Isolation of an infectious endogenous retrovirus in a proportion of live attenuated vaccines for pets

Abstract

The genomes of all animal species are colonized by endogenous retroviruses (ERVs). Although most ERVs have accumulated defects that render them incapable of replication, fully infectious ERVs have been identified in various mammals. In this study, we isolated a feline infectious ERV (RD-114) in a proportion of live attenuated vaccines for pets. Isolation of RD-114 was made in two independent laboratories using different detection strategies and using vaccines for both cats and dogs commercially available in Japan or the United Kingdom. This study shows that the methods currently employed to screen veterinary vaccines for retroviruses should be reevaluated.

FIG. 1.

Detection of RD-114 in pet vaccines. A total of 14 vaccine brands acquired from 7 different vaccine manufacturers were tested (also see Table 1 for a complete summary of the data obtained). Four of the 14 vaccines tested in both Japan and the United Kingdom showed evidence of RD-114 in at least one of their batches. Two of the vaccines (J-Cd and J-Co) from which RD-114 was isolated were commercially available only in Japan, while the remaining two were present in both markets (J-Aa, J-De, UK-Aa, and UK-De). Consequently, the presence of RD-114 was revealed in two different laboratories in the same vaccines (J-Aa/UK-Aa and J-De/UK-De), using independent samples, reagents, and detection strategies. (A to E) LacZ marker rescue assays were performed as already described (19). Panels A and C show representative examples of TE671 cells exposed to a vial containing vaccines J-De1 and J-Co1 respectively. The same vaccine preparations were not able to infect TE671-RD, a cell line chronically infected with RD-114, as shown in panels B and D. As a control, both TE671 and TE671-RD were infected by a murine leukemia virus-based vector pseudotyped with the FeLV-B Env (E and F). (G) RD-114 provirus was amplified from genomic DNA (200 ng/reaction) extracted from TE671(LacZ) cells exposed to the indicated vaccines. Reactions were carried out using the primers indicated in the text in a 25-μl standard PCR. The PCR cycles employed were 94°C for 10 min and 30 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 90 s, with a final extension of 72°C for 10 min. Specific PCR products were obtained only from those samples for which there was a positive result in the LacZ assay, as indicated schematically in the figure. PCR products were sequenced and confirmed to represent RD-114 env. (H) Visualization of retroviral particles by electron microscopy. TE671(LacZ) cells infected with vaccine J-Aa1 were passaged for 4 weeks. Cells were then fixed, dehydrated, embedded, and sectioned for electron microscopy by using standard methods. The microphotograph shows mature viral particles with a retroviral morphology in the vicinity of the cell membrane (bar, 100 μm).

FIG. 2.

Detection of RD-114 by Western blotting and reverse transcriptase (RT) assay. (A) Vaccines obtained from the United Kingdom were passaged in TE671 cells for 3 to 6 weeks. The samples represented in the figure were taken at the end of the experiment at 6 weeks after the initial exposure of TE671 cells to the indicated vaccines. TE671 cells were exposed to two vials of vaccines in each experiment. Approximately 10 ml of culture supernatants was collected, filtered through a 0.45-μm filter, and ultracentrifuged at 100,000g for 1 h. Virus pellets were resuspended in 50 μl Tris-EDTA (TE) buffer. Twelve microliters was then analyzed by Western blotting using a goat antiserum against the RD-114 major capsid protein (serum 72-S-781 from the U.S. National Cancer Institute). Note the presence of RD-114 CA in supernatants of cells infected with vaccines UK-De2 and UK-Aa4. As a positive control (lanes labeled “RD-114”), we utilized supernatants obtained from the feline cell line FER that expresses RD-114 (2). The specificity of the antibody used was also tested by transfecting 293T cells transfected with an infectious molecular clone of RD-114 as previously described (13). (B and C) RT assays were performed using the C-type RT activity kit (Cavidi) as recommended by the manufacturer. Panel B show representative data from some of the vaccines tested. Values are normalized against a Moloney murine leukemia virus recombinant RT standard and expressed in milliunits (mU) per ml. Note that culture supernatants were taken either undiluted (“neat”) or diluted as indicated in the graph. All samples that contained detectable RD-114 by Western blotting also had detectable RT activity. Panel C shows RT assays of supernatants of TE671 cells infected with vaccine UK-Aa4 and collected at different times postinfection. Note that RT activity is above the detection limit only after 3 weeks postinfection. An aliquot of the same sample collected at day 41 postinfection is the Uk-Aa4 sample shown also in panel B. o.d., optical density.