An outbreak in three-yellow chickens with clinical tumors of high mortality caused by the coinfection of reticuloendotheliosis virus and Marek's disease virus: a speculated reticuloendotheliosis virus contamination plays an important role in the case.

Both reticuloendotheliosis and Marek's disease are neoplastic diseases of chickens caused by reticuloendotheliosis virus (REV) and Marek's disease virus (MDV), respectively. The infection of REV or MDV may lead to clinical tumors and also result in immunosuppression and easily allow secondary infection by other pathogens. Here, we investigated a breeder flock of three-yellow chickens in southern China that had been vaccinated with CVI988/Rispens at hatching and had experienced depression, weakness, reduction in weight gain, and an increased death rate after 120 d of age. The morbidity and mortality were 20% and 10%, respectively, at 140 d of age when this infection was diagnosed. The necropsy of the birds revealed significant tumor-like lesions in the heart, liver, spleen, and ceca. Peripheral blood lymphocytes and tumor-like tissues were sampled for PCR detection and for histopathological observation, for virus isolation and the subsequent immunofluorescent assay on the cell cultures and for gene sequencing of the isolated viruses. A REV isolate GX18NNR1 and a MDV isolate GX18NNM5 were both recovered from the sampled bird. Further phylogenetic analysis based on the env gene of REV and the meq gene of MDV demonstrated that GX18NNR1 was closely related to the reference REV strain MD-2, which was isolated from a contaminated commercial turkey herpesvirus vaccine. In addition, the GX18NNM5 was found to belong to the Chinese very virulent MDV strains' cluster. The coinfection of REV and MDV may contribute to tumor outbreaks with high morbidity and mortality in three-yellow chicken flocks.

Genetic characterization of reticuloendotheliosis virus in chickens in Thailand.

Reticuloendotheliosis virus (REV) causes an immunosuppressive, runting, and oncogenic disease in poultry, posing a significant threat to the poultry industry. In Thailand, an unidentified disease associated with runting-stunting syndrome and neoplasia, resembling REV infection, has been continuously observed in several chicken farms. However, REV infection in Thailand has never been reported. In this study, we investigated the occurrence and genetic characteristics of REVs in chickens in Thailand from 2013 to 2016. Of the 130 clinical samples obtained from 29 chicken farms from 9 provinces located in the major chicken-raising regions of Thailand, including the central, eastern, northern, and northeastern parts of Thailand, 51 samples (39.23%) and 21 farms (72.41%) were REV-positive. REV-positive samples were detected in all 9 provinces tested. Our results demonstrated that REV was extensively distributed in the major chicken-raising regions of Thailand. Phylogenetic analysis of the whole genome sequence showed that Thai REV was most closely related to Chinese, Taiwanese, and the US REV strains isolated from different avian species and clustered into REV subtype III. This finding indicates that REV subtype III was predominantly circulated in Thai chicken flocks. This study is the first report on REV infection in chickens in Thailand. Our findings raise the awareness of REV as another causative agent of runting and oncogenic disease in chickens in Thailand and highlight the wide distribution of REV infection among chickens worldwide.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

Emergence of reticuloendotheliosis virus in pigeons in Guangdong Province, Southern China.

Reticuloendotheliosis virus (REV), an important immunosuppressive pathogen, has many hosts, including chickens, ducks, geese, turkeys, and wild birds. Clinically, REV may lead to increased susceptibility to other pathogens, resulting in serious tissue damage (especially tumors) and the death of its host. In this study, we encountered a disease outbreak resulting in a large number of deaths of pigeons in Guangdong Province, Southern China. Histopathological analysis revealed apparent tumor-like lesions in multiple organs of pigeons. PCR assays for detection of tumor-associated pathogens (REV, avian leukosis virus, and Marek's disease virus) in poultry revealed the presence of REV sequences only. Moreover, fowlpox virus (FPV) with an insertion of REV long terminal repeat (LTR) sequences was also considered, but it was excluded using a specific PCR assay. To gain more genetic information, two full-length REV genome sequences were determined and found to have the highest nucleotide sequence similarity (99.9 %) and the closest genetic relationship to a vaccine strain (MD-2) and had a more distant genetic relationship (94.3 %) to a duck-origin strain (ATCC-VR775). To confirm the presence of REVs in pigeons, specific-pathogen-free (SPF) chickens and healthy pigeons were inoculated with microfiltered tumor tissue homogenates and were found to be susceptible to infection with REV. To our knowledge, this is the first report of REV in pigeons, and the data suggest that pigeons may be the natural host of REV.

