This full-length genome sequence of a feline leukemia virus Kawakami-Theilen strain (designated KT-FeLV-UCD-1), produced from the chronically infected FL74-UCD-1 cell line, was obtained using high-throughput sequencing. It consisted of 8,464 bp and had a genomic organization similar to that of other gammaretroviruses, containing long terminal repeats and open reading frames for Gag, Pol, and Env.
ABSTRACT
This full-length genome sequence of a feline leukemia virus Kawakami-Theilen strain (designated KT-FeLV-UCD-1), produced from the chronically infected FL74-UCD-1 cell line, was obtained using high-throughput sequencing. It consisted of 8,464 bp and had a genomic organization similar to that of other gammaretroviruses, containing long terminal repeats and open reading frames for Gag, Pol, and Env.
ANNOUNCEMENT
Feline leukemia virus (FeLV) infections are widespread in cats due to horizontal species-to-species transmission (1–3) and can remain chronic or result in a variety of disorders (4). Although humans have close contact with cats, cross-species infections have not been reported (5). An FeLV vaccine for cats was developed using the FL74 feline lymphoblastoid cell line (6), originally derived by long-term laboratory culture from the Kawakami-Theilen (KT) cell line (7, 8). These cells are chronically infected with FeLV strain KT, which has attenuated virulence for cats (9). The cell line was designated FL74-UCD-1 (6), and the virus was designated KT-FeLV-UCD-1.
A high-titer virus stock of KT-FeLV-UCD-1 was prepared by harvesting and concentrating supernatant from expansion of FL74-UCD-1 cells (catalogue number CRL-8012; American Type Culture Collection, Manassas, VA). Nucleic acid was extracted from 200 μl of the virus stock using the QIAamp MinElute virus spin kit and eluted in 60 μl RNase-free water. cDNA was obtained using random primers and Superscript II enzyme (Invitrogen) to obtain cDNA, followed by purification using magnetic beads (AMPureXP beads; Beckman Coulter). Double-strand cDNA was quantified with a Qubit 3.0 fluorometer (Life Technologies) using a Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Thermo Fisher Scientific). The library was obtained using the Nextera XT DNA library preparation kit (Illumina) using 5 ng as the starting material quantity. Library quality control was checked using an Agilent 2100 bioanalyzer instrument to calculate library average size, while quantification was verified using a Qubit 3.0 fluorometer. Sequencing was performed using the HiSeq 2500 platform (Illumina) with 101-bp paired-end mode.
Read trimming and assembly were done using the CLC BIO Genomics Workbench software version 11.0.1 (Qiagen Aarhus A/S, Denmark). The total number of trimmed, paired-end reads was 505,153,300, and the average read length was 88 bp. The 8,464-bp complete viral genome sequence was obtained by mapping the raw reads (using the default parameters) to the FeLV Rickard strain (GenBank accession number NC_001940.1) as a reference genome, and the consensus sequence was extracted using default parameters. Open reading frames were identified using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) (10). The genomic structure was similar to those of other retroviruses encoding for Gag, Pol, and Env, with 5′ and 3′ long terminal repeats (LTRs). Nucleotide sequence analysis using NCBI BLASTN indicated 94% sequence identity with the FeLV Rickard strain. Comparative nucleotide sequence analysis indicated that the most remarkable difference was an insertion of 9 nucleotides resulting in an in-frame addition of 3 amino acids in the N-terminal leader region of the glycoGag protein (gPr80) in KT-FeLV-UCD-1 (11). Further analysis of Gag using a CLC BIO Genomics Workbench version 11.0.1 antigenicity plot (12) indicated a potential reduction in the antigenicity of N-terminus Gag in KT-FeLV-UCD-1 compared with that in the FeLV Rickard strain. The availability of the full genomic sequence of KT-FeLV-UCD-1 may help identify the attenuation determinants in this strain versus the pathogenic FeLV Rickard strain.
Data availability.
