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.

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.

Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil.

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks (Cairina moschata), Wild Turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Serological and molecular identification of Reticuloendotheliosis virus (REV) in chickens in Sudan.

BACKGROUND: Reticuloendotheliosis virus (REV) is a gammaretrovirus that belongs to the family of Retroviridae. The infection can result in immunosuppression, runting syndrome, high mortality, acute reticular cell neoplasia or T- and/ or B-cell lymphoma, in a variety of domestic and wild birds. The disease is widespread around the world. No related data have been reported in Sudan about the disease. The present study was conducted to determine the prevalence of REV antibodies and DNA in local and commercial breeds of chickens older than 20 weeks from June 2014 to February, 2017. METHODS: A total of 460 sera samples and 150 (50 liver and 100 spleen) tissue samples were collected from local and commercial breeds of chickens older than 20 weeks and screened for anti-REV antibodies in four states of Sudan using a commercial REV antibody ELISA test kit (IDEXX). Polymerase chain reaction (PCR) was performed to detect REV DNA in tissue samples in Khartoum State. RESULTS: The results revealed that the overall seroprevalence of REV was 74.6% among local and commercial chicken breeds, but in commercial it was 79.5% (190/239) and 69.2% in local breeds (153/221). One hundred and fifty tissue samples of chickens (50 liver, 100 spleen) were tested using PCR for detection of REV using primer sets of the conserved region in envelope glycoprotein (env) gene with a band length of 850 bp. Five out of 50 (10%) liver samples were RE provirus DNA positive detected by PCR, whereas 15 out of 100 (15%) spleen samples were PCR positive. Univariate analysis revealed there was a difference (p ≤ 0.05) between locality and breed of chickens and seropositivity to REV. CONCLUSIONS: The prevalence of the disease was high in Sudan and more studies are needed to evaluate the epidemiology and pathogenesis of the virus.

A duplex real-time polymerase chain reaction assay for the simultaneous detection of long terminal repeat regions and envelope protein gene sequences of Reticuloendotheliosis virus in avian blood samples.

The Reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The objective of the present study was to develop a highly sensitive and specific diagnostic test for the detection of REV in whole blood samples. In order to increase the diagnostic sensitivity, a duplex real-time polymerase chain reaction (PCR) that detects both the envelope protein gene (env) and the long terminal repeat (LTR) region of REV was designed. This assay demonstrated greater analytical and diagnostic sensitivity than the gel-based PCR assay when using DNA extracted from whole blood by both phenol-chloroform and magnetic bead methods. In general, threshold cycle values in the duplex real-time PCR assay were lower from DNA extracted using the magnetic bead system compared to DNA extracted by the phenol-chloroform method. Data presented herein show the successful development of a rapid and accurate test procedure, with high-throughput capability, for the diagnosis of REV infection using avian blood samples.

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.

Simultaneous endemic infections with subgroup J avian leukosis virus and reticuloendotheliosis virus in commercial and local breeds of chickens.

Epidemiological studies of subgroup J avian leukosis virus (ALV-J) and reticuloendotheliosis virus (REV) infections were conducted during 1999 to 2009 on 29 chicken flocks of various commercial and local breeds located in six provinces in China. Samples were typically from chickens with myelocytomas or proventricular lesions. ALV-J was isolated from 25 flocks including seven out of seven flocks containing "yellow chickens" or other local breeds and several flocks of layer chickens. REV was isolated from 19 flocks. Co-infection of ALV-J and REV was confirmed in 11/38 birds of 8/15 flocks with myelocytomatosis and in 11/24 birds of 3/5 flocks with proventricular lesions. Both ALV-J and REV were vertically transmitted in four breeder flocks of local chicken breeds. One experiment in specific pathogen free chickens indicated that co-infection at hatch strongly depressed antibody responses to ALV-J but not to REV. The results suggest that infections with both ALV-J and REV have become endemic in local breeds, including breeder flocks where both viruses appear to be perpetuated by vertical transmission. In addition, both viruses were present in at least some commercial broiler, layer and breeder flocks. Eradication programmes are needed but will be difficult, especially in local breeds. This unprecedented concurrence of simultaneous endemic infections with ALV-J and REV may have serious economic consequences and needs to be better understood.

