Long nucleotide insertions between the HN and L protein coding regions of human parainfluenza virus type 3 yield viruses with temperature-sensitive and attenuation phenotypes

Recombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus v accine. In the present study, we examined the effect of inserted foreign se quence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additi onal gene unit (GU insert) between the HN and L genes, so that one addition al mRNA would be made. in a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the n umber of encoded mRNAs remained unchanged. In each case, the foreign sequen ce was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3 918 nt in length, the largest size tested, were viable and replicated effic iently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39 degrees C and 40 degrees C. The rPIV3 with a 3918-nt GU inse rtion was restricted in replication in the upper (fivefold) and lower (25-f old) respiratory tracts of hamsters. Although a 1908-nt GU insertion did no t significantly modify replication of wild-type PIV3 in vitro or in vivo, i ts introduction significantly augmented the level of temperature sensitivit y (Ls) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype , that is, efficient replication in vitro but restricted replication in ham sters, especially in the lower respiratory tract. Furthermore, these effect s were greatly enhanced when the rPIV3 backbone contained other ts or att m utations. The implications of these findings for the use of single-stranded , negative-sense RNA viruses as Vectors for vaccines are discussed. (C) 200 0 Academic Press.