We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydrodroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.

Atypical rotavirus obtained from fecal specimens of six patients with diarrhea from Thailand, Nepal, and England were characterized by using polyacrylamide gel electrophoresis and immune electron microscopy. The electropherotypes were characteristic of the porcine reference group C rotavirus strain but demonstrated considerable strain-to-strain variation. Human convalescent group C sera had a high titer (1:320) when tested against the human isolates and a low titer (1:40) when tested against a porcine reference strain (Cowden). When porcine antiserum (Cowden) was tested against the human isolates, the titers ranged from 1:40 to 1:320, indicating significant antigenic diversity between strains. Group C rotavirus appears to have a worldwide distribution as an agent associated with diarrhea in children and adults.

Rotavirus diarrhea in 453 pediatric patients (29.8% of 1,518) was studied in greater Bangkok during 1985 to 1987. The disease persisted all year, increasing in incidence from August to January (30 to 50%). Polyacrylamide gel electrophoresis of rotavirus RNA from these patients and from an additional 46 patients of a 1982 to 1983 epidemic revealed 26 electropherotypes, 4 with short (S) and 22 with long (L) RNA profiles. Of the analyzed specimens, 85.5% were L forms. Only one or a few electropherotypes predominated in each epidemic, whereas others appeared sporadically at low frequencies. Shifts in the predominant electropherotypes were observed in every epidemic. Of these, 126 strains were tested for subgroup and serotype by monoclonal antibody enzyme immunoassay. Serotype 4 prevailed from 1982 to 1983, while serotype 1 was encountered more frequently than serotypes 2 and 4 from 1985 to 1987. A complete correlation was found between the electrophoretic migration of segments 10 and 11 and the serologically defined subgroup specificity. Distinct electropherotypes occurred within the same serotype, and strains with the identical electropherotype always showed the same serotype specificity. No specific electropherotype or serotype correlated with patient age. In this study, atypical rotaviruses and mixed infections with different rotaviruses were identified.

The replication of type 2 dengue (D-2) virus in Aedes albopictus (Aal) mosquito cell cultures differed from that in vertebrate (LLC-MK2) rhesus monkey kidney cells. Virus readily replicated in Aal cells at either 30 or 37 C, but had no apparent effect on the host cell. Persistent infection was established with continual virus production for at least 6 months, although the virulence of progeny virus for both suckling mice and LLC-MK2 cells became attenuated. Density gradient analysis of infected Aal cell supernatant products indicated that only complete virus was released, in contrast to infected LLC-MK2 cells which also released incomplete virus: The surface antigens of the virus produced in Aal cells appeared to be considerably modified in that antiserum to vertebrate cell-produced D-2 virus did not block hemagglutination, whereas anti-Aal cell antiserum did. Virus infectivity could be neutralized by the antiserum to D-2 virus grown in vertebrate cells, however. Virus produced in LLC-MK2 cells did not demonstrate a similar host-cell modification. These results may reflect a difference in the mechanism by which D-2 virus matures in Aal cells.

To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Revertants did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern. Viral pathogenesis involves not only the direct interaction of a virus particle with its host cell but also depends on a large number of known and unknown host factors in the whole animal. One approach in extending the knowledge obtained on the molecular level to mechanisms of pathogenesis is to establish infected cultures in such a way as to permit almost continuous monitoring ofviral and cellular populations over a period of time. Using a highly virulent strain of vesicular stomatitis virus (VSV) and diluting the infected culture daily with fresh uninfected cells, it is evident that, rather than continuous cell destruction, cells survive at some passages and not at others. In fact, virus production and cell survival alternate in a cyclic fashion-a balance which is maintained by the production ofdefective interfering (DI) particles in the viral population (11). The interfering ability, the dependence on helper standard virus, and the lack ofcytotoxicity are the essential properties of VSV DI particles that result in the cyclic nature of these interactions (see reference 5). ' Present address: