--- id: "edwards-2022-prairie-chicken" title: "Near-Complete Proviral Genome Sequence of Reticuloendotheliosis Virus Isolated from an Attwater’s Prairie Chicken (Tympanuchus cupido attwateri)" authors: - "Bianca Willis" - "Brittany Stewart" - "Faith Cox" - "Tiffany Lujan" - "Holly Haefele" - "Camila Romano" - "Jeff Brady" - "Dustin Edwards" venue: "Microbiology Resource Announcements" year: 2022 date: "2022-04-21" doi: "10.1128/mra.00173-22" url: "/research/publications/10-1128-mra-00173-22/" pdf: "/research/publications/10-1128-mra-00173-22/dustin-edwards-10-1128-mra-00173-22.pdf" pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC9022497/" openAccess: true license: "cc-by" accessions: - "genbank:OL857288" - "sra:SRX11820216" - "sra:SRX13461851" citedBy: 0 citedBySource: "OpenAlex, read 2026-09-12" --- # Near-Complete Proviral Genome Sequence of Reticuloendotheliosis Virus Isolated from an Attwater’s Prairie Chicken (Tympanuchus cupido attwateri) Genome of the reticuloendotheliosis virus from the 2016 to 2017 outbreak in captive Attwater's prairie chickens. ## Abstract We report the near-complete proviral genome sequence of a reticuloendotheliosis virus isolated and propagated from an endangered Attwater’s prairie chicken (Tympanuchus cupido attwateri) during a 2016–2017 outbreak at a captive breeding facility. ## Full text Machine-extracted from the PDF linked above. It carries the artifacts that come with reading a typeset two-column page: running heads, figure captions in the flow of the prose, and words broken across line ends. The abstract above is the registry's deposit and is the authoritative text. Near-Complete Proviral Genome Sequence of Reticuloendotheliosis Virus Isolated from an Attwater’s Prairie Chicken (Tympanuchus cupido attwateri) Bianca Willis,a,d Brittany Stewart,a Faith Cox,a Tiffany Lujan,a,d Holly Haefele,b Camila Romano,c Jeff Brady,d Dustin Edwardsa aDepartment of Biological Sciences, Tarleton State University, Stephenville, Texas, USA bFossil Rim Wildlife Center, Glen Rose, Texas, USA cInstituto de Medicina Tropical de São Paulo e Hospital das Clínicas da Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil dTexas A&M AgriLife Research, Stephenville, Texas, USA ABSTRACT We report the near-complete proviral genome sequence of a reticuloendo- theliosis virus isolated and propagated from an endangered Attwater’s prairie chicken (Tympanuchus cupido attwateri) during a 2016–2017 outbreak at a captive breeding facility. Reticuloendotheliosis virus (REV), a member of the Retroviridae family and Gammaretrovirus genus, is an immunosuppressive avian virus that infects B lymphocytes and has been detected in Anseriformes, Galliformes, and Passeriformes (1). In 2016 to 2017, the virus was the leading cause of death among adult endangered Attwater’s prairie chickens (APCs) (Tympanuchus cupido attwateri) at the Fossil Rim Wildlife Center (FRWC) captive breeding facility in Glen Rose, Texas (2). REV was previously isolated and sequenced from APC-566 (GenBank accession number DQ387450) during an outbreak at the facility 10 years earlier (3). We performed next-generation sequencing of a 2017 isolate for comparison between outbreaks. Blood from APC-2794, diagnosed as REV positive by quantitative PCR (qPCR) at the Texas A&M Veterinary Medical Diagnostic Laboratory (College Station, TX), was drawn into EDTA-coated tubes by FRWC veterinarians in January 2017 as part of normal practices. Blood was received and diluted 1:3 in phosphate-buffered saline (PBS), and mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation. The buffy coat layer was aspirated, washed in PBS, and cocultivated for 5 days with chicken embryonic fibro- blast DF-1 cells (CRL-12203; ATCC) with 5% CO2 at 39°C in Dulbecco's modified Eagle me- dium (11965-092; Gibco) supplemented with 10% fetal bovine serum (20082-147; Gibco) (4). After incubation, supernatant containing released REV was collected, passed through 0.22- mm filters, and placed on fresh DF-1 cells for 5 days, which was repeated for a total of three passages. After the final passage, cells were washed with PBS, pelleted by centrifuga- tion, resuspended in cell culture freezing medium (12646-010; Gibco), aliquoted, and stored in liquid nitrogen. Supernatant containing REV was stored at 280°C, and a frozen aliquot was submitted to Charles River Avian Services (Wilmington, MA) and determined to contain a 50% tissue culture infective dose (TCID50) of 6.8 105 virions/mL. Genomic DNA containing provirus was extracted by incubating approximately 1 106 REV-infected DF-1 cells with 75 mL HotSHOT alkaline lysis reagent