Complete Genome Characterization of Reticuloendotheliosis Virus Detected in Chickens with Multiple Viral Coinfections.

Reticuloendotheliosis virus (REV) is a retroviral pathogen capable of infecting several avian hosts and is associated with immunosuppression, anemia, proventriculitis, neoplasia, and runting-stunting syndrome. Its genome contains the three major genes, gag, pol, and env, and two flanking long terminal repeat (LTR) regions. Complete genome sequences of REV are limited in terms of geographical origin. The aim of this study was to characterize the complete genome of REV detected in Brazilian chickens with multiple viral coinfections and analyze the polymorphisms in the deduced amino acids sequences corresponding to its encoded proteins. We tested the presence and completeness of REV as well as other viral pathogens in samples from Brazilian poultry farms by qPCR. The complete genomes of two REV strains were sequenced by overlapping fragments through the dideoxy method. Phylogenetic analysis, pairwise identity matrix, polymorphism identification and protein modeling were performed along the entire genome. We detected REV in 65% (26/40) of the tested samples. Concomitant viral infections were detected in 82.5% (33/40) of the samples and in 90% (9/10) of the farms. Multiple infections included up to seven viruses. Phylogenetic analysis classified both Brazilian strains into REV subtype 3, and the pairwise comparison indicated that strains from the USA and fowlpox virus (FWPV)-related strains were the most identical. The subdomain p18 in gag, the reverse transcriptase/ribonuclease H in pol, and the surface (SU) in the env protein were the most polymorphic in genomic comparisons. The relevant motifs for each protein were highly conserved, with fewer polymorphisms in the fusion peptide, immunosuppression domain, and disulfide bonds on the surface (SU) and transmembrane (TM) of env. This is the first study to include complete genomes of REV in Brazil and South America detected in farms with multiple viral coinfections. Our findings suggest an involvement of REV as an immunosuppressor and active agent in the emergence and progression of multiple infectious diseases. We also found a possible etiological relationship between Brazilian strains and the USA and FWPV recombinant strains. This information highlights the need for epidemiological vigilance regarding REV in association with another pathogens.

Detection and Molecular Characterization of a Natural Coinfection of Marek's Disease Virus and Reticuloendotheliosis Virus in Brazilian Backyard Chicken Flock.

Marek's disease virus (MDV) and the reticuloendotheliosis virus (REV) are two of the primary oncogenic viruses that significantly affect chickens. In Brazil, there have been no previous published reports on the presence of field REV alone or in coinfection. This retrospective study analyzes samples from a case of lymphoproliferative lesions from a backyard chicken flock. MDV and REV were detected by PCR and classified as MDV1 and REV3, respectively, through sequencing and phylogenetic analysis based on the glycoprotein B (gB) genes for MDV and the polymerase (pol) and envelope (env) genes for REV. Real-time PCR reactions were performed for MDV to rule out the presence of the Rispens vaccine strain. This is the first report of the presence of REV in coinfection with a MDV clinical case in Brazil and the first molecular characterization of REV in South America. This study highlights the importance of molecular diagnosis for REV and MDV in poultry. In addition, this study highlights the distribution of these two viruses worldwide and the latent risk of them solely or in coinfection to this part of the world.

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.

Protection induced by a gp90 protein-based vaccine derived from a Reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine.

BACKGROUND: Reticuloendotheliosis is an immunosuppressive disease caused by avian reticuloendotheliosis virus (REV). It is commonly found in poultry farms and has caused a notable economic loss worldwide. Despite this, there is currently no effective vaccine available to protect against REV infection. METHOD: In this study, gp90 protein derived from an REV isolated from a contaminated vaccine was co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) adjuvant to hens to determine if it protects their chicks against REV infection. To synthesize the gp90 protein, the gp90 gene was amplified using polymerase chain reaction, expressed in Escherichia coli, and purified. The resulting recombinant protein was injected intramuscularly into breeder hens along with CpG-ODN adjuvant and then serum antibody levels were regularly evaluated. After the fertilized eggs from these vaccinated hens had hatched, the resulting chicks were challenged with a 102.7 50% tissue culture infectious dose (TCID50) of REV at 1 day old and the REV antibody levels in these hatched chickens were evaluated before and after the challenge. Viremia and growth rate were measured weekly and statistically analyzed. RESULTS: The results suggest that the gp90 recombinant protein was successfully prepared and, when used with CpG-ODN adjuvant to immunize breeder hens, induced serological antibody production against REV in both hens and their hatched chicks. In addition, the maternal antibodies induced by the gp90 protein vaccine effectively protected majority of the chicks from REV infection. CONCLUSIONS: Overall, we found the gp90 protein obtained in this study may be a potential vaccine candidate that had good immunogenicity and could be an auxiliary measure to accelerate the eradication of REV.

Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

Synergetic effects of subgroup J avian leukosis virus and reticuloendotheliosis virus co-infection on growth retardation and immunosuppression in SPF chickens.

To further understand the effect of co-infection of subgroup J avian leukosis virus (ALV-J) and reticuloendotheliosis virus (REV) in specific-pathogen-free (SPF) white leghorn chickens, the experiment was made to study the pathogenicity, the weight of body and immune organs, response to newcastle disease virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. Chickens were randomly divided into four groups, which includes injection groups (REV, ALV-J, REV plus ALV-J), and negative control group. The pathogenesis experiments indicated that chickens co-infected with REV and ALV-J had significantly higher mortality rate than those of the chickens infected with REV or ALV-J alone (P<0.05). Chickens inoculated with REV and ALV-J had significantly lower weights than chickens in all other groups (P<0.05). There were no significant differences between the two single infection groups and co-infection group (P>0.05) on bursa and thymus over body wt ratios, however, chickens co-infected with REV and ALV-J had significantly lower titers than REV-infected chickens and ALV-J-infected chickens on HI antibody titers to ND and AIV-H9 after vaccination (P<0.05). These findings suggested that the co-infection of REV and ALV-J caused more serious growth retardation and immunosuppression in SPF chickens.