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PubMed1993

Murine intestinal mucins inhibit rotavirus infection.

BACKGROUND: Mucin, a population of polymeric glycoproteins, constitutes the primary component of the mucus layer that overlies the gastrointestinal tract. These studies aimed to determine whether murine intestinal mucins inhibit rotavirus infection. METHODS: Murine intestinal mucins were obtained by scraping segments of mouse intestine and purification via CsCl gradient centrifugation and sepharose 4B chromatography. Inhibition of infection was determined by quantitation of immunoperoxidase-stained cells after infection with mucin-rotavirus mixtures. RESULTS: Crude and purified intestinal mucins from suckling and adult mice are potent inhibitors of replication of a simian rotavirus, rhesus rotavirus (RRV), but weak inhibitors of other rotaviruses. In all preparations, colonic mucins were more potent inhibitors of RRV than small intestinal mucins. Suckling mucins neutralized RRV more effectively than adult mucins. In a panel of rotavirus reassortants, susceptibility to mucin inhibition correlated with the ability to hemagglutinate human type O erythrocytes and with RRV gene 4. Murine intestinal mucin inhibited RRV binding to MA104 cells, suggesting inhibition of virus-cell attachment to be the mechanism for neutralization. Mercaptoethanol or neuraminidase inhibited mucins' anti-RRV activities, implying the functional importance of mucins' polymeric structure and sialic acid content. CONCLUSIONS: These findings suggest that intestinal mucins represent a barrier to certain rotavirus infections.

Animals↗
PubMed1987

Variation in neutralization epitopes of human rotaviruses in relation to genomic RNA polymorphism.

Fecal rotaviruses collected from October 1983 to September 1984 from outpatients and inpatients attending the Royal Children's Hospital, Melbourne, Australia, with acute gastroenteritis were serotyped by enzyme immunoassay using rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1 to 4. Application of three different serotype 1-neutralizing antibodies indicated variations in neutralization epitopes between serotype 1 rotaviruses on the major outer capsid glycoprotein, including a site shared with serotype 3 rotaviruses. The three different binding patterns observed were termed "monotypes." Comparison of monotype and serotype with genomic RNA profiles generated by gel electrophoresis of ds viral RNA indicated that each RNA electropherotype corresponded to only one monotype (1a, 1b, 1c) or serotype (3, 4). However, serotypes 1 (a and c) and 4 contained multiple electropherotypes. Greater numbers of RNA segment variations, including alteration in mobility of gene segments 7, 8, and 9, were evident between rotaviruses of different serotype or monotype than within those groups. Within limits of time and location, rotavirus neutralization epitope variations appear to correlate with RNA polymorphism. Where rotavirus epidemiology has been analyzed by year using RNA electropherotypes, only limited numbers of each RNA pattern need to be serotyped to ascertain the major serotypes in circulation.

Antibodies, Monoclonal↗
PubMed1996

Evaluation of different methods (ELISA, IF, EM, PAGE) for the diagnosis of rotavirus infection in piglets.

The study on the prevalence of rotaviral infections among diarrhoetic piglets in 11 commercial farms in Poland was carried out on 531 faecal samples tested using an ELISA to screen for rotavirus. Of these, 169 were found to contain rotavirus antigen (32% of all cases of diarrhoea tested). Those pig herds managed using the Bisprol system had a lower prevalence of rotavirus in pigs with diarrhoea than those faecal samples obtained from Gi-Gi or Agrokomplex Agard farms. To further establish the presence of rotavirus antigen, 28 of those positive in ELISA samples were taken for isolation of the virus using tissue culture; 18 (64%) isolates were successfully adapted into MA-104 cells and the presence of rotavirus confirmed by immunofluorescence (IF) and electron microscopy (EM). In addition, an analysis of the band patterns visualised on PAGE showed 9 distinct electropherotypes for rotaviral dsRNA among the tested specimens. These findings suggest that rotavirus may represent an important contribution to the incidence of diarrhoea in Polish pig herds. The use of ELISA technology provided an efficient and effective means of evaluating the presence of rotavirus antigen in faecal samples and indicates that this procedure is a very useful tool in epidemiological studies, but that other techniques are required to confirm the presence of virus.

Animals↗
PubMed1984

The isolation, propagation and characterization of tissue-cultured equine rotaviruses.

