Preparation of a cell line persistently infected with maedi/visna virus and production of viral antigens

SUZUKI, Kazuya; OGUMA, Keisuke; SENTSUI, Hiroshi

PCR was performed using the GoTaq Green Master Mix (Promega, Madison, WI, U.S.A.) to amplify a partial doi: 10.1292/jvms. sequence of the long terminal repeat (LTR) [1] . DNA was extracted using a Gentra Puregene Cell Kit (QIAGEN, Hilden, Germany). The primer pair used to amplify LTR was LTR 2s (5′-CAGAAATCATAGTCAGGATGACAC) and LTA 2a (5′-CCACGTTGGGCGCCAGCTGCGAGA). Amplification of LTR was performed as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. The MVV-positive control was prepared from FGL infected with the MVV Iwate strain. The amplification of LTR was not observed when cells of early passages were analyzed (i.e., passages 3 through 6) after virus inoculation. Nonetheless, ZZ-R cells became MMV-positive (according to PCR) after several additional passages and continued to be virus-positive after 18 passages (Fig. 1) . As for the PCR products, a slightly large clear-cut band was noticed together with a band of expected size. Amplification of LTR was not observed in other cell lines, including FLK-N3 (after 24 passages) and CCL-88 (after 20 passages). The LTR gene sequences from passage 18 of ZZ-R cells were analyzed using the BigDye Terminator v3.1 Cycle Sequencing Kit on an Applied Biosystems 3130 Genetic Analyzer (Life Technologies). It was confirmed that there was no difference in the sequence between the product of expected size and those of the Iwate strain. The large clear-cut PCR product was also sequenced, and we found that the 82 bases including the primer part in the amplicon of expected size were tandemly repeated at the 5′ end. The redundant parts were almost identical in sequence except their five bases at the 3′ end were different between the two viral strains. Therefore, the clear-cut band was also specific, and the mutant virus might be able to grow well (as compared to the original virus) in ZZ-R cells. The MVV antigen for the AGID test was prepared using culture fluids of ZZ-R cells infected with MVV and cultivated for more than 18 passages. The culture fluids were concentrated using ammonium sulfate, dialyzed with PBS and further concentrated with polyethylene glycol 6000 according to published procedures [8] . These concentrated virus-containing fluids were treated with 1.0% Triton X-100 and used as antigens for antiviral antibodies. The AGID tests were performed according to the OIE manual. The gel consisted of 1.0% Noble agar, 8.5% NaCl and 10 mM phosphate buffer. The wells were 5 mm in diameter, and six circumferential wells were placed at a distance of 3 mm from the central well. The central well was filled with the control antiserum, and two exterior wells were filled with the positive-control antigen and PBS, respectively. The positive-control MVV antigen and antiserum were provided by the National Institute of Animal Health (Tsukuba, Japan). The MVV antigen was serially diluted and placed in the remaining four wells. The gel diffusion plate was allowed to stand at room temperature for 48 hr and was examined for precipitation lines. The antigen concentrated approximately 50-fold from the culture fluids formed a precipitation line until 1:8 dilutions and connected to a line produced between the positive reference serum and reference antigen (Fig. 2) .