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Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults.

Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.

Acute Disease↗

[Rotavirus diarrhea. Impact in a pediatric hospital of Buenos Aires].

Anticipating the use of the rotavirus vaccine, we performed this study in order to estimate the rotavirus disease burden in a pediatric hospital. We studied 648 children < 3 years of age between September/97 and August/98, assisted at the Outpatient Diarrhea Unit, or hospitalized due to acute diarrhea in our Hospital. We found rotavirus associated to 36% of the diarrhea cases studied in the Outpatient Clinics, and in 45% of the hospitalized children. We estimate the assistance of 1674 rotavirus diarrheas per year in the Outpatient Clinics, but only 14 of them required hospitalization. The study describes a peak of rotavirus diarrheas between March and June, and another peak of rotavirus-negative diarrheas between January and March (probably due to bacterial diarrheas). Rotavirus disease presented a higher frequency between 6 to 23 months of age; only 10% of the 233 rotavirus cases occurred in children older than 24 months and 13% in infants less than 6 months of age. The situation described is significant because the recently licensed rotavirus vaccine is being used in 3 doses at 2, 4 and 6 months and could have prevented most of the rotavirus cases observed during this study.

Acute Disease↗

[Nosocomial infections due to rotavirus and respiratory syncytial virus in pediatric wards: a 2-year study].

Rotavirus and respiratory syncytial virus (RSV) infections represent up to 30% of the totality of nosocomial infections in paediatric wards. We studied the importance of these infections in the paediatric wards of the University Hospital Center of Poitiers, France, from October 1996 to September 1998. We defined as nosocomial an infection acquired after 3 days of hospitalization for rotavirus and after 7 days for RSV. The 274 cases of children presenting rotavirus gastroenteritis or RSV infection within this period were studied. Rotavirus was detected in stools by using an agglutination test and RSV was diagnosed in nasopharyngeal aspirations by direct examination with an immunofluorescence assay (IFA), cell culture and serotyping with IFA. We noted 50 rotavirus and 224 RSV infections, with a first epidemic of RSV subgroup B (49.5%) and a second epidemic of subgroup A (44.9%). 19 (38%) were rotavirus nosocomial infections and 5 (2.2%) were RSV nosocomial infections. The majority of the nosocomial infections occurred before the age of one year and particularly before the age of 6 months (42.2% for rotavirus, 60% for RSV). In comparison to community-acquired infections, children with rotavirus nosocomial infections were younger (9 months versus 12.5 months) which was the opposite for RSV nosocomial infections (10.8 months versus 6.5 months). The sex-ratio of children with community-acquired infections was 2.1 that was not reported in nosocomial infections. The length of stay in hospital was always longer in nosocomial infections (11.7 days versus 3.6 days for rotavirus; 38.8 days versus 4.8 days for RSV). Diarrhea (p = 0.007) and vomiting (p = 0.013) for enteric infections and wheezing (p = 0.02) for respiratory infections were more often observed in community-acquired infections. This study emphasizes the frequency and the consequences of rotavirus and RSV nosocomial infections in paediatric wards and the importance of the hygienic rules to prevent these infections.

Age Factors↗

[Epidemiological study of rotavirus diarrhea in Beijing, China - a hospital-based surveillance from 1998 - 2001].