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.

Animals↗
PubMed1987

Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela.

The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983-February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4-6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.

Animals↗
PubMed1990

Hybridization probes for the detection and differentiation of two serotypes of porcine rotavirus.

A dot hybridization assay was developed to detect and differentiate two serotypes of porcine rotavirus by hybridization of specific probes to rotavirus RNA dotted onto nylon membranes. The assay was conducted using radiolabeled probes prepared from cDNA clones of OSU (serotype 5) and Gottfried (serotype 4) rotavirus gene 9 segments. The probes were prepared by excision of full length cDNA copies of gene 9 inserts from recombinant plasmids followed by nick translation and 32P-dCTP labeling. Rotavirus RNA samples used in the assay were prepared from tissue culture, and intestinal contents from gnotobiotic and conventional pigs. Optimal conditions for hybridization consisted of 52 degrees C, 50% formamide, and 5 x SSC. Hybridization studies with the OSU and Gottfried gene 9 probes have shown them to be specific with only low levels of cross-reactivity with heterologous rotavirus preparations. Sensitivity studies have demonstrated the ability of the probes to detect at least 2 ng of rotavirus RNA. Further development and refinement of this technique may provide a useful tool for the detection and differentiation of porcine rotavirus serotypes.

Animals↗
PubMed1993

Survey of rotavirus infection in a dairy herd: comparison between polyacrylamide gel electrophoresis and two commercial tests.

A survey of rotavirus infection in a dairy herd with a history of neonatal diarrhoea was carried out. Faecal samples taken from 15 cows before and after calving as well as faeces taken from their calves daily from birth to two weeks of life were tested for rotavirus by polyacrylamide gel electrophoresis (PAGE) and compared with an ELISA and a latex agglutination commercial test. Rotavirus excretion was not detected in faeces from cows around parturition by any of the three tests. However, all of their calves shed rotaviruses during the observation period. The onset of rotavirus excretion determined by PAGE ranged from day 2 to day 8 of life (day 4.8 +/- 1.8 on average) and lasted for 4 to 7 days (5.3 +/- 1.1 days on average). Chi-square test showed a significant association (P = 0.0001) between the presence of rotavirus and the altered consistency of calves faeces. All the three tests showed similar results (overall agreement 92.5%) but discrepancies were detected mainly at the beginning or at the end of the rotavirus excretion period. Results obtained with both commercial kits closely paralleled each other and parameters other than sensitivity, specificity, diagnostic accuracy or predictive values have to be considered as selection criteria.

Animals↗
PubMed2005

Characterisation of rotavirus strains among hospitalised and non-hospitalised children in Guinea-Bissau, 2002 A high frequency of mixed infections with serotype G8.

BACKGROUND: In a previous community-based cohort study in Guinea-Bissau from 1996 to 1998, characterisation of rotavirus strains showed a high frequency of less common genotypes such as G8 and G9 and a high proportion of mixed infections. OBJECTIVES AND STUDY DESIGN: In the present study, we examined the prevalence of rotavirus genotypes among 81 hospitalised and 23 non-hospitalised Guinean children with rotavirus associated diarrhoea during the 2002 seasonal rotavirus outbreak. G- and P-types were determined in a two-step procedure using reverse transcription followed by a standard multiplex PCR. The multiplex PCR for G-types was furthermore supplemented with a single locus PCR including the MW8 primer for the G8-genotype. RESULTS: The dual infection G2/P[4]P[6] (24%) appeared to be the most frequent cause of rotavirus infections followed by G2P[4] (19%), G2P[6] (16%) and G8P[6] (13%). Overall 38% of the infections were mixed and 18% of the samples had the genotype G8. However, by subjecting all samples and not only the strains, which according to the standard multiplex PCR procedure were non-typeable, to a single locus G8-PCR, we found that the genotype G8 appeared in 62% of the infections, either as a single G-strain or in combination with other G-types, especially G2. Including these results, more than 63% of infections emerged as mixed. Neither genotype (including the presence of G8) nor the presence of mixed infections, seem to influence the severity of the rotavirus infection. CONCLUSION: We found a high frequency of mixed infections especially due to G8-genotypes, which might have implications for development of rotavirus vaccine candidates for use in Africa. Our results do not suggest that a single genotype is associated with severity, but the present study is based on a modest number of samples and results should be interpreted with caution.

