An unpassaged, safety-tested strain (CJN) of human rotavirus from a stool specimen of a hospitalized child was administered orally to 62 adult volunteers for determination of the dose required to produce infection with or without illness. Subjects ingested doses ranging from 9 X 10(-3) to 9 X 10(4) focus-forming units in buffered salt solution after consumption of 50 ml of 4% NaHCO3. The amount of virus in the inoculum required to cause infection (shedding of virus, seroconversion, or both) in study subjects was comparable to the minimum detectable in cultures of primary monkey kidney cells. Seventeen of 30 infected subjects became ill with doses equivalent to that required for infection. Although the preinoculation titers of serum neutralizing antibody to the challenge virus in study subjects ranged from less than 1:2 to 1:1,600, the concentration of serum antibody could not be correlated with protection from infection or illness in subjects given an infectious dose of virus.

A large placebo-controlled efficacy trial of the rhesus tetravalent (RRV-TV) and serotype G1 monovalent (RRV-S1) rotavirus vaccines was conducted in 1991-1992 at 24 sites across the United States. Protection was 49% and 54% against all diarrhea but 80% and 69% against very severe gastroenteritis for the two vaccines, respectively. Post-vaccination neutralizing antibody titers to the G1 Wa strain, whose VP7 protein is nearly identical to that of the D strain of rotavirus contained in both vaccines, did not correlate with protection against subsequent illness with G1 strains. This result raised the possibility that in infants who developed post-vaccination neutralizing antibody to Wa, breakthrough (i.e., vaccine failure-the occurrence of rotavirus diarrhea after immunization) may have been due to infection by G1 strains that were sufficiently antigenically distinct from the vaccine strain to evade the neutralizing antibodies elicited by vaccination. To test this hypothesis, we initially compared post-vaccination neutralizing antibody titers of vaccinees against Wa and G1 breakthrough strains using sera from subjects who experienced breakthrough. Post-immunization neutralizing antibody titers to Wa elicited by vaccination were significantly (P< 0.001) greater than to the breakthrough strains subsequently obtained from these subjects. This difference did not, however, correlate with lack of protection since similar differences in titer to Wa and breakthrough strains were found using post-vaccination sera from vaccinees who either experienced asymptomatic rotavirus infections or no infections. To determine the genetic basis for these differences, we compared the VP7 gene sequences of Wa with vaccine strain D, 12 G1 breakthrough strains, and 3 G1 control strains isolated during the same trial from placebo recipients. All breakthrough strains were distinct from Wa and D in antigenically important regions throughout the VP7 protein, but these differences were conserved between breakthrough and placebo strains. Furthermore, a comparative analysis of the deduced amino sequences form VP7 genes of G1 rotaviruses from 12 countries indicated that four distinct lineages have evolved. All breakthrough and control strains from the U.S. vaccine trial were in a lineage different from strain D, the serotype G1 vaccine strain. Although the overall results do not support our original hypothesis that immune selection of antigenically distinct escape mutants led to vaccine breakthrough in subjects with a neutralization response to Wa, it cannot be excluded that breakthrough could be partially due to antigenic differences in the VP7 proteins of currently circulating G1 strains.

This study was designed to find methods to reproducibly propagate human rotaviruses from fecal specimens and to determine the relationship between particle numbers and infectivity. Growth of virus was initially compared in primary and continuous lines of monkey kidney cells. Primary cells (African green and cynomolgus monkey kidney) supported virus growth directly from fecal specimens much more efficiently than did continuous lines of African green (CV-1) or rhesus (MA104) monkey kidney cells. Rotaviruses were grown in primary cells from 14 of 14 fecal specimens of different individuals collected over a 3-year period. Although rotaviruses in fecal samples could not always be grown in the continuous cell lines, two passages in primary cells appeared to fully adapt the viruses for propagation in the continuous cell line tested (MA104). The efficiency of rotavirus growth was quantified with five of the fecal isolates. It was calculated that, on the average, 1 out of every 46,000 particles in fecal specimens infected monkey kidney cells. After three passages in primary cells, an average of 1 out of every 6,600 progeny virus particles appeared to be infectious. Thus, rotaviruses in fecal specimens were consistently grown in primary cells, and passage in these cells both increased virus infectivity and adapted the viruses for growth in continuous cell lines.

Of 335 rotavirus isolates associated with diarrheal disease in Bangladesh that were culture adapted and subsequently characterized for electropherotype, subgroup, and serotype, 9 had properties that suggested they may be natural reassortants between human rotaviruses belonging to different "genogroups." Two of these were examined in greater detail by RNA-RNA hybridization with prototype strains representative of each of the three proposed human rotavirus genogroups. One subgroup II isolate, 248, with a "long" electrophoretic pattern was neutralized by hyperimmune antisera to both serotype 2 and 4 strains. Consistent with these results, seven RNA segments of this isolate formed hybrids with human strains belonging to the Wa genogroup and four segments hybridized with strains belonging to the DS-1 genogroup. The second isolate examined, 456, belonged to subgroup II and had a long electrophoretic pattern but was found to be a serotype 2 strain. This isolate also appeared to be an intergenogroup reassortant because three of its segments formed hybrids with strains belonging to the Wa genogroup and eight hybridized with viruses of the DS-1 genogroup. On the basis of the relative migration rates of these RNA-RNA hybrids during gel electrophoresis, a suggested origin for each gene segment was proposed which was consistent with the results expected from electrophoretic, subgroup, and serotypic analyses.

Fresh water obtained from nine sources was shown to cause inactivation of poliovirus. Further testing with four of these water samples showed that enteric viruses from different genera were consistently inactivated in these freshwater samples. Studies on the cause of inactivation were conducted with echovirus type 12 as the model virus. The results revealed that the virucidal agents in the waters tested could not be separated from microorganisms. Any treatment that removed or inactivated microorganisms caused loss of virucidal activity. Microbial growth in a sterilized creek water seeded with a small amount of stream water resulted in concomitant production of virucidal activity. When individual bacterial isolates obtained from a stream were grown in this sterilized creek water, most (22 of 27) produced a large amount of virucidal activity, although the amount varied from one isolate to the next. Active and inactive isolates were represented by both gram-positive and gram-negative organisms. Examination of echoviruses inactivated in stream water revealed that loss of infectivity first correlated with a slight decrease in the sedimentation coefficient of virus particles. The cause appeared to be cleavage of viral proteins, most notably, VP-4 and, to a lesser extent, VP-1. Viral RNA associated with particles was also cleaved but the rate was slower than loss of infectivity. These results suggest that proteolytic bacterial enzymes inactivate echovirus particles in fresh water by cleavage of viral proteins, thus exposing the viral RNA to nuclease digestion.