OBJECTIVE: To provide information on epidemiology of rotavirus infection in Beijing, China. METHODS: An ongoing hospital-based surveillance was conducted among children < 5yr old with acute diarrhea according to WHO generic protocol (CID-98). During a 3-year study (Apr. 1998 to Mar. 2001), a total of 484 stool samples were collected from 1 457 patients, including 275 samples from 1 048 outpatients and 209 samples from 409 inpatients. RESULTS: The overall detection rate of rotavirus infection was 25.4%. Rotavirus was responsible for 27.3% of diarrhea inpatients on a yearly base, and 46.2% during rotavirus season. Two peaks of diarrhea were observed each year, one in the summer (June-Sep.) due to bacterial dysentery (16.7%) and another in fall winter (Oct.-Dec.) due to rotavirus infection (23.0%). The detection rate on rotavirus was the highest in age group of 6 - 11 months (38.2%), followed by 1 - 2 years old (28.5%). Ninety six point eight percentage of children were infected under 3 years of age. The number of deaths, possibly caused by rotavirus diarrhea were accounted for 40% of all diarrhea deaths and 11.1% of the total deaths. Serotyping of 123 rotavirus isolates showed that serotype G1 (55.3%) was predominant, followed by G2 (26.8%), G3 (9.8%), G4 (0.8%), and 10 isolates (8.1%) remained non-typeable. Mixed infections (0.8%) seemed to be rare. CONCLUSION: Rotavirus diarrhea was an important infectious disease among children in Beijing. Safe and effective rotavirus vaccines for the prevention of severe diarrheas and the reduction of treatment costs are of significant importance to China.

Age Factors↗

Immune response of pregnant cows to bovine rotavirus immunization.

Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the group II cows (ELISA IgG1 GMT = 39; VN GMT = 33) or group III cows (ELISA IgG1 GMT = 21; VN GMT = 19). These results indicate that IM plus IMm immunization of pregnant cows, using modified-live bovine rotavirus with added adjuvant, may significantly enhance serum, colostrum, and milk rotavirus antibody titers, whereas IM vaccinal inoculation of pregnant cows with a commercial modified-live rotavirus-coronavirus vaccine may not.

Adjuvants, Immunologic↗

Rotavirus excretion in suckling pigs followed under field circumstances.

The enzootic behaviour of porcine rotavirus in swine breeding farms was investigated by determination of the rotavirus excretion pattern in faeces using an enzyme linked immunosorbent assay (ELISA). Three herds were followed for 23, 14 and 9 consecutive weeks respectively and faeces were collected once a week from suckling piglets, weaned pigs and sows. The piglets of nearly all litters were found to excrete rotavirus in faeces before the age of 5-6 weeks. Weaned pigs rarely excreted rotavirus and faeces of sows were always negative. About half of the litters excreted rotavirus during more than one week or showed a recurrent excretion period 3-4 weeks after a first one. The presence of rotavirus group-specific antibodies in milk of sows, as detected by ELISA, did not appear to prevent infection of their piglets. These data may be an indication of the presence of serologically different porcine rotavirus strains within the same herd. Subclinical rotavirus infection occurred often in suckling pigs of all ages. Rotavirus was also found in a high number of fecal samples from pigs with diarrhea, but it was impossible to demonstrate the irrefutable association between the rotavirus infection and the presence of diarrhea.

Animal Population Groups↗

Laboratory-based surveillance for rotavirus--United States, July 1997-June 1998.

Rotavirus infections are the leading cause of severe gastroenteritis among infants and young children worldwide. Each year in the United States, rotavirus causes an estimated 2.7 million cases of gastroenteritis among children aged <5 years, resulting in approximately 500,000 outpatient clinic and emergency department visits and 49,000 hospitalizations. In addition, rotavirus accounts for an estimated $264 million in health-care costs and approximately $1 billion in total medical and nonmedical costs. The large disease burden and cost associated with rotavirus have led to the development of rotavirus vaccines. In August 1998, the first live attenuated rotavirus vaccine (Rotashield [Wyeth Lederle Vaccines and Pediatrics]) was approved for use in infants by the Food and Drug Administration. The Advisory Committee on Immunization Practices has recommended that this vaccine be given as a three-dose schedule to infants aged 2, 4, and 6 months. Since 1991, rotavirus activity in the United States has been prospectively monitored by the National Respiratory and Enteric Virus Surveillance System (NREVSS), a voluntary, laboratory-based system. This report summarizes surveillance data from NREVSS during the 1997-1998 rotavirus season and reviews issues related to rotavirus surveillance that are important for a national rotavirus vaccine program.

Clinical Laboratory Techniques↗

Rotavirus vaccine and the news media, 1987-2001.