Detection of reticuloendotheliosis virus by immunohistochemistry and in situ hybridization in experimentally infected Japanese quail embryos and archived formalin-fixed and paraffin-embedded tumours.

Reticuloendotheliosis virus (REV) infection can result in immunosuppression, a runting syndrome, high mortality, acute reticulum cell neoplasia, or T-cell and/or B-cell lymphomas, in a variety of domestic and wild birds. Histopathological changes in REV infection are not sufficient to differentiate it from avian lymphoid leukosis and Marek's disease, and currently there are no available in situ diagnostic methods for detection of active REV presence in pathologic specimens. To develop immunohistochemistry and in situ hybridization assays for detection of REV active infections, experimentally inoculated Japanese quail embryos, and archived formalin-fixed paraffin-embedded tissues from natural and experimental reticuloendotheliosis cases in chickens and turkeys, were examined. The in situ hybridization and immunohistochemistry assays proved to be efficient for the detection of several REV strains in Japanese quail embryos during active infection, whereas these assays were much less sensitive when applied to archived tissue samples from chronically infected birds with lymphoid tumours. The diagnostic assays developed in this study have potential as diagnostic tools for detection of active REV infections.

Detection and differentiation of re-emerging fowlpox virus (FWPV) strains carrying integrated reticuloendotheliosis virus (FWPV-REV) by real-time PCR.

Current strains of fowlpox virus (FWPV) carrying circulating reticuloendotheliosis virus (FWPV-REV) sequence are becoming more pathogenic to poultry. This is evidenced by the fact that vaccination with current available FWPV vaccines provides limited protection against them. To characterize REV insertions in a collection of both older and more recent field isolates, we developed three different types of adjacent oligoprobes and primer sets from specific genomic locations of FWPV and REV: REV-ENV (accession no. K02537, 1382-2260), FWPV-REV integration site (accession no. AF006064, 86-1328), FWPV (accession no. AF198100, 232461-232670), and REV-LTR (accession no. V01204, 305-496). The data indicated that the primers from the REV-ENV region and the TaqMan probes specifically targeted REV-ENV sequences of FWPV-REV strains. Furthermore, the strains were differentiated based on quantitative melting temperature (T(m)) of their amplified products using FRET-based probes. The amplified products were further characterized by sequencing and multiple sequence alignment analysis. The results suggest that integrated REV-ENV sequences are both common and mostly conserved in field isolates. However, the minor variations found within the short-targeted ENV sequence from FWPV-REV strains suggest that these strains could have either undergone periodic point mutational changes or integration with different REV-ENV subtypes.

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.

Occurrence of reticuloendotheliosis in Chinese partridge.

Two commercial flocks of Chinese partridge experienced increased mortality associated with a wasting disease at 120-day old in June 2006. Postmortem examination of dead chickens consistently showed visceral tissues mainly proventriculus, liver and spleen were diffuse enlargement. Microscopic examination revealed masses of immature lymphocytes with frequent mitotic figures were seen in various tissues including proventriculus, liver, spleen, kidney, heart, lung, thymus and intestine. Reticuloendotheliosis virus (REV) was isolated from each of four blood samples. Viral antigens were observed in cultured CEF (SPF embryos came from the Ji-nan poultry institute) inoculated blood samples via on indirect immunofluorescent assay. Three hundred bp fragments of LTR of REV obtained from liver samples of six chickens by PCR. This disease has not previously been reported in Chinese partridge. Chinese partridge may represent a potential reservoir of infection for other Chinese local chickens.

Detection of reticuloendotheliosis virus in live virus vaccines of poultry.

