The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100-1000 fold restricted in replication in the respiratory tract of non-human primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children yet is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host-range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA and the level of viral replication in vitro and in the respiratory tract of rhesus monkeys was determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tract of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host-range restriction of replication of BPIV3 for primates are polygenic in nature, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher HPIV3 hemagglutination-inhibiting (HAI) serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild type HPIV3. The high level of attenuation of rHPIV3-L bearing an adventitious ts mutation in L in rhesus monkeys reflects the additivity of the restriction of replication specified by host-range sequences of its L gene and one or both of the T1711I and A425V point mutations. The T1711I mutation was shown to confer the ts phenotype in vitro and thus likely also is responsible for the attenuation phenotype in vivo, although a contribution by the A425V mutation cannot be excluded at this time. Although the individual contributions of these two mutations to attenuation in vivo remain to be defined, the increased attenuation of rHPIV3-L mutant versus rHPIV3-LB illustrates that ts and host-range determinants of attenuation can be combined to fine-tune the level of attenuation of a candidate vaccine virus. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccines candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3. Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide (nt) length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. HPIV2 is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the rule of six. To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains, Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998 (V98). Each of these strains was found to have a genome length of 15,654 nucleotides, thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0-5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the non-polyhexameric antigenomic cDNAs. These were found to contain small nt insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the HN and L genes or the intervening intergenic region, and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.
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