Base Sequence↗
PubMed2006

Prevalence and molecular characterization of human group C rotaviruses in Hungary.

BACKGROUND: Group C rotaviruses are recognized enteric pathogens of humans and animals. Human group C rotaviruses have been associated with sporadic episodes and large outbreaks of gastroenteritis in children and adults but their epidemiology and ecology are still unexplored. OBJECTIVES: To collect epidemiological data on group C rotavirus infections among children with gastroenteritis in Hungary and perform molecular characterization on the identified strains. STUDY DESIGN: Fecal samples were collected during the 2003 surveillance in Baranya County, Hungary. The presence of group C rotavirus RNA was investigated by polyacrylamide gel electrophoresis and by reverse transcription-nested polymerase chain reaction for the VP6 gene. The identified strains were further characterized by sequencing and phylogenetic analysis of the VP7, VP6, VP4, and NSP4 genes. RESULTS: Three of 472 samples (0.6%) tested positive for group C rotavirus. Two samples were selected for molecular analysis. Strains BaC 6104/03 and BaC 11549/03 displayed an overall identity of >99.8% and 99.3% at the nucleotide and amino acid level, respectively. The VP7 of the strain BaC 6104/03 was most closely related (99.5% aa) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1% aa) to strains Belem and 208, detected in Brazil and China, respectively. CONCLUSIONS: Based on this 1-year study, we conclude that group C rotaviruses are not of epidemiological relevance in the etiology of childhood acute gastroenteritis in Hungary. The low sequence divergence between the Hungarian strains suggested that a single group C rotavirus strain circulated in this period in the study area.

Adolescent↗
PubMed2006

Detection and characterization of human group C rotavirus in the pediatric population of Barcelona, Spain.

BACKGROUND: The role of group C rotavirus as a cause of childhood diarrhea is not well defined. OBJECTIVES: To determine the prevalence of human group C rotavirus in stools of children in Barcelona, Spain, and to describe the genetic diversity of the rotavirus capsid proteins - VP6, VP7 and VP4 - in these samples. STUDY DESIGN: Stool specimens were assayed for rotavirus C RNA by an RT-PCR/southern-blot technique that included controls to indicate the presence of inhibitors of RT-PCR in the samples. RESULTS: Human rotavirus C was detected in 3 of 467 samples. One hundred and forty-five (31%) of these samples showed the presence of inhibitors of the RT-PCR assay. Thus, the corrected estimation for detection of group C rotavirus in Barcelona was of 1%. The entire VP4, VP6 and VP7 sequences were determined for all three isolates, revealing the relatedness of two of them to strains circulating in Europe, while the third was very close to sub-Saharan African strains. CONCLUSION: The low rate of detection of group C rotavirus suggests that it is not an emerging pathogen in children in our region.

Antigens, Viral↗
PubMed2005

In vitro anti-rotavirus activity of some medicinal plants used in Brazil against diarrhea.

Acute diarrhea, especially in children, is a very common disease with worldwide distribution and with a significant public health impact. Rotaviruses have been recognized as the major agents of diarrhea in infants and young children in developed as well as developing countries. In Brazil, diarrhea is one of the principal causes of death, mainly in the infant population. To fight diarrhea, traditional Brazilian medicine uses a great variety of plants. In this work, 12 medicinal plant species were screened for simian (SA-11) and human (HCR3) rotaviruses inhibition in vitro. At non-cytotoxic concentrations, the extracts from Artocarpus integrifolia L. (Moraceae) bark (480 microg/ml) and Spondias lutea L. (Anacardiaceae) leaves (160 microg/ml) had antiviral activity against both viruses. They showed inhibition of 99.2% and 97%, respectively, for human rotavirus, and 96.4% and 96.2% for simian rotavirus. The extracts from Myristica fragrans Houtt (Myristicaceae) seeds (160 microg/ml) and Spongias lutea bark (40 microg/ml) inhibited human rotavirus (90% and 82.2% inhibition, respectively), whereas the extracts from Anacardium occidentale L. (Anacardiaceae) leaves (4 microg/ml) and Psidium guajava L. (Myrtaceae) leaves (8 microg/ml) showed activity only against simian rotavirus (82.2% and 93.8% inhibition, respectively). Our results indicate that the extracts of Artocarpus integrifolia, Myristica fragrans and Spongias lutea can be useful in the treatment of human diarrhea if the etiologic agent is a rotavirus.

