Detection of FeLV <i>env</i> genes by PCR, and strategies for analysis of these genes.

<p>(A) Strategy used for generating PCR products. Schematics of coding sequences for the FeLV <i>env</i> gene are shown. FeLV proviral <i>env</i> sequences were amplified by PCR with the primer pairs Fe-8S/Fe-4S and Fe-3R, PRB-1 and Fe-3R, Fe-9S and Fe-7R. The lengths of the expected products from amplifications using each primer pair are shown. The abbreviations s.p., SU and TM represent, respectively, signal peptide, surface glycoprotein, and transmembrane subunit. (B) The DNA templates for PCR amplifications were as follows: neg. (genomic DNA isolated from FeLV-negative AH927 cells), GA5 (DNA from FeLV-A Glasgow-1-infected AH927 cells), GB (DNA from Gardner-Arnstein FeLV-B-infected AH927 cells), SC (DNA from FeLV-C Sarma-infected AH927 cells), DNA from FT-1 cell line, DNA from sample SN5, DNA from sample MZ29, DNA from a FeLV-positive cat (SN22), and DNA from a FeLV-negative cat (FS23). c-<i>myc</i> was amplified as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061009#pone.0061009-Anai1" target="_blank">[25]</a>. PCR products were electrophoresed and stained with ethidium bromide. Asterisk indicates atypical bands of <i>env</i> gene. (C) Each detected PCR fragment was cloned into a cloning plasmid, and full-length <i>env</i> gene libraries were constructed. Several unique <i>env</i> genes were isolated from these full-length <i>env</i> gene libraries using information on fragment size, or by screening using FeLV-B specific PCR. In addition to non-recombinant <i>env</i> genes, FeLV-B-type and other recombinants were isolated and analyzed. Furthermore, <i>env</i> genes smaller than full length were also analyzed. N indicates negative and P positive for FeLV-B detection or recombination detection. (D) For the most part, only non-recombinant sequences were used for phylogenetic analysis, but where recombinant sequences were included, their endogenous-derived regions were removed from the alignment. However, partial sequences from some recombinants as well as representatives of the FeLV-C and FeLV-B subgroups were included in the analyses after excluding their recombinant regions, to determine the FeLV genotypes from cats from which non-recombinants were not identified.