CONTEXT: In August 1998, the US Food and Drug Administration licensed the first vaccine against rotavirus, the most important cause of severe childhood diarrhea. Fourteen months later, amid intense media activity, the vaccine was withdrawn after an association was found with intussusception. OBJECTIVES: To examine the character of news media stories about rotavirus vaccine before and after intussusception became an issue, to evaluate what prompted the stories, and to assess the extent to which they evoked public reaction. DESIGN AND SETTING: We searched Lexis-Nexis and Video Monitoring Services of America databases for rotavirus vaccine stories from the first US clinical trials (January 1, 1987) until 17 months after withdrawal (March 31, 2001) and examined calls to the National Immunization Hotline during the period in which rotavirus vaccine information was captured (July 1-December 31, 1999). MAIN OUTCOME MEASURES: Mention of vaccine benefits and adverse events, classification of stories as positive, negative, or neutral toward the vaccine, story stimuli, and public response. RESULTS: We included 280 newspaper (primary subject of analysis), 49 wire service, and 257 television stories. Prior to identification of the intussusception association (January 1, 1987-July 14, 1999), 21% of 188 newspaper stories mentioned vaccine adverse events and only 2 stories were negative toward the vaccine. Ninety-nine percent of stories mentioned vaccine benefits. During the period surrounding withdrawal (July 15-December 31, 1999), 93% of 90 stories mentioned adverse events and 77% were negative toward the vaccine. Eighty-four percent mentioned vaccine benefits. The rate of stories per month was 14-fold greater than the preceding period (P<.001); temporal and geographic patterns of media and hotline activity were similar. Thereafter (January 1, 2000-March 31, 2001), only 2 stories focused on rotavirus vaccine. Scientific research or public health actions prompted 80% of stories. Wire service and television stories showed similar patterns. The increase in rotavirus stories in July 1999 was followed by an increase in calls to the National Immunization Hotline regarding rotavirus but not other topics. The number of rotavirus calls that month was 57% higher than for any other childhood vaccine for any month since the hotline began in 1997. Rotavirus calls ceased almost completely after withdrawal of the vaccine in October 1999. CONCLUSIONS: In response to reports about an adverse event, news media stories about vaccines can change abruptly from positivity to negativity. Since most vaccine stories may be stimulated by research and public health actions, opportunities exist to provide the media with accurate information necessary to avoid the "early idealization-sudden condemnation" pattern seen with rotavirus vaccine.

Hotlines↗

Growth of group A rotaviruses in a human liver cell line.

Recent observations in children with rotavirus gastroenteritis and in infant mice given rotavirus vaccine by oral administration suggest that this well-known gastrointestinal pathogen may infect the liver. To examine this possibility, the susceptibility of Hep G2 cells to infection with a variety of rotavirus strains was tested. These cells were used because they are considered to be well differentiated and exhibit many liver-specific functions. The Hep G2 cells supported the growth of the simian strain rhesus rotavirus (MMU 18006), a strain currently being used in vaccine trails, but did not support the growth of any human strain (D, DS1, Price or ST3). The rhesus rotavirus infection was cytopathic and resulted in release of lactate dehydrogenase. Rhesus rotavirus growth in Hep G2 cells displayed trypsin-enhanced infectivity and was inhibited by pretreatment of cells with Arthrobacter ureafaciens neuraminidase but not with neuraminidase from Clostridium perfringens. Hep G2 cells were also permissive for another simian strain (SA11), a bovine strain (UK) and single gene substitution reassortants containing VP7 (the major outer capsid neutralization protein) from a human rotavirus strain and the remaining 10 genes from either rhesus rotavirus or UK. In general, UK and its reassortants produced lower levels of antigen than did rhesus rotavirus and its reassortants. Hep G2 cells and other hepatic cell lines may prove to be useful tools to explore the hepatotropic potential of wild-type rotaviruses and candidate vaccine strains.

Antigens, Viral↗

Entry of rotaviruses is a multistep process.

