A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal aspirates and discriminates subtypes A and B.

We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.

Epidemiology of rotavirus in infants and protection against symptomatic illness afforded by primary infection and vaccination.

This study assessed the frequency of symptomatic and asymptomatic primary and secondary infections with rotavirus in children under 24 months and determined protection against symptomatic illness afforded by rhesus and human-rhesus rotavirus reassortant vaccines. Successive cohorts of children (n 236) were followed through five winter rotavirus seasons with cultures of each reported episode of diarrheal disease and serologic determination of rotavirus exposure on paired sera bracketing the winter. An average of 46% of children experienced rotavirus infection in each season with almost all infected by two years of age. The relative risk of rotavirus associated gastroenteritis in naive children versus naturally immune children was 2.4 (1.1, 5.3). The relative risk of rotavirus associated gastroenteritis in naive children versus vaccinees was 4.1 (1.6, 10.7). In a community with predominantly serotype G1 rotavirus rhesus rotavirus-based vaccines are as protective against rotavirus gastroenteritis as prior natural infection.

Twenty years of outpatient respiratory syncytial virus infection: a framework for vaccine efficacy trials.

BACKGROUND: Respiratory syncytial virus (RSV) is the most important viral respiratory pathogen of infancy and childhood. Much has been written about inpatients with severe disease. Inpatients, however, represent only a minority of RSV-infected children. We studied the characteristics of symptomatic outpatient RSV infection in healthy children to gain a better understanding of RSV disease and to provide a background for the testing of intervention strategies in children without high-risk conditions. METHODS: A total of 1113 children were followed during 20 consecutive RSV seasons. Signs and symptoms of respiratory infection were monitored. Cultures were obtained for febrile upper respiratory infection, acute otitis media, and lower respiratory infection (LRI). Rates of febrile upper respiratory infection, acute otitis media, LRI, and hospitalization were calculated. Given those rates, numbers of children needed to demonstrate efficacy of a vaccine product were calculated. RESULTS: Mild disease from RSV infection lacked some of the classic features of RSV infection seen in hospitalized children. Involvement of the lower respiratory tract was, however, noted to be much higher in RSV infection than it was in infection with other viral respiratory pathogens. LRI was, therefore, considered the best candidate endpoint for vaccine trials. A product with 60% efficacy could be proven, with a power of 0.8, to be efficacious with as few as 1500 infants. CONCLUSIONS: RSV infection is common and often involves the lower respiratory tract, even in outpatients. Our 20-year study of RSV infection provides a basis for calculation of sample sizes to be used in trials of vaccine candidates.

Tetravalent rhesus rotavirus vaccine in young infants.

A study of a tetravalent live oral rhesus rotavirus vaccine was conducted in 26 healthy infants 6-22 weeks of age to evaluate safety, viral shedding, and immunogenicity in a double-blind, placebo-controlled fashion. The tetravalent rhesus rotavirus vaccine (RRV-TV) contained equal amounts of RRV (serotype 3) and the VP7 reassortants DxRRV (serotype 1), DS1xRRV (serotype 2), and ST3xRRV (serotype 4). RRV-TV was highly infectious; 16 of 18 recipients shed virus. However, the recovered virus was almost exclusively the parent rhesus strain, RRV. Humoral immune responses were largely limited to the RRV strain. The most consistent response, seen in 14 of 18 vaccines, was an ELISA IgM response to purified RRV. Mucosal antibody responses were not seen. The limited immune response may be a function of age, presence of maternal antibody, timing of the convalescent specimens, or the polyvalent nature of the vaccine.

Sequential administration of two human-rhesus rotavirus reassortant strains of VP7 serotype 1 and 2 specificity to infants and young children.

Rotavirus vaccine strains representing serotypes 1 and 2 have been derived by reassortment between genes of a rhesus rotavirus master strain, MMU18006, and the gene from human viruses coding for VP7, a surface protein with neutralizing determinants. As simultaneous administration of these two human rotavirus reassortants had resulted in disappointing strain-specific immunity, these two vaccines were administered sequentially to infants and young children to assess whether immunity to both serotypes could be achieved with two monovalent doses. Following initial immunization with RRVxDS-1, a serotype 2 vaccine, 12 of 13 shed virus and showed serologic responses to multiple rotavirus proteins. However, with subsequent administration of RRVxD, serotype 1, only 2 of 12 shed virus, and an enhancement or broadening of the immune response was not uniformly seen. Although rhesus rotavirus vaccines are immunogenic with primary administration of monovalent vaccine in seronegative children, and supplemental seroresponses are seen with a second dose, other strategies or new vaccine candidates must be sought to induce immunity against the multiple serotypes of human rotavirus.

Simultaneous administration of two human-rhesus rotavirus reassortant strains of VP7 serotype 1 and 2 specificity to infants and young children.

Two rotavirus vaccine strains representing VP7 serotypes 1 and 2 derived by reassortment between a rhesus rotavirus master strain, MMU18006, and either of two human rotavirus strains were administered simultaneously to infants and young children to assess potential interactions between strains. Children were observed in a day care setting for 10 days after vaccine administration for clinical symptoms, evidence of vaccine transmission, and patterns of viral shedding. Serum and local antibody responses were measured. The ratio of input virus strongly influenced the amount of each strain recovered from the child. Regardless of dose of virus administered, the neutralizing antibody response to the VP7 glycoprotein, the serotype determinant, was diminished in a bivalent preparation compared with a monovalent vaccine. Additional strategies must be sought to induce immunity against the multiple serotypes of human rotavirus before broad immunity will be established.

Evaluation of a reassortant rhesus rotavirus vaccine in young children.

A candidate live attenuated rotavirus vaccine representative of serotype 1 was administered orally to 26 children, 14 of whom were undergoing primary exposure to rotavirus. The vaccine was derived by reassortment between rhesus rotavirus strain, MMU 18006, and the human serotype 1 strain Wa. The resultant virus has the gene coding for the major surface glycoprotein VP-7 from the human strain and all other genes from the attenuated rhesus parent which is a serotype 3 strain. Prior natural exposure to rotavirus determined the infectivity and immunogenicity of the vaccine. Only two of 12 seropositive children had evidence of reinfection while all 14 seronegative children were infected. Mild febrile illness was seen in vaccinees, however there was no evidence of gastrointestinal disease. As determined by neutralization of the human strains, the resultant serum antibody was entirely strain specific. However, heterotypic neutralization was seen when the rhesus strains were used, suggesting that neutralizing antibody can be directed to shared components of the donor and reassortant strain presumably VP-4, the other major surface protein.