Replication of an adeno-associated virus in canine and human cells with infectious canine hepatitis virus as a "helper"

Adeno-associated viruses (AAV) are small, defective deoxyribonucleic acid viruses requiring adenoviruses for replication (J. L. Melnick et al., J. Bacteriol. 90:271, 1965; R. W. Atchison et al., Science 149:754, 1965; M. D. Hoggan et al., Proc. Natl. Acad. Sci. U.S. 55:1467, 1966; K. 0. Smith et al., J. Immunol. 97:754, 1966). M. D. Hoggan et al. (Proc. Natl. Acad. Sci. U.S. 55:1467, 1966) found that infectious canine hepatitis virus (ICHV) could "help" one strain of human AAV replicate in canine cells. The present paper confirms this observation with another AAV strain. Experiments were also designed to determine whether ICHV, which undergoes an abortive or low-level replicative cycle in human cells, would potentiate AAV in human cells. We used ICHV from the American Type Culture Collection (verified as ICHV by neutralization), AAV type 1 (K. 0. Smith et al., J. Immunol. 97:754, 1966), primary canine kidney (DK) cells, and human amnion (HA, strain FL) cells. AAV particles were counted (K. 0. Smith, Bacteriol. Proc., p. 130, 1964), and AAV infectivity was titrated by a fluorescent-focus technique (J. F. Thiel and K. 0. Smith, Proc. Soc. Exptl. Biol. Med., in press); complement fixation (CF) tests were then performed (K. 0. Smith, J. Immunol. 94:976, 1965). Virus suspensions were sonictreated for 30 sec before titration. AAV suspensions were heated at 60 C for either 5 or 30 min before passage. This reduced the concentration of infectious adenovirus below recoverable levels without significantly decreasing the infectivity of AAV (M. D. Hoggan et al., Proc. Natl. Acad. Sci. U.S. 55:1467, 1966; K. 0. Smith et al., J. Immunol. 97:754, 1966). AAV was serially passed in the following way. A virus suspension, containing approximately 1014 TCID50 per ml of adenovirus 4 and 108.0 fluorescing-focus units ofAAV per ml, was heated at 60 C for 30 min, to insure destruction of adenovirus, and diluted to 10-2; 2.0-ml volumes were inoculated on DK and HA cells (contained in 900-ml bottles). After 1 hr of incubation, inocula were removed, cultures were washed three times with Eagle's medium, and cells were inoculated with 2.0 ml of ICHV which contained 107.0 TCID50 per mnl. Inocula were again removed after 1 hr, cell cultures were washed three times with Eagle's medium, medium was replaced, and cultures were incubated at 36 C for 84 to 108 hr. Cultures were frozen-thawed; some inocula were removed for subpassage, and the remainder were stored at -20 C until needed. Material for subpassage was heated at 60 C for 5 min, and was inoculated (undiluted) on DK and HA cells (as before). Cells were then superinfected with ICHV, washed three times, and treated as before. AAV was thus serially subpassed three times, with fresh ICHV as "helper" each time, in DK and HA cells. Stored passages were later thawed, AAV particles were counted and titrated, and AAV CF antigen was measured by chessboard titration. Table 1 shows the results obtained from these serial passages in DK cells. Passages 1, 2, and 3 gave AAV particle increases of 200-, 78-, and 1.5-fold. Infectivity and CF assays gave similar results. The poor third passage in this series appears to be a technical error, since a repeat subpassage at this level gave about a 100-fold increase in infectious AAV. Table 2 shows the results obtained from three serial passages in HA cells. Particle increases were 1,125-, 14-, and 65-fold, respectively. Again, infectivity and CF antigen increases were of about the same order of magnitude as the particle increases. The particle-infectivity ratio of AAV in these passages (Tables 1 and 2) ranged from about 600 to 1,600. Approximately 5 x 109 to 2 X 1010 particles of AAV were required for minimal CF reaction, which is similar to our previous findings (K. 0. Smith et al., J. Immunol. 97:754, 1966). Passage of AAV in canine cells without adenovirus as a helper resulted in a substantial loss of the AAV introduced at the time of inoculation. We originally found this AAV strain associated