In vitro and in vivo assays have been used for the detection of reticuloendotheliosis virus (REV) in live virus vaccines of poultry. The presence of REV is confirmed by the demonstration of viral antigen or provirus in chicken embryo fibroblasts (CEFs) or in specific-pathogen-free chickens inoculated with vaccine. Using REV polyclonal or monoclonal antibodies, CEFs inoculated with vaccines can be examined for REV by immunofluorescence or immunoperoxidase staining methods. Cell lysates from such inoculated CEFs can also be used for detection of REV major group-specific antigen (p30) by an enzyme-linked immunoassay. Detection of proviral DNA by polymerase chain reaction (PCR) assays that amplifies the 291 base pairs product of REV LTR has been shown to be a sensitive and specific method for detection of various strains of REV in infected CEFs and in the blood of SPF chickens inoculated with contaminated fowlpox virus (FPV) vaccines. Recently, using PCR tests that amplify REV envelope and REV 3' LTR sequences provided a more accurate assessment of the insertion of REV provirus in FPV than PCR assays that amplify the REV 5' LTR. This paper reviews the most common methods used for testing live virus vaccines of poultry for contamination with REV.

Phylogenetic analyses indicate little variation among reticuloendotheliosis viruses infecting avian species, including the endangered Attwater's prairie chicken.

Reticuloendotheliosis virus infection, which typically causes systemic lymphomas and high mortality in the endangered Attwater's prairie chicken, has been described as a major obstacle in repopulation efforts of captive breeding facilities in Texas. Although antigenic relationships among reticuloendotheliosis virus (REV) strains have been previously determined, phylogenetic relationships have not been reported. The pol and env of REV proviral DNA from prairie chickens (PC-R92 and PC-2404), from poxvirus lesions in domestic chickens, the prototype poultry derived REV-A and chick syncytial virus (CSV), and duck derived spleen necrosis virus (SNV) were PCR amplified and sequenced. The 5032bp, that included the pol and most of env genes, of the PC-R92 and REV-A were 98% identical, and nucleotide sequence identities of smaller regions within the pol and env from REV strains examined ranged from 95 to 99% and 93 to 99%, respectively. The putative amino acid sequences were 97-99% identical in the polymerase and 90-98% in the envelope. Phylogenetic analyses of the nucleotide and amino acid sequences indicated the closest relationship among the recent fowl pox-associated chicken isolates, the prairie chicken isolates and the prototype CSV while only the SNV appeared to be distinctly divergent. While the origin of the naturally occurring viruses is not known, the avian poxvirus may be a critical component of transmission of these ubiquitous oncogenic viruses.

Serological and virological surveys of reticuloendotheliosis in chickens in Taiwan.

Reticuloendotheliosis virus (REV) is widespread in the world. No related data has been reported in Taiwan. To determine the REV infection status, antibody determination and virus isolation were performed on chickens in Taiwan. The results revealed that serological flock prevalence for the REV antibody reached 92.8% (39/42) amongst breeders (> 16 weeks old). Two different REV isolates were identified by reverse transcriptase-polymerase chain reaction, electron microscopic, immunofluorescent, and western blot assays after isolation. One of these viruses was isolated from a broiler breeder farm and the other was isolated from a Taiwan Country Chicken farm. Despite their different origins, the percent identity of the nucleotide sequences of the env gene of these two isolates was 99.7%. These two strains were similar to the FPV-UI-REV strain, featuring 99.7% and 99.8% percent identity. Indeed, REV infection would appear to be quite common amongst chickens.

Novel criteria for the diagnosis of Marek's disease virus-induced lymphomas.

Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 10(2)-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.

Insect contribution to horizontal transmission of Reticuloendotheliosis virus.