The infection of epithelial cells by some animal rotavirus strains requires the presence of sialic acid (SA) on the cell surface. Recently, we isolated rhesus rotavirus variants, named nar, whose infectivity, like that of human rotaviruses, is not dependent on SA. In this work, we have determined the binding properties of these SA-dependent and -independent rotavirus strains to MA104 cells. The half-time of attachment of the SA-dependent porcine rotavirus YM and reassortant virus DS1xRRV was found to be about 10 times longer in neuraminidase-treated cells than in untreated cells. On the other hand, human rotaviruses Wa and DS1, and the variant nar3, bound to cells two to three times more rapidly in the absence of SA. To investigate whether the SA-independent cellular structure recognized by the variant and human rotaviruses was the same, we used an infection assay designed to detect competition for cell surface molecules at both attachment and post-attachment steps. In this assay, human rotavirus Wa efficiently competed the infectivity of YM in untreated cells and that of the variant nar3 in untreated, as well as neuraminidase-treated, cells. This competition was nonreciprocal, since YM and nar3 did not compete, but rather increased three- to fivefold the infectivity of Wa. In contrast, a two-direction competition between the variant nar3 and DS1xRRV was found. Similar results were obtained when psoralen-inactivated viruses were used as competitors, indicating that the competition observed was during the early stages of infection. Altogether, these results suggest the existence of multiple interactions between rotaviruses and the cell surface and revealed the existence of common steps during the entry of human and animal rotavirus strains.

Adsorption↗

Comparative studies of the pathogenesis, antibody immune responses, and homologous protection to porcine and human rotaviruses in gnotobiotic piglets.

Gnotobiotic piglets serve as a useful animal model for studies of rotavirus pathogenesis and immunity. An advantage over laboratory animal models is the prolonged susceptibility of piglets to rotavirus-induced disease, permitting an analysis of cross-protection and active immunity. Studies from our laboratory of the pathogenesis of human rotavirus infections in gnotobiotic piglets have confirmed that villous atrophy is induced in piglets given virulent but not attenuated human rotavirus (Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. To facilitate and improve rotavirus vaccination strategies, it is important to identify correlates of protective immunity. Comparison of antibody immune responses induced by infection with virulent porcine and human rotaviruses (mimic host response to natural infection) with those induced by live attenuated human rotavirus (mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Our results indicate that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the site of viral replication (small intestine). Secondly there was a direct association between the degree of protection induced and the level of the intestinal immune response, with primary exposure to virulent rotaviruses inducing significantly higher numbers of IgA ASC and complete protection against challenge. These studies thus have established basic parameters related to immune protection in the piglet model of rotavirus-induced disease, verifying the usefulness of this model to apply new strategies for the design and improvement of rotavirus vaccines.

Animals↗

Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies.

Monoclonal antibodies (MAbs) were prepared to analyze antigens on the major inner capsid protein, VP6 of avian group A rotavirus. Based on the results of a competitive binding assay using 15 MAbs directed against VP6 of the PO-13 rotavirus strain, isolated from a pigeon in Japan, it was found that VP6 of avian rotavirus possesses at least four spatially distinct antigenic sites. Two antigenic sites (I and II) were topologically distinct from the other two (III and IV), which were in close proximity. From the reaction of MAbs in indirect immunofluorescent antibody tests to a series of known rotaviruses, epitopes representing common antigens of all group A rotavirus including avian rotavirus were localized in sites II and III. One epitope in site IV appeared to have a subgroup antigenic specificity that reacted only with rotaviruses belonging to subgroup I. Interestingly, avian rotaviruses isolated from turkeys and chickens in Northern Ireland also reacted only with these subgroup I specific MAbs, but not with subgroup II specific MAb. This indicates that avian rotavirus has subgroup I specific antigen, which is antigenically similar to that of other mammalian rotavirus strains.

Animals↗

Influence of immunomodulatory agents on bovine humoral and cellular immune responses to parenteral inoculation with bovine rotavirus vaccines.

