immunoglobulin fraction of immune serum. The serum did not influence the development of an unrelated malignancy. Serotherapy studies in Bx and Tx recipients indicate that the B-cell or T-cell system of the host does not significantly contribute to the curative activity of immune serum. Absorption studies show that the curative effect is mediated by antibodies to tumor-associated transplantation antigens on tumor cells, not by antiviral antibodies.

Spontaneously occurring lymphoproliferative disease in ducks.

A naturally occurring lymphoproliferative disease was observed in a domestic flock of Muscovy ducks. Gross lesions were observed only in one of three ducks necropsied (in the small intestine). Microscopic lesions consisted of mononuclear cell infiltrates composed primarily of reticuloendothelial cells, mature lymphocytes, and plasma cells in livers, spleens, small intestines, peripheral nerves, and brains. Antibodies to reticuloendotheliosis virus were detected in sera of four ducks, whereas all six ducks examined lacked antibodies ot Marek's disease virus.

Complement-fixation test for reticuloendotheliosis viruses: limits of sensitivity in infected avian cells.

A specific micro-complement-fixation procedure for assay of avian reticuloendotheliosis viruses (REV), designated by use as the COFAR test, was compared with an assay based on immunofluorescent (IF) antibody staining of infected chick embryo fibroblasts. Endpoint titrations in which REV strain T, chick syncytial virus, and spleen necrosis virus were used indicated that cultures infected with limiting dilutions of each strain were positive by both procedures within 6 days. Depending on cell density, infection of 2 to 9% of the cells cultured produced an unambiguous positive response (titer greater than or equal to 1:2) with COFAR test. When both tests were used in a study on the transmission and in vivo status of ducks infected with spleen necrosis virus, COFAR was no less, possibly more, sensitive than IF for detecting infection in cultures inoculated with plasma.

Pathogenesis of abnormal feathers in chickens inoculated with reticuloendotheliosis virus.

Abnormal feathers, characterized by thinness and increased transparency of the calamus and rachis, and loss of barbs, were induced at a high frequency by inoculating day-old chicks with reticuloendotheliosis virus (REV) propagated in chicken-embryo fibroblast (CEF) cultures. The few birds that survived inoculation with oncogenic stock of REV derived from liver tissue of an infected chick developed similar abnormalities. Lesions of an inflammatory-degenerative type were observed in close association with the presence of viral antigen and numerous c-type virus particles, characteristic of REV, in the intermediate and cylindrical cell layers of all abnormal feathers examined. These findings, first detected in the intermediate and cylindrical cell layers of developing feathers 6--9 days after infection, suggest that degeneration and necrosis of feather-forming cells result from productive infection of REV, resulting in the development of the abnormal feathers.

Effect of reticuloendotheliosis virus on the response of chickens to Salmonella typhimurium infection.

Groups of 25 chickens free of maternal antibody to reticuloendotheliosis virus (REV) were inoculated with either third or seventh passage REV at either one or seven days of age. Some of the birds inoculated at day 1 with REV were inoculated with Salmonella typhimurium either concurrently or six or 13 days later while some of those inoculated with REV at day 7 were inoculated concurrently with S typhimurium. At day old, infection with S typhimurium alone caused the death of 12 of 25 chicks whereas in the dual infection, using the third passage REV, 18 of 25 birds died. Similarly no seven or 14 day old chickens died when challenged with S typhimurium alone, but previous day-old infection with REV caused a respective mortality of eight of 25 and five of 25 birds. With the seventh passage REV a similar pattern was seen. At day old S typhimurium infection alone killed seven of 25 birds whereas combined with virus the mortality was 14 of 25 and while S typhimurium alone killed none of 25 chicks infected at seven days old, the mortality in birds also infected with REV was 14 of 25. Combined virus and bacterial infections did not increase the proportion of feathering defects in birds surviving S typhimurium infections. There was a significantly higher proportion of feathering defects in birds infected with third passage virus compared with seventh passage virus. Although a higher proportion of birds had antibody responses to REV in the seventh than in the third passage group, there was no discernible difference in the effect the different viruses had on chickens' susceptibility to S typhimurium.

