Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo

Respiratory syncytial virus (RSV) encodes several proteins that lack well-d efined functions; these include NS1, NS2, SH, and M2-2. Previous work has d emonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can rep licate efficiently when two or more transcriptional units are deleted, we r emoved NS1, NS2, SH, and M2-2 genes individually and in different combinati ons from an infectious cDNA clone derived from human RSV A2 strain. The fol lowing six mutants with two or more genes deleted were obtained: Delta NS1N S2, Delta M2-2SH, Delta M2-2NS2, Delta SHNS1, Delta SHNS2, and Delta SHNS1N S2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were de leted. All of the double and triple deletion mutants exhibited reduced repl ication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 s ingle and multiple gene deletion mutants obtained, Delta M2-2NS2 was most a ttenuated, Delta M2-2NS2 formed barely visible plaques in HEp-2 cells and h ad a reduction of titer of 3 log(10) compared with the wild-type recombinan t RSV in infected HEp-2 cells. When inoculated intranasally into cotton rat s, all of the deletion mutants were attenuated in the respiratory tract. Ou r data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispens able for virus replication in vitro, provide auxiliary functions for effici ent RSV replication, (C) 2000 Academic Press.