Sodium diethyldithiocarbamate (DTC), mycobacterium cell wall extract (MCWE, Regressin), killed Corynebacterium parvum (C. parvum, Immunoregulin) and muramyldipeptide (MDP) were each combined with purified, live bovine rotavirus and inoculated into 3 month-old Holstein-Friesian calves in order to examine their ability to potentiate specific humoral and cellular immune responses. The vaccinated calves were boosted twice at 3 and 6 weeks after initial vaccine inoculation. The rotavirus was administered intramuscularly either in an aqueous suspension or in a water-in-oil (WIO) emulsion, prepared with incomplete Freund's adjuvant (IFA). DTC and C. parvum were given by the intravenous route, while MCWE and MDP were incorporated directly in the rotavirus suspension. Two groups of calves were also vaccinated either with rotavirus and IFA or with rotavirus emulsified in mineral oil and a mannide oleate compound (MOC, Montanide 888). A control group of calves was given phosphate-buffered saline (PBS) solution emulsified with IFA. The different vaccine preparations were then compared by studying the kinetics of serum rotavirus-neutralizing antibody production and of proliferative response by blood lymphocytes following in vitro stimulation with bovine rotavirus. The results showed that: (1) the bovine rotavirus should be incorporated in a WIO emulsion in order to induce a cell-mediated immune response as detected by the rotavirus-specific in vitro stimulation test with blood lymphocytes, and to produce higher neutralizing antibody titers in the serum; (2) the vaccines prepared with the mineral oil-MOC complex or IFA both induced comparable levels of humoral and cellular immune responses. The use of mineral oil and MOC as adjuvant may be preferred to IFA, because of the facility of preparing the vaccine and of the low viscosity of the resulting WIO emulsion: (3) the addition of MDP to the WIO emulsion prepared with IFA resulted in a higher cell-mediated immune response as determined by the in vitro blood lymphocyte transformation index specific for bovine rotavirus.

Acetylmuramyl-Alanyl-Isoglutamine↗

Rotavirus infection in children in Japan.

Currently, a high morbidity of rotavirus diarrhea has been seen in children in developed and developing countries. Improvement of the vaccines is necessary in order to reduce the burden of diarrhea caused by rotavirus. A survey of rotavirus infection from diarrheal stool specimens in children of seven regions in Japan was conducted from 1984 to 1999. The present study discusses the survey results and reviews the national and international data of more than 23 papers and congress proceedings about rotavirus infection in Japan. We analyze the prevalence of rotavirus infection in acute diarrheal in- and outpatients, the distribution of rotavirus G-serotypes and surveillance data for seasonality and age groups in Japan. The data indicated that rotavirus is the most important cause of diarrhea in Japan among young children, with the prevalence ranging from approximately 9.7 to 88%. The most common rotavirus strains belonged to serotype G1, specifically since 1993. Serotypes G2, G3 and G4 had also been documented to be predominantly based in the area and year before 1992. However, untypeable rotavirus strains had been found each year, with a prevalence up to 56.7% which suggests that rare serotypes (except G1-4) or new serotypes might exist. Unexpectedly, in Tokyo and Sapporo from 1998 to 1999, G9 was found to be the first most prevailing serotype with a high prevalence of 52.9 and 71.4%, respectively. Despite these data from different geographic areas, the year under investigation was relatively clear in respect to seasonality, with a peak of rotavirus activity in late winter (February) through early spring (March). Age distribution had also characterized that the infection was predominant among children aged 1-2 years of age, although it was also common in children of 2-3 years. In addition, mixed infection with bacteria was documented.

Adolescent↗

Epidemiologic features of rotavirus infection in Taiwan: a review.

