Equine infectious anemia virus (EIAV) is a lentivirus that infects members of the family Equidae. Despite extensive study, there is no method for prevention or treatment of this disease. Novel mechanisms, such as RNA interference (RNAi), have been used to decrease replication of many viruses. Results of previous in vitro studies have indicated that in the process of generating transgenic cells, EIAV replication was inhibited. Thus, these data were inconclusive as to the efficacy of RNAi against EIAV. To further investigate the effectiveness of RNAi, it is essential to discern which aspects of generating transgenic cell lines affects viral replication. Here, we have investigated 2 common reagents: polybrene (Pol), a polycationic polymer used to increase initial cell transduction, and puromycin (Pur), an antibiotic selecting agent. Non-transgenic feline adenocarcinoma cells, persistently infected with EIAV₁₉ (a laboratory adapted strain of the virus), were exposed to high and low concentrations of Pol (8 and 16 µg/mL), and Pur, (1 and 2 µg/mL). Control cell lines were exposed to neither reagent. Supernatants were collected for 17 d and viral replication was quantified using a reverse transcriptase (RT) Assay. The RT values were analyzed using R (R, v. 2.15.3, R Development Core Team, 2012) to generate 9999 permutations of the data. Mean RT values (Table 1) for experimental groups were not significantly different from the mean RT values for the control groups. These data indicate that Pol and Pur alone have no effect on viral replication in these cells. Additional studies are needed to determine which reagents used in establishing transgenic cells is responsible for the observed decreases in viral replication. Table 1. Mean RT values per 100,000 cells per day of viral accumulation

Day	Control	Pol High	Pol Low	Pur High	Pur Low
2	3.2E6 ± 0.5E6	3.8E6 ± 0.4E6	4.9E6 ± 0.9E6	13.6E6 ± 0.4E6	13.4E6 ± 4.9E6
4	12.7E6 ± 2.7E6	13.7E6 ± 1.5E6	15.2E6 ± 1.3E6	720.6E6 ± 341.6E6	59.1E6 ± 21.8E6
7	8.6E6 ± 1.7E6	13.8E6 ± 2.5E6	17.4E6 ± 3.3E6	527.1E6 ± 210.1E6	21.6E6 ± 5.9E6
9	27.9E6 ± 1.8E6	27.7E6 ± 1.5E6	28.4E6 ± 6.5E6	529.4E6 ± 104.4E6	27.4E6 ± 1.5E6
13	9.7E6 ± 0.8E6	12.0E6 ± 3.1E6	9.9E6 ± 0.9E6	215.0E6 ± 13.5E6	6.5E6 ± 0.8E6
17	5.2E6 ± 1.0E6	7.0E6 ± 1.7E6	6.4E6 ± 0.8E6	198.6E6 ± 11.7E6	4.2E6 ± 0.3E6