Isolation, Identification, Genomic Analysis, and Rescue of a Field-Derived Isolate of Reticuloendotheliosis Virus from Hubei, China

Reticuloendotheliosis virus (REV) is an oncogenic retrovirus that threatens the global poultry industry through immunosuppression and neoplastic diseases. However, functional studies on field isolates have long been hampered by co-infections with other avian tumor viruses and complex genetic backgrounds. To address these issues, a field-derived REV strain, designated HB2301, was obtained from chickens in Hubei Province, China. These chickens exhibited emaciation, depression, and decreased egg production, and tumor-like nodules were observed in visceral organs at necropsy. The isolate was confirmed as REV-positive by multiplex PCR and IFA, with no detectable ALV (A/B/J/K) or MDV co-infection. Sequence analysis of the 8285 bp consensus proviral genome classified HB2301 into antigenic Subtype III. The REV genome proved highly conserved overall; the long terminal repeat (LTR) region exhibited greater variation than the coding regions, while the gag and pol genes remained the most conserved. Further analysis of the REV gp90 protein identified a unique valine-to-methionine point mutation at amino acid position 72 (V72M). This mutation is exclusively present in HB2301 and a previous Hubei isolate (HB2015021), suggesting it functions as a candidate regional marker rather than a random variation. Following consensus genome sequencing, a full-length infectious clone plasmid (pMD18-REV ABC) was constructed utilizing seamless cloning and enzymatic digestion, incorporating a targeted synonymous mutation (A2649C) as a specific molecular marker. Transfection of the infectious clone into DF-1 cells successfully rescued the recombinant virus rHB2301, which retained the A2649C genetic marker after three serial passages. The reactivity with an anti-REV gp90 monoclonal antibody and the in vitro replication kinetics of rHB2301 were comparable to those of the parental field-derived HB2301 stock. This study expands the epidemiological data on REV genetic evolution in China and provides a pure, genetically defined infectious clone platform that will facilitate future investigations into REV pathogenesis and support the development of novel prevention strategies.