Group A rotaviruses are a major cause of severe gastroenteritis in children under 4 years of age worldwide. Group A rotaviruses have been identified in many animal and bird species; they are antigenically complex, and multiple serotypes infect humans. Reassortant rotavirus vaccines are now available that confer protection against severe illness due to rotavirus serotypes G1-4. Before vaccines are introduced, it is necessary to establish their efficacy and to establish a baseline for future surveillance strategies. The purpose of the present review is to describe current knowledge of the diversity of rotavirus in Taiwan. The present review is based on the available published studies and data from China Medical College Hospital (Taiwan). In Taiwan, rotavirus has been detected year-round, with the epidemic peak swinging from November-December in 1984 to January-March in 1988-95 and then back to December-March in 1996-99. Most affected patients are under 2 years of age. Only group A rotavirus has been found. Neither group B nor group C rotavirus have been identified. G1 strains have been the predominant strains except for the year 1992-93, when G2 strains were prevalent. There has been no G4 strain detected in published studies. The electropherotype 'baba' has been observed every year. Five other electropherotypes have been sporadically found. Half of the strains that have been studied were genotype P[8] and their VP7 serotype was G1 or G3. All genotype P[4] strains have shown serotype G2. It is expected that the reassortant rotavirus vaccine will be effective in prevention of severe illness caused by rotavirus in Taiwan.

Adolescent↗

Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region.

cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.

Animals↗

VP4 differentially regulates TRAF2 signaling, disengaging JNK activation while directing NF-kappa B to effect rotavirus-specific cellular responses.

Rotaviruses rapidly activate NF-kappaB and induce the secretion of selected chemokines after infection. The ability of rotavirus particles lacking genomic RNA to activate NF-kappaB suggested that rotavirus proteins direct cell signaling responses. We identified conserved TNFR-associated factor (TRAF) binding motifs within the rotavirus capsid protein VP4 and its N-terminal VP8* cleavage product. TRAFs (-1, -2, and -3) are bound by the rhesus rotavirus VP8* protein through three discrete TRAF binding domains. Expression of VP4 or VP8* from rhesus or human rotaviruses induced a 5-7-fold increase in NF-kappaB activity and synergistically enhanced TRAF2-mediated NF-kappaB activation. Mutagenesis of VP8* TRAF binding motifs abolished VP8* binding to TRAFs and the ability of the protein to activate NF-kappaB. Expression of pathway-specific dominant negative (DN) inhibitors DN-TRAF2 or DN-NF-kappaB-inducing kinase also abolished VP8*-, VP4-, or rotavirus-mediated NF-kappaB activation. These findings demonstrate that rotavirus primarily activates NF-kappaB through a TRAF2-NF-kappaB-inducing kinase signaling pathway and that VP4 and VP8* proteins direct pathway activation through interactions with cellular TRAFs. In contrast, transcriptional responses from AP-1 reporters were inhibited 5-fold by VP8* and were not activated by rotavirus infection, suggesting the differential regulation of TRAF2 signaling responses by VP8*. VP8* blocked JNK activation directed by TRAF2 or TRAF5 but had no effect on JNK activation directed by TRAF6 or MEKK1. This establishes that fully cytoplasmic rotaviruses selectively engage signaling pathways, which regulate cellular transcriptional responses. These findings also demonstrate that TRAF2 interactions can disengage JNK signaling from NF-kappaB activation and thereby provide a new means for TRAF2 interactions to determine pathway-specific responses.

Animals↗

Rotavirus gastro-enteritis in hospitalized children with acute diarrhoea in Zambia.

The clinical and epidemiological aspects of rotavirus diarrhoea were studied in hospitalized children with acute diarrhoea in Lusaka, Zambia. Two hundred and fifty-six (24.0%) of 1069 children admitted to the study were shedding rotavirus. The rotavirus-positive rate was highest in children less than 1 year of age (37.0%) and it was also high in those less than 6 months old. Rotavirus diarrhoea was seen throughout the year with a higher rotavirus-positive rate in the dry season. In rotavirus-positive diarrhoea patients, more children were dehydrated (82.4%) than in the rotavirus-negative group (56.2%). Rotavirus infection was more common in the children with normal nutritional status (27.6%, 162/588) than in those with malnutrition (19.3%, 93/482). The associated case fatality rate in the rotavirus-positive group was 6.4%, significantly less than in the rotavirus-negative group (OR 0.44, 95% CI 0.24-0.79), and mortality cases were seen only in children less than 2 years old.

Acute Disease↗