Depression of vaccinal immunity to Marek's disease by infection with reticuloendotheliosis virus.

The effect of infection with low-virulence, tissue culture-propagated strains of reticuloendotheliosis virus (REV) on protective vaccinal immunity against Marek's disease (MD) lymphomas was investigated. Vaccinated chickens inoculated at hatching with greater than 10(4) focus-forming units of REV and challenged with MD virus were poorly protected against MD lesion development as indicated by protective indices of 53 to 79% for strain CS (P less than 0.05) and 42 to 49% for strain T (P less than 0.01) compared to 78 to 100% for REV-free controls. Furthermore, the response of blood lymphocytes to mitogen stimulation and the antibody response to sheep erythrocytes and Brucella abortus were less in REV-inoculated chickens than in controls. The REV-induced depression of immune responses was more severe in chickens infected with mildly pathogenic strain T than in chickens infected with the apathogenic strain CS and was generally transient with both virus strains. Little or no depression of immune responses was observed in chickens inoculated with less than 10(3) focus-forming units of REV. These studies extend knowledge on the immunodepressive ability of low-virulence REV strains and establish that infection with these viruses depresses certain parameters of MD vaccinal immunity, an important model for cellular immunity against virus-induced neoplasia in the chicken.

Infection with chicken anaemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes.

Infection with chicken anaemia virus (CAV), a circovirus, can result in immunosuppression and subsequent increased susceptibility to secondary infections. This is the first report of impairment of pathogen-specific cytotoxic T lymphocytes (CTL) after natural and experimental infection of chickens with CAV and Marek's disease virus (MDV) or reticuloendotheliosis virus (REV). MDV- and REV-specific CTL were generated at 7 days post infection by 9-30-day-old-chickens that were positive for maternal antibodies to CAV at 9-17 days of age. Replication of CAV could not be demonstrated in these chickens using quantitative real-time polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR assays. In contrast, REV-specific CTL failed to develop when chickens negative for maternal antibodies at 9-17 days of age were infected. Infection with CAV at 45 days of age after CAV maternal antibodies had waned also caused a decreased REV-specific CTL response. In these chickens increased levels of CAV DNA of up to 107 copy numbers per micro g DNA and increased relative transcript levels of CAV by up to a factor of 106 were detected by quantitative real-time PCR and RT-PCR. Interleukin (IL)-1beta and IL-2 mRNA levels were not significantly affected by CAV infection at 7 or 14 days p.i. Similar assays for interferon-gamma (IFN-gamma) transcripts demonstrated a 10-fold increase in IFN-gamma mRNA levels at 7 days post infection following REV or REV + CAV infection, while CAV alone caused a two- to fourfold increase. These results show a strong link between CAV antibody status, CAV replication, and the ability to generate REV-specific CTL. It is likely that the immunosuppressive effects of subclinical infection have previously been underestimated.

Isolation of a reticuloendotheliosis virus from chickens inoculated with Marek's disease vaccine.

Over a period from spring to fall in 1974, a disease with delayed growth, anemia, abnormal feathers, and leg paralysis as main symptoms broke out in flocks of chickens inoculated with Marek's disease vaccine. A virus was isolated from affected birds in the field and the same lot of Marek's disease vaccine as inoculated into these birds. It had a common antigenicity to the T strain of reticuloendotheliosis virus (REV) and could not be discriminated from this strain on the basis of morphology or property. When chicks were inoculated with it, they presented essentially the same symptoms as the birds affected in the field. Since the disease was reproduced in this manner, it was presumed to have been caused by REV contained in the vaccine as contaminant. The virus persisted in the body for long time and also induced horizontal infection.

Separation of reticuloendotheliosis virus from avian tumor viruses.

Velocity sedimentation and isopycnic density gradient centrifugation indicate that reticuloendotheliosis virus has a different mass and buoyant density than members of the avian tumor virus group. The group-specific antigen of the avian tumor virus group was not detected in concentrated and purified reticuloendotheliosis virus preparations.