The involvement of insects in Reticuloendotheliosis virus (REV) transmission was examined by testing insects trapped at commercial farms and by controlled feeding experiments using mosquitoes, Culex pipiens L. and house flies, Musca domestica L. We established sensitive methods of REV detection, including reverse transcription-polymerase chain reaction (PCR) for REV-LTR and REV-gag genes, REV antigenemia measurements by enzyme-linked immunosorbent assay, and virus isolation in tissue cultures. A variety of blood-sucking species of insects were trapped at farms with infected poultry and tested, but none were positive. To rule out the possibility of PCR inhibition by insect RNA, spiking experiments were conducted and no interference was observed. Because Cx. pipiens mosquitoes were trapped frequently at farms, we performed feeding experiments with mosquito females fed on a REV-containing tissue culture medium and chicken blood mixture. Virus was detected in the mosquitoes up to 5 h postfeeding, compared with 96 h in the feeding mixture, indicating that Cx. pipiens can only harbor REV for a short period. House flies were suspected to be involved in the virus transmission because they frequently were trapped on positive farms. In contrast to mosquitoes, REV was harbored within the house fly digestive tract for up to 72 h and could infect chickens, as demonstrated by seroconversion and by detection of viral gag-sequence in the cloaca. The current study is supportive for the role of house flies as a mechanical vector of REV among poultry.

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.

Characterization of T-cell lymphomas associated with an outbreak of reticuloendotheliosis in turkeys.

Increased mortality and decreased egg production associated with disseminated lymphoma were observed in a turkey breeding flock for more than 20 weeks. A few unrelated meat turkey flocks, from the same integrator, experienced increased condemnation due to neoplasia in a few organs. Lymphoma was characterized by a uniform population of large lymphocytes with large vesicular nuclei containing one or two nucleoli and with little, faintly staining, basophilic cytoplasm. Neoplastic cells replaced normal tissue and were consistent with lesions seen with reticuloendotheliosis virus (REV) infection. Immunoperoxidase and fluorescent antibody staining characterized the neoplastic cells as CD3+, CD4+ and CD8- lymphocytes. Infection with REV was confirmed by virus isolation, polymerase chain reaction, serology and indirect fluorescent antibody. Poults hatched from these breeders tested positive for REV antibodies at hatch, but the performance of these flocks was normal and lymphoma was not observed. The origin of REV infection in this outbreak could not be determined. This is the first documented report of T-cell lymphomas associated with REV in commercial flocks. Furthermore, this is the first time that lymphomas have been characterized as T helper cells (CD3+ CD4+ and CD8-) in an outbreak of REV in turkeys.

Development of the hot spot-combined PCR assay for detection of retroviral insertions into Marek's disease virus.

A two-step PCR, the Hot Spot-combined PCR assay, was developed for the identification and characterization of recombinant viruses in Marek's disease (herpes) and retrovirus co-infections. In the first PCR the herpesvirus genomic fragment, that was recognized in previous studies as a hot spot site for retroviral integration was amplified [reviewed in Bronovskis, P., Kung, H.-J., 1996. Retrotransposition and herpesvirus evolution. Virus Genes 11, 259-270]. The products served for a second amplification step, performed in six PCR sets, using the six possible combinations of the two herpes and the retrovirus primer sets. Development of the assay employed DNA of the recombinant virus, RM1, which was created by in vitro co-cultivation of Marek's disease and reticuloendotheliosis viruses [Isfort et al., 1992. Proc. Natl. Acad. Sci. 89, 991-995; Witter et al., 1997. Avian Dis. 41, 407-421]. As the retroviral insertion site and junction sequences were determined previously [Jones et al., 1996. J. Virol. 70, 2460-2467], RMI served in the present study as a test virus for the development of the new assay. It is shown now that the Hot Spot-combined PCR can detect the retroviral insert in RM1. the MDV integration site and the insert orientation. For confirmation the herpes and retrovirus chimeric PCR products were sequenced and the results were similar to those published previously [Jones et al., 1996. J. Virol. 70, 2460-2467]. This assay might be adopted in additional systems to detect foreign inserts at suspected genomic sites.