First isolation of reticuloendotheliosis virus from mallards in China.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission.

The extraordinary evolutionary history of the reticuloendotheliosis viruses.

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs-unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events.

Molecular characterization and phylogenetic analysis of the reticuloendotheliosis virus isolated from wild birds in Northeast China.

To analyze the status of reticuloendotheliosis (RE) infection of wild birds in China, 585 samples from wild birds collected in Liaoning, Jilin and Heilongjiang provinces China were investigated and analyzed. The sampled birds represent 3 orders and more than 40 species. Virus isolation and PCR amplification showed that some of the wild birds were infected with REV, and 10 REV strains were isolated. The gp90 gene from each of the 10 REV strains was amplified, cloned, and sequenced. Sequence analysis indicated that the gp90 genes of the 10 REV strains isolated in this study were more similar at the nucleotide level with the northeast Chinese strains HLJR0901 and HLJR0801 and some REV strains found in the US and Taiwan than with the early Chinese REV isolate HA9901. Furthermore, phylogenetic analysis indicated that the gp90 genes of the 10 REV strains were more similar to the REV subtype III-representing strain (CSV) than to strains 170A (subtype I) or SNV (subtype II). This is the first study to investigate the status of wild birds infected with REV. The results of this paper will not only provide necessary information for further understanding the evolution of REV, but they also identify the potential role of wild birds in REV transmission and furthers our understanding of the ecology of REV in wild bird species.

The immunodominant proteins of reticuloendotheliosis virus.

The antigenic profiles of three REV prototype strains, CSV, SNV and REV-T and eight Israeli isolates were analysed by SDS-PAGE and immunoblotting with convalescent chicken serum, three mAbs, 11A25, 11C237 and 11C100, a rabbit antiserum to REV-T whole virus (Cui et al., 1986) and a rabbit antiserum to REV-A p30 gag protein (Tsai et al., 1985). Under both reducing (+DTT) and non-reducing conditions of SDS-PAGE, a major immunodominant 75-100 kDa band was shared by all strains examined. In contrast to the chicken serum that recognized both continuous and discontinuous epitopes on the 75-100 kDa band of all the isolates, the mAbs and the two rabbit sera behaved otherwise. Only the DTT-resistant epitopes on the 75-100 kDa band of REV-T were recognized by the rabbit antisera and the mAb 11C237, and only the DTT-labile epitopes of REV-T 75-100 kDa antigen were detected by mAb 11C100. The two mAbs 11A25 and 11C237 detected discontinuous epitopes of all the strains except SNV, while the rabbit antisera recognized the discontinuous epitopes on the 75-100 kDa band of all the 11 strains. The rabbit antisera and mAb 11C237 detected additional lower molecular weight proteins and the mAb 11C237 also detected three proteins of high molecular weight under non-reducing conditions only. The p30 antiserum detected the low molecular weight proteins demonstrating their gag gene-encoded identity. From these results we conclude that the major immunogen of REV is the 75-100 kDa protein that contains both continuous and discontinuous epitopes. With this panel of antibodies the eight new isolates appeared to belong antigenically to REV subtype 3 (Chen et al., 1987).

Serologic differences among nondefective reticuloendotheliosis viruses.

Antigenic relationships among 26 isolates of reticuloendotheliosis virus (REV) obtained from several avian species were compared by cross neutralization tests with polyclonal chicken sera and by immunofluorescent assays with monoclonal antibodies to REV strain T. The isolates were all strongly related by neutralization assays and thus probably constitute a single serotype. However, 3 antigenic subtypes were suggested by minor but distinct differences in neutralization titers. The validity of these 3 subtype designations was confirmed by differential reactivity of viral isolates to selected monoclonal antibodies. Subtype-associated differences in serum antibody titers were noted following the inoculation of chickens with the REV isolates.