The complete genome sequence of KT-FeLV-UCD-1 has been deposited in NCBI/GenBank under the accession number MT129531. The raw data were deposited under SRA accession number SRR11242952, BioSample number SAMN14273276, and BioProject number PRJNA610048.
ACKNOWLEDGMENTS
The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Food and Drug Administration.
REFERENCES
- 1.Francis DP, Essex M, Gayzagian D. 1979. Feline leukemia virus: survival under home and laboratory conditions. J Clin Microbiol 9:154–156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Hardy WD Jr, Old LJ, Hess PW, Essex M, Cotter S. 1973. Horizontal transmission of feline leukaemia virus. Nature 244:266–269. doi: 10.1038/244266a0. [DOI] [PubMed] [Google Scholar]
- 3.Jarrett W, Jarrett O, Mackey L, Laird H, Hardy W Jr, Essex M. 1973. Horizontal transmission of leukemia virus and leukemia in the cat. J Natl Cancer Inst 51:833–841. doi: 10.1093/jnci/51.3.833. [DOI] [PubMed] [Google Scholar]
- 4.Powers JA, Chiu ES, Kraberger SJ, Roelke-Parker M, Lowery I, Erbeck K, Troyer R, Carver S, VandeWoude S. 2018. Feline leukemia virus (FeLV) disease outcomes in a domestic cat breeding colony: relationship to endogenous FeLV and other chronic viral infections. J Virol 92:e00649-18. doi: 10.1128/JVI.00649-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Terry A, Kilbey A, Naseer A, Levy LS, Ahmad S, Watts C, Mackay N, Cameron E, Wilson S, Neil JC. 2017. Barriers to infection of human cells by feline leukemia virus: insights into resistance to zoonosis. J Virol 91. doi: 10.1128/JVI.02119-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Theilen GH, Penderson NC. 1979. Vaccine for preventing persistent feline leukemia viremia in cats. Patent CA1155763A, Canada. https://patents.google.com/patent/CA1155763A.
- 7.Jarrett W, Essex M, Mackey L, Jarrett O, Laird H. 1973. Antibodies in normal and leukemic cats to feline oncornavirus-associated cell membrane antigens. J Natl Cancer Inst 51:261–263. doi: 10.1093/jnci/51.1.261. [DOI] [PubMed] [Google Scholar]
- 8.Theilen GH, Kawakami TG, Rush JD, Munn RJ. 1969. Replication of cat leukemia virus in cell suspension cultures. Nature 222:589–590. doi: 10.1038/222589b0. [DOI] [PubMed] [Google Scholar]
- 9.Pedersen NC, Theilen GH, Werner LL. 1979. Safety and efficacy studies of live- and killed-feline leukemia virus vaccines. Am J Vet Res 40:1120–1126. [PubMed] [Google Scholar]
- 10.Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Federhen S, Feolo M, Fingerman IM, Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Lu Z, Madden TL, Madej T, Maglott DR, Marchler-Bauer A, Miller V, Mizrachi I, Ostell J, Panchenko A, Phan L, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Slotta D, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Wang Y, Wilbur WJ, Yaschenko E, Ye J. 2011. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 39:D38—D51. doi: 10.1093/nar/gkq1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li S, Ahmad I, Shi J, Wang B, Yu C, Zhang L, Zheng YH. 2018. Murine leukemia virus glycosylated gag reduces murine SERINC5 protein expression at steady-state levels via the endosome/lysosome pathway to counteract SERINC5 antiretroviral activity. J Virol 93:e01651-18. doi: 10.1128/JVI.01651-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kolaskar AS, Tongaonkar PC. 1990. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172—174. doi: 10.1016/0014-5793(90)80535-q. [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The complete genome sequence of KT-FeLV-UCD-1 has been deposited in NCBI/GenBank under the accession number MT129531. The raw data were deposited under SRA accession number SRR11242952, BioSample number SAMN14273276, and BioProject number PRJNA610048.