at 95°C for 15 min and was cooled on ice for 5 min before the addition of 75 mL of HotSHOT neutralization solution (5). A genomic sequencing library was prepared at Texas A&M AgriLife Research (Stephenville, TX) using KAPA HiFi HotStart ReadyMix (07958927001; Roche) and primers with Illumina adapters to amplify three overlapping sections, i.e., nucleotide positions 1 to 5075, 1500 to 7295, and 7052 to 8295 (3). Resolved PCR products were excised, pooled, tagmented using the Nextera XT DNA library preparation kit (FC-131-1096; Illumina), and sequenced with an Illumina MiSeq instrument at Texas A&M Genomics and Bioinformatics Services (College Station, TX). Sequencing produced a total of 35,372 paired-end reads of 300-base read Editor Simon Roux, DOE Joint Genome Institute Copyright © 2022 Willis et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Dustin Edwards, dcedwards@tarleton.edu. The authors declare no conflict of interest. Received 21 February 2022 Accepted 9 March 2022 Published 23 March 2022 April 2022 Volume 11 Issue 4 10.1128/mra.00173-22 1 GENOME SEQUENCES Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207. length. Primer sequences and low-quality and short (,30-bp) reads were trimmed. A consen- sus sequence was assembled from sequence reads of two libraries for high coverage, prepared from the same DNA extraction, and a reference (GenBank accession number DQ387450) using the CLC Genomics Workbench v6 assembly to reference tool (mismatch cost=1, indels cost=2, minimum fraction of similarity between reads and reference=90%). A total of 92.4% of the reads were mapped (average coverage, 1,948). Genome annotation was formatted with SeqBuilder Pro v17.2.1. All tools were run with default parameters unless indicated other- wise. The assembled sequence is 8,246 bp, representing 99.5% of the genome, and has a G1C content of 52.3%. Nucleotide alignment of entire genome sequences with BLASTn (https://blast.ncbi.nlm.nih.gov) (6) showed that the virus is well conserved, with 99.9% nu- cleotide identity to strain APC-566 with a single nucleotide polymorphism at position 6653 (Table 1). APC-2794 shared 100% nucleotide identity with strain 104865 (GenBank accession number KJ756349), which was isolated from a domestic backyard turkey. Data availability. This sequence was deposited under GenBank accession number OL857288. Raw reads are available under SRA accession numbers SRX11820216 and SRX13461851. ACKNOWLEDGMENTS We thank FRWC and the U.S. Fish and Wildlife Service for APC blood samples. Support for this research was provided by Tarleton State University and the College of Science and Technology for Undergraduate Research Assistantships and Student Research Grants. REFERENCES 1. Ferro PJ, Morrow ME, Flanagan JP, Ortego B, Chester RE, Mueller JM, Lupiani B. 2017. Wild birds, a source of reticuloendotheliosis virus infection for the endangered Attwater’s prairie-chicken (Tympanuchus cupido attwa- teri)? J Wildl Dis 53:586–590. https://doi.org/10.7589/2016-07-169. 2. Stewart B, Trautman C, Cox F, Spann H, Hardin J, Dittmar R, Edwards D. 2018. Survey of reticuloendotheliosis virus in wild turkeys (Meleagris gallopavo) in Texas, USA. J Wildl Dis 55:689–693. https://doi.org/10.7589/2018-08-187. 3. Barbosa T, Zavala G, Cheng S, Villegas P. 2007. Full genome sequence and some biological properties of reticuloendotheliosis virus strain APC-566 isolated from endangered Attwater’s prairie chickens. Virus Res 124:68–77. https://doi .org/10.1016/j.virusres.2006.10.002. 4. Himly M, Foster DN, Bottoli I, Iacovoni JS, Vogt PK. 1998. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology 248:295–304. https://doi.org/10.1006/viro.1998.9290. 5. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. 2000. Preparation of PCR-quality mouse genomic DNA with hot sodium hydrox- ide and Tris (HotSHOT). Biotechniques 29:52–54. https://doi.org/10.2144/ 00291bm09. 6. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local align- ment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022 -2836(05)80360-2. TABLE 1 Reference REV isolates used for strain comparisons GenBank accession no. Host Country Year of isolation Identity (%)a OL857288 APC USA 2017 KJ756349 Turkey USA 2014 100.00 DQ387450 APC USA 2005 99.99 MF631845 Chicken Thailand 2013 99.93 GQ415646 Chicken China 2009 99.89 FJ439119 Goose Taiwan 2006 99.89 JQ804915 Duck China 2011 99.84 a Percent identity is based on nucleotide alignments obtained from BLASTn. Announcement Microbiology Resource Announcements April 2022 Volume 11 Issue 4 10.1128/mra.00173-22 2 Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207.