Animals↗
PubMed2004

The zoonotic potential of rotavirus.

Rotaviruses are generally species-specific, but cross-species transmission is possible, as has been demonstrated experimentally. Several case studies have indicated infection of humans by animal rotaviruses. Comparison of genetic sequences of human and animal rotaviruses often reveals close identity. Surveillance of circulating rotaviruses in the human population has revealed the presence of several uncommon genotypes. Many of these have been found in domestic animals, and it is possible that they arose in the human population through zoonotic transmission. The low incidence of uncommon strains would suggest that such transmission, or at least the establishment of an animal rotavirus or a human/animal reassortant virus in the human population, does not happen with any great frequency. However, many millions of people will be exposed year on year to animal rotaviruses. This happens within farming communities, and potentially to visitors to the countryside. There may be some measure of environmental contamination through livestock excrement. This exposure may not result in high levels of infection, but some infection could occur. There may be a continual input of rotavirus strains or sequences into the human population from the animal population albeit at a very low level.

Animals↗
PubMed2004

Burden of hospitalizations attributable to rotavirus infection in children in Spain, period 1999-2000.

The aim of this study is to provide estimates on the incidence of rotavirus infection requiring hospitalization in children <or=5 years of age during the 1999-2000 period in Spain and the associated direct cost of these hospitalizations. Hospital admissions attributable to rotavirus were estimated by using data on confirmed laboratory reports of rotavirus and hospital admissions due to acute gastroenteritis. During the study period a total of 32541 cases of pathogens responsible for acute gastroenteritis were reported to the microbiological information national system (SIM) and 14.0% were rotavirus. The proportion of laboratory-confirmed rotavirus cases was extrapolated to the total number of hospitalizations for acute gastroenteritis in children <or=5 years of age. An annual incidence of 1.0 hospital admissions attributable to rotavirus per 1000 persons <or=5 years of age was found, although during the winter season it reached 2.5 hospitalizations per 1000. Overall, the estimated annual number of days of hospitalization attributable to rotavirus was 8742 days that results in a cost of 3.6 million per year.

Acute Disease↗
PubMed2006

The epidemiology and burden of rotavirus in China: a review of the literature from 1983 to 2005.

We reviewed studies of rotavirus in MEDLINE and the Chinese literature to get a preliminary estimate of the burden of rotavirus gastroenteritis in China and the epidemiology of the disease. Studies were selected if they were conducted for a period 1 year or more, had more than 100 patients enrolled, and used an accepted diagnostic test. Overall, in 27 reports of children hospitalized for diarrhea in urban areas and 3 in rural areas, 44 and 33%, respectively, had rotavirus identified as the etiologic agent. Rotavirus was less commonly detected in children with milder illness seen in clinics (26% in urban and 28% in rural areas) and those cared for in the community (9%). The four main strains of rotavirus in circulation worldwide were also found in China and while G1 was the predominant strain overall, G3 emerged to be the most common strain in 9 of the 12 most recent studies. The disease has a distinct winter seasonal pattern and affects most children in their first 2 years of life. Although further studies are required to fully assess the burden of rotavirus diarrhea before decisions can be made about vaccine use, this review suggests that development and implementation of rotavirus vaccines should be a national priority.

Age Factors↗
PubMed2006

Protection against rotavirus shedding after intranasal immunization of mice with a chimeric VP6 protein does not require intestinal IgA.

Intranasal immunization of mice with chimeric VP6 and the adjuvant LT(R192G) consistently elicits >95% reductions in fecal rotavirus shedding following challenge. To determine the association between mucosal antibody and protection, we immunized BALB/c wt and J chain knockout (Jch-/-) mice with VP6 and either LT(R192G) or cholera toxin (CT). Both strains developed nearly equal levels of serum rotavirus IgG, but Jch-/- mice, which cannot transport dimeric IgA across epithelial cell surfaces, developed >4-fold higher levels of serum rotavirus IgA. Stool rotavirus IgA was present in wt but undetectable in Jch-/- mice. When challenged with rotavirus strain EDIM, reductions in rotavirus shedding were nearly identical in VP6-immunized wt and Jch-/- mice (i.e., 97% and 92%, respectively; P > 0.01). Th1 CD4 T cell responses were also detected in VP6-immunized animals based on high levels of IFN-gamma and IL-2 found after in vitro VP6 stimulation of spleen cells. Therefore, protection induced by intranasal immunization of mice with VP6 and adjuvant does not depend on intestinal rotavirus IgA antibody but appears to be associated with CD4 T cells.

Adjuvants, Immunologic↗
PubMed2003

Human rotavirus strains bearing VP4 gene P[6] allele recovered from asymptomatic or symptomatic infections share similar, if not identical, VP4 neutralization specificities.

A rotavirus VP4 gene P[6] allele has been documented in a number of countries to be characteristically associated with an endemic predominantly asymptomatic infection in neonates in maternity hospital nurseries. The mechanisms underlying the endemicity and asymptomatic nature of such neonatal infections remain unknown. Rotavirus strains sharing this same P genotype, however, have more recently been recovered from an increasing number of symptomatic diarrheal episodes in infants and young children in various parts of the world. Previously, we have shown that an asymptomatic P[6] rotavirus neonatal infection is not associated with a unique VP7 (G) serotype but may occur in conjunction with various G types. Although amino acid sequence comparisons of the VP4 gene between selected "asymptomatic" and "symptomatic" P[6] rotavirus strains have been reported and yielded information concerning their VP4 genotypes, serotypic comparisons of the outer capsid spike protein VP4 of such viruses have not been studied systematically by two-way cross-neutralizations. We determined the VP4 neutralization specificities of four asymptomatic and four symptomatic P[6] strains: two each of asymptomatic and symptomatic strains by two-way tests, and two each of additional asymptomatic and symptomatic strains by one-way tests. Both asymptomatic and symptomatic P[6] strains were shown to bear similar, if not identical, VP4 neutralization specificities. Thus, P[6] rotavirus strains causing asymptomatic or symptomatic infections did not appear to belong to unique P (VP4) serotypes. In addition, a close VP4 serotypic relationship between human P[6] rotavirus strains and the porcine P[6] rotavirus Gottfried strain was confirmed.

Alleles↗
PubMed2006

Rotavirus vaccines: current prospects and future challenges.

Rotavirus is the most common cause of severe diarrhoea in children worldwide and diarrhoeal deaths in children in developing countries. Accelerated development and introduction of rotavirus vaccines into global immunisation programmes has been a high priority for many international agencies, including WHO and the Global Alliance for Vaccines and Immunizations. Vaccines have been developed that could prevent the enormous morbidity and mortality from rotavirus and their effect should be measurable within 2-3 years. Two live oral rotavirus vaccines have been licensed in many countries; one is derived from an attenuated human strain of rotavirus and the other combines five bovine-human reassortant strains. Each vaccine has proven highly effective in preventing severe rotavirus diarrhoea in children and safe from the possible complication of intussusception. In developed countries, these vaccines could substantially reduce the number and associated costs of child hospitalisations and clinical visits for acute diarrhoea. In developing countries, they could reduce deaths from diarrhoea and improve child survival through programmes for childhood immunisations and diarrhoeal disease control. Although many scientific, programmatic, and financial challenges face the global use of rotavirus vaccines, these vaccines-and new candidates in the pipeline-hold promise to make an immediate and measurable effect to improve child health and survival from this common burden affecting all children.

Child↗
PubMed1997

Rotavirus vaccines against diarrhoeal disease.

Rotaviruses are responsible for more diarrhoeal disease-associated mortality than any other single agent. Vaccination may therefore hold the key to combating diarrhoeal disease worldwide. Natural immunity to rotavirus infection indicates that rather than protection from reinfection such immunity gives rise to less severe and less frequent attacks of diarrhoea. Early attempts to design a rotavirus vaccine with bovine rotavirus failed because of poor efficacy in some developing countries. Research into rhesus rotavirus, particularly the high-titre rhesus rotavirus tetravalent (RRV-TV) vaccine, has given slightly better results. A stumbling block to truly effective oral vaccines seems to be immunogenicity in developing countries. If efficacy can be ensured by trials in the developing countries, money spent on rotavirus vaccines will be well spent.

Animals↗