A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52͞ 2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription-PCRamplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.Respiratory syncytial virus (RSV), the leading cause of severe viral respiratory illness in pediatric populations throughout the world (reviewed in ref. 1), accounts for approximately 90,000 hospitalizations in infants and children in the United States each year (2). The importance of RSV as a respiratory pathogen makes development of a safe and effective RSV vaccine a public health priority (3). Although a number of approaches to RSV vaccine development have been taken, live RSV vaccines may provide the best alternative for immunizing young infants, because a live vaccine would mimic natural infection, induce a balanced cellular and humoral immune response, and be unlikely to produce enhanced disease (4).RSV exists as two antigenically distinct subgroups, A and B, and both RSV A and RSV B infections are capable of inducing severe lower respiratory tract disease (5-7). For this reason, a bivalent live RSV vaccine containing attenuated RSV A and RSV B components would be most desirable. Recently, a live attenuated RSV A candidate vaccine has been identified that appears to be safe and immunogenic in infants and children over 6 months of age (8). In addition, a cold-passaged (cp) RSV B candidate vaccine, designated RSV B1 cp-52͞2B5 (cp-52), was derived by passage of the RSV B1 wild-type (wt) virus 52 times at low temperature (21-32°C) (9). Cp-52 was shown to be restricted in replication in vivo but still able to induce RSV serum-neutralizing antibody responses in cotton rats, African green monkeys, and chimpanzees (9). Also, it was found to be phenotypically stable after prolonged replication in cotton rats (9). Here, we describe the phase I evaluation of the cp-52 candidate vaccine in adults, children, and infants. Although this virus mutant grew to high titer (Ͼ10 7....

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.

A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%v. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100o, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%v.