We previously showed that the genome of HRSV is a single negative-sense strand of RNA of 15,222 nucleotides that encodes 10 mRNAs and 11 proteins. We also identified functions for a number of the proteins. Several recombinant vaccine candidates have been constructed and evaluated in clinical trials (see B.R. Murphy report). The most promising virus, called rA2cp2484041030delSH, was safe and immunogenic in young infants. However, approximately one-third of the vaccine virus isolates recovered from vaccinees had evidence of a partial loss of the temperature-sensitive (ts) phenotype, implying a partial loss of the attenuation (att) phenotype. Sequence analysis indicated that this was associated with reversion and loss of either the 248 or the 1030 point mutation (Tyr-1321-Asn and Gln-831-Leu, respectively) in the large polymerase L protein. Reversion can readily occur because each amino acid substitution is based on a single nucleotide substitution relative to wild type. We are presently working to genetically stabilize the 248 and 1030 mutations. Briefly, this involves constructing separate panels of viruses for each mutation representing all 20 possible amino acid assignments, in viruses that are completely sequenced to preclude adventitious mutations. The viruses are then analyzed for ts (in cell culture) and att (in mice) phenotypes, thus identifying the various assignments as att, intermediate, and wt. Deletions of 1 and 2 amino acids at each locus also were evaluated but were not recoverable. Taking the degeneracy of the genetic code into account, one then chooses an att assignment that differs from all possible wt-like assignments by as many nt as possible, on the premise that an att-to-wt reversion involving a change of 1 nt has a frequency of approximately 10(-4) (the viral mutation frequency) whereas the frequencies associated with changes of 2 or 3 nt would be 10(-8) and 10(-12), respectively. The analysis for the 248 and 1030 mutations is four-fifths complete. This should enable us to construct an improved version of rA2cp2484041030delSH with increased genetic and phenotypic stability. Since the att phenotype conferred by certain of the alternative assignments is greater or less than that conferred by the original mutant assignment, it may be possible to marginally increase or decrease the att phenotype if necessary. We also have been preparing additional attenuated versions of HRSV under conditions suitable for making clinical material for evaluation in humans. These involve deletion of the type I interferon (IFN) antagonist NS1 protein or the M2-2 protein that is involved in regulating viral RNA synthesis. A seed of the delM2-2 virus has been prepared and is presently being amplified to make clinical lot material. [unreadable] Pneumonia virus of mice (PVM) is a murine relative of HRSV with the same genome organization and array of encoded viral RNAs and proteins. It replicates efficiently in the respiratory tract of mice and causes severe respiratory tract disease. Thus, it provides a model for studying severe disease caused by an HRSV-like virus in a natural host that is easily manipulated and has extensive genetic and immunological reagents. We developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Remarkably, deletion of the cytoplasmic tail of the envelope G protein resulted in a virus (called Gt) that retained the ability to replicate in mice as efficiently as its wild type parent but was completely non-pathogenic at doses that otherwise would be lethal. Thus, attenuation was not due to a reduction in virus load. The mechanistic basis of attenuation is under investigation. We presently are constructing HRSV and human metapneumovirus versions of the Gt mutant to see if this phenotype can be recreated. This would be desirable for vaccine development since it would provide a virus that is attenuated but retains a high level of virus replication and hence a high level of antigen production and immunogenicity. [unreadable] We deleted the nonstructural NS1 and NS2 protein genes individually and in combination from PVM. Deletion of NS1 resulted in a virus that was substantially attenuated in mice, identifying NS1 as a virulence factor whose mechanistic basis is unknown. Deletion of NS2 was highly attenuating, an effect that was associated with the loss of the ability of PVM to suppress the host IFN response. Wild type PVM replicated efficiently and increased over a 6-day period, resulting in a strong up-regulation of IFN and a wide array of representative pro-inflammatory cytokines/chemokines and T cell-related cytokines and the appearance of overt disease on day 6. This also was observed for the &#8710;NS1 mutant, although disease was substantially less. In contrast, virus lacking NS2 was modestly attenuated for replication on day 3 concurrent with an early up-regulation of pulmonary IFN and CXCL10 (IP-10). By day 6, the viral titer was declining, the expression of IFN and CXCL10 was returning to baseline, and no other cytokines or chemokines were induced. These results provide evidence that severe PMV disease is associated with high and seemingly poorly-controlled virus replication driving the expression of high levels of pulmonary type I IFN and an array of cytokines/chemokines. In contrast, in the absence of NS2, there was an early innate response involving moderate levels of IFN and CXCL10 that restricted virus replication early and prevented disease. This illustrated protective versus pathogenic host responses. [unreadable] Dendritic cells (DC) are potent antigen presenting cells that play a major role in initiating and modulating the immune response. We compared the effects of HRSV on human monocyte-derived DC in a side-by-side comparison with HMPV and HPIV3 using GFP-expressing viruses. Previous studies have tended to highlight apparently dramatic findings, such as the preliminary finding that HRSV-inoculated DC were highly deficient in activating CD4+ T cells or that HMPV seemed to induce poor DC maturation. We could not confirm these dramatic effects. We found that all three viruses infected DCs poorly (whereas a Newcastle disease virus-GFP control infected very efficiently) with only a few percent of cells being GFP+ and the remainder having low, abortive levels of viral RNA synthesis. Low infectivity and low intracellular antigen synthesis of these viruses likely reduces activation of CD8+ T cells important for host defense. The three viruses induced low-to-moderate maturation of DC and cytokine/chemokine responses, which also might reduce the immunological footprint of theses viruses. Infection at the individual cell level tended to be relatively benign, such that GFP+ cells were neither more nor less able to mature compared to GFP- bystanders. However, HPIV3 infection did down-regulate expression of CD38, an effect that was at the RNA level. We are presently evaluating the ability of DC inoculated with HRSV, HMPV, HPIV3, or influenza A virus to activate autologous CD4+ T cells. [unreadable] We also analyzed the maturation of DC following exposure to HRSV lacking the NS1 or NS2 genes. Deletion of NS1, whose major effect (in HRSV) is to block IFN induction, resulted in a substantial increase in the level of maturation and cytokine/chemokine expression, effects that were somewhat augmented by deletion of NS2. This up-regulation was largely abrogated by pre-treatment with a blocking antibody against the type I IFN receptor. Thus, maturation of human DC in response to HRSV infection is dependent in large part on secreted type I IFN and is partially suppressed by the IFN antagonists encoded by the virus. While HRSV does not appear to directly impair the ability of the DC to mature (as noted above), it inhibits maturation indirectly by blocking production and action of this key maturation stimulus.