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In vitro studies related to various viral pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. The susceptibility to porcine rotavirus infection was evaluated by using a newly established porcine intestinal epithelial cell line. Immunohistochemical staining for cytokeratin confirmed that the cultured cells were epithelial cells. Measurement of cell viability and detection of infected cells confirmed that these epithelial cells were susceptible to porcine rotavirus infection. This study describes the cytopathic changes in cultured porcine intestinal epithelial cells during virus invasion. Following infection with porcine rotavirus, the cell cultures contained viral protein at 16 h post-infection as detected by direct immunofluorescence. The epithelial cell cultures provided competent target cells for studying host cell responses to porcine rotavirus and a homologous system for investigating the response of intestinal epithelial cells during viral infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Anti-rotaviral activities of Sophora flavescens extract (SFE) and stevioside (SV) from Stevia rebaudiana Bertoni either singly or in various combinations were examined in vitro and in vivo using a porcine rotavirus G5[P7] strain. Combination of SFE and SV inhibited in vitro virus replication more efficiently than each single treatment. In the piglet model, SV had no effect on rotavirus enteritis, whereas SFE improved but did not completely cure rotaviral enteritis. Interestingly, combination therapy of SFE and SV alleviated diarrhea, and markedly improved small intestinal lesion score and fecal virus shedding. Acute toxicity tests including the piglet lethal dose 50, and body weight, organ weight and pathological changes for the combination therapy did not show any adverse effect on the piglets. These preliminary data suggest that the combination therapy of SV and SFE is a potential curative medication for rotaviral diarrhea in pigs. Determination of the efficacy of this combination therapy in other species including humans needs to be addressed in the future., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Swine production is an important economic activity in Brazil, and there is interest in the development of clean production mechanisms to support sustainable agro-industrial activities. The biomass derived from swine manure has good potential to be used as a biofertilizer due to its high nutrient concentration. However, the land application of manure should be based on safety parameters such as the presence of pathogens that can potentially infect animals and people. This study was designed to assess the presence of porcine circovirus-2 (PCV2), porcine adenovirus (PAdV), rotavirus-A (RV-A) and Salmonella spp. in liquid manure, as well the infectivity of two genotypes of circovirus-2 (PCV2a and PCV2b) present in liquid manure. Three swine farms were evaluated: 1) a nursery production farm (manure analyzed before and after anaerobic biodigestion), 2) a grow-finish production farm (analyzed before and after anaerobic biodigestion), and 3) a second grow-finish production farm (raw manure-affluent). PCV2, PAdV and RV-A were present before and after anaerobic biodigestion (either affluent or effluent) at all farms. Salmonella spp. were detected at farm 1 (affluent and effluent) and farm 3 (raw manure-affluent) but not farm 2 (affluent and effluent). When the ability of the anaerobic biodigestion process to reduce viral concentration was evaluated, no significant reduction was observed (P>0.05). Both the PCV2a and PCV2b genotypes were detected, suggesting viral co-infection in swine production. The results revealed infectious PCV2 even after anaerobic biodigestion treatment. The presence of Salmonella spp. and enteric viruses, especially infectious PCV2, in the final effluent from the anaerobic biodigester system suggests that the process is inefficient for pathogen inactivation. Due to the prevalence and infectivity of PCV2 and considering the successful use of molecular methods coupled to cell culture for detecting infectious PCV2, we suggest that this virus can be used as a bioindicator in swine manure treatment systems to check the efficiency of pathogen inactivation and ensure the production of safe biofertilizers from swine manure., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aims: To determine the magnitude of microbial risks from waterborne viruses and bacteria in Bwaise III in Kampala (Uganda), a typical slum in Sub-Saharan Africa., Methods and Results: A quantitative microbial risk assessment (QMRA) was carried out to determine the magnitude of microbial risks from waterborne pathogens through various exposure pathways in Bwaise III in Kampala (Uganda). This was based on the concentration of Escherichia coli O157:H7, Salmonella spp., rotavirus (RV) and human adenoviruses F and G (HAdV) in spring water, tap water, surface water, grey water and contaminated soil samples. The total disease burden was 680 disability-adjusted life years (DALYs) per 1000 persons per year. The highest disease burden contribution was caused by exposure to surface water open drainage channels (39%) followed by exposure to grey water in tertiary drains (24%), storage containers (22%), unprotected springs (8%), contaminated soil (7%) and tap water (0.02%). The highest percentage of the mean estimated infections was caused by E. coli O157:H7 (41%) followed by HAdV (32%), RV (20%) and Salmonella spp. (7%). In addition, the highest infection risk was 1 caused by HAdV in surface water at the slum outlet, while the lowest infection risk was 2.71 × 10(-6) caused by E. coli O157:H7 in tap water., Conclusions: The results show that the slum environment is polluted, and the disease burden from each of the exposure routes in Bwaise III slum, with the exception of tap water, was much higher than the WHO reference level of tolerable risk of 1 × 10(-6) DALYs per person per year., Significance and Impact of the Study: The findings of this study provide guidance to governments, local authorities and nongovernment organizations in making decisions on measures to reduce infection risk and the disease burden by 10(2) to 10(5) depending on the source of exposure to achieve the desired health impacts. The infection risk may be reduced by sustainable management of human excreta and grey water, coupled with risk communication during hygiene awareness campaigns at household and community level. The data also provide a basis to make strategic investments to improve sanitary conditions in urban slums., (© 2013 The Society for Applied Microbiology.)

Abstract :  The persistence of Giardia, Cryptosporidium, Rotavirus, and Adenovirus in samples of raw and treated sewage collected monthly in 2010 at the Biological Wastewater Treatment Plant of Ribeirão Preto, SP, Brazil, was analyzed. The USEPA Method 1623 was used to detect and quantify Giardia and Cryptosporidium. An enzyme immunoassay was carried out to test Rotavirus and Adenovirus antigen optical density (Rotascreen® and Adenoscreen®). The results show a significant decrease in the concentrations of Giardia, Rotavirus and Adenovirus (P < 0.05) and a trend of decreasing Cryptosporidium densities, without statistical significance. Giardia concentrations ranged from 120 to 2,200 cysts/L in raw sewage and from 0.45 to 3.5 cysts/L in treated sewage. Cryptosporidium concentration ranged from undetectable to 28.9 oocysts/L in raw sewage and undetectable to 1.05 oocysts/L in treated sewage. Rotavirus presented absorbance values that ranged from 1.17 ± 0.81 in raw sewage to 0.46 ± 0.32 in treated sewage. Adenovirus, in turn, presented absorbance values of 0.64 ± 0.20 in raw sewage and of 0.45 ± 0.04 in treated sewage. There was no significant seasonal tendency observed in the distribution of protozoa (oo)cysts and in the viral antigen density in the monthly sewage samples during 2010 (P > 0.05). Even though these pathogenic agents decreased after treatment, the remaining loads observed in treated sewage can reach the watercourses receiving it. Giardia, Cryptosporidium, Rotavirus, and Adenovirus are pathogens with very low infectious doses, representing a public health risk especially for vulnerable groups, such as children living near these watercourses and homeless people using this water for various purposes. Studies addressing the environmental persistence of opportunistic pathogens in watercourses are hugely important in the public health sphere, especially in developing countries, where economic, social, cultural, and environmental factors still persist that are favorable to population's exposure to diarrhea-causing agents.

Food-borne viruses such as human Noroviruses (NoVs), hepatitis A virus (HAV), Rotaviruses (RoVs) are a public health concern worldwide. Biochemical substances, which occur naturally in plants, animals or microorganisms, might possess considerable antimicrobial properties. In this study, the reported effects of biochemical substances on food-borne viruses are reviewed. The biochemical