No abstract

The efficacy of a rhesus rotavirus vaccine (MMU 18006, serotype 3) against infantile diarrhea was evaluated by active home surveillance of a group of 320 children 1-10 months of age in Caracas, Venezuela. During a 1 year period following oral administration of vaccine or placebo under a double-masked code, over 600 diarrheal episodes were detected. Etiologic studies revealed that heat-stable toxin (ST) producing enterotoxigenic E. coli (ETEC) was the most common diarrheal agent detected (34%) followed by enteropathogenic E. coli (EPEC, 10.9%), heat-labile toxin (LT) producing ETEC (7.6%), rotavirus (6.9%), Cryptosporidium (4.8%) and Campylobacter (1.3%). ST-producing ETEC were also recovered from over 20% of control stool specimens obtained during diarrhea-free periods, whereas EPEC, rotavirus, Cryptosporidium, and Campylobacter were rarely detected in such control specimens. Rotavirus was responsible for about one-half of the more severe cases of diarrhea. Twenty-two of 151 infants who received placebo (14.6%) and eight of 151 receiving a 10(4) PFU dose of vaccine (5.3%) had rotavirus diarrhea during the follow-up period for an efficacy level of 64% against any rotavirus diarrhea. However, vaccine efficacy reached 90% against the more severe cases of rotavirus diarrhea and was noticeably high in the 1-4 month age group. Serotypic analysis of the rotaviruses detected suggests that the resistance induced by the vaccine was type specific since significant protection was only evident against serotype 3 rotaviruses. A 10(3) PFU dose tested initially in 18 children did not appear to protect against rotavirus diarrhea.

We have developed a hybridization assay that permits distinction of rotavirus serotypes 1, 2, 3, and 4. The serotype of rotaviruses from stool samples or tissue culture was recognized by hybridization of specific probes to (i) blots of viral double-stranded RNAs electrophoresed in agarose gels (Northern blots) or (ii) heatdenatured double-stranded RNAs directly dotted on nylon membranes. The probes consisted of 32P-labeled cDNA synthesized by reverse transcription of in vitro derived rotavirus mRNA from rotavirus serotypes 1 to 4. To prepare these probes, mRNAs were primed with a 17-mer nucleotide common to all four serotypes whose sequence is complementary to bases 375 to 391 of the rotavirus gene encoding the VP7 glycoprotein (gene 8 or 9 depending on the rotavirus strain). The resulting downstream transcripts encompassed areas of major sequence divergence among the four serotypes. Hybridization at high stringency (50°C, 50% formamide, 4x SSC [lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) was performed for 16 to 48 h. Autoradiograms of the washed membranes allowed recognition of the rotavirus serotype present in the blotted or dotted specimens since each of them hybridized preferentially to one of the four probes. Twenty-four laboratory specimens and 103 clinical specimens from Washington, D.C., Venezuela, and Chile were "serotyped" with this assay. The results were similar to those obtained with a monoclonal antibody serotyping assay.

The reactions to and antigenicity of two human-rhesus rotavirus (RRV) reassortants (human rotavirus strain D x RRV and human rotavirus strain DS1 x RRV) with the VP7 neutralization specificity of a serotype 1 or serotype 2 rotavirus were evaluated in a placebo-controlled double-blind trial in 116 1to 5-month-old infants in Caracas, Venezuela. The children were randomly divided into five groups to receive orally the following inocula: (i) 104 PFU of D x RRV reassortant; (ii) i04 PFU of DS1 x RRV reassortant; (iii) 1 PFU of RRV; (iv) 5 x 103 PFU of D x RRV and 5 x 103 PFU of RRV; and (v) placebo. The children were examined daily for 7 days following vaccine administration; 8 to 26% of the vaccinated infants developed a mild febrile reaction which in most cases lasted only 1 day. Seroresponses to rotavirus were observed in 39 to 65% of the vaccinees by plaque neutralization assay and in 57 to 88 % by an immunoglobulin A enzyme-linked immunosorbent assay. Vaccine shedding was detected in 53 to 86% of the vaccinees. Analysis of neutralization antibody responses indicates that the VP4 protein represents an important component of the response induced by the vaccines.

The rotavirus VP4 protein elicits the production of neutralizing antibodies and is known to play a role in inducing resistance to disease. At least five human rotavirus VP4 gene alleles have been described on the basis of antigenic polymorphism and/or nucleotide sequence differences. In the present study, we developed cDNA probes directed at the hyperdivergent region of the VP4 gene of the five described human rotavirus VP4 alleles (Wa, DS1, M37, AU228, and 69M) and used them in hybridization assays with human rotavirus strains from Latin America and Europe to determine the distribution of the VP4 gene alleles in nature. The Wa-like allele was detected most fiequently, occurring in 57% of the 402 rotavirus strains tested, and the DS1-like allele was the next most common, occurring in 14% of the strains tested. The M37-and AU228-like alleles were detected in only 4 and 3% of the rotavirus strains tested, respectively, whereas the 69M-like VP4 gene allele was not detected. Several rotavirus strains from Europe did not react with any of the VP4 gene probes, although they did hybridize to a probe generated from a representative strain from the group. These data indicate the global distribution of various VP4 gene alleles and raise the possibility that other, unrecognized human VP4 alleles exist in nature because almost one-fourth of the strains could not be classified into any of the established VP4 groups.

We have synthesized 32P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

We have used a recently developed monoclonal antibody enzyme-linked immunosorbent assay (K. Taniguchi, T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa, J. Infect. Dis. 155:1159-1166, 1987) for serotyping rotaviruses recovered from 134 Venezuelan infants over a period of 15 months. One hundred and nine of the specimens were typed with the following distribution: serotype 1, 48%; serotype 2, 16%; serotype 3, 22%; and serotype 4, 14%. Three specimens reacted with two different monoclonal antibodies. In addition, 6 specimens (5%) containing enough outer capsid antigen could not be typed; partial RNA sequence analysis of the glycoprotein gene from three of these six strains failed to reveal sequence differences with prototype strains that could be serotyped with the monoclonal antibodies. Variations in the recovery rates of the different serotypes were observed. Serotypes 2, 3, and 4 predominated at the beginning of the study, and serotype 1 predominated at the end of the study. Diarrheal illness appeared to be more prolonged in infants shedding rotavirus serotypes 1 and 3 than in those shedding serotypes 2 and 4.

The ability of p53 species (wild-type and mutant) to modulate the "differentiated" response of human hepatoma cell lines Hep3B and HepG2 to interleukin-6 (IL-6) was investigated. Transient transfection experiments were carried out in Hep3B and HepG2 cell cultures in which IL-6 was used to activate a ␤-fibrinogen (␤Fib) enhancer/reporter construct containing two copies of the 36-base pair IL-6-response element (IL-6RE) (p␤FibCAT). Cotransfection with constitutive expression vectors for wild-type (wt) human or murine p53 inhibited the activation of the p␤FibCAT reporter by IL-6 in both Hep3B and HepG2 cells. Several mutant p53 species either did not inhibit the activation of p␤FibCAT or up-regulated the response. Hepatoma cell lines stably expressing the Val-135 temperature-sensitive mutant of murine p53 (wt-like at 32.5°C and mutant-like at 37°C) were derived from Hep3B cells and tested for the temperature-sensitive phenotype of their ability to synthesize and secrete fibrinogen and ␣ 1 -antichymotrypsin in response to IL-6. In an experimental protocol in which the parental Hep3B cells did not show a significant difference in plasma protein secretion at the two temperatures, hepatoma line 3 (p53Val-135 ؉ ) had a greater response to IL-6 at 37°C than parental Hep3B cells, while line 3 cells had a reduced response to IL-6 at 32.5°C. Similarly, hepatoma lines 1 and 2 (both p53Val-135 ؉ ) had reduced IL-6 responsiveness at 32.5°C, whereas line 22 (transfected with pSVneo alone) and the parental Hep3B cells did not. These data indicate that mutations in p53 contained in tumor cells can modulate the "differentiated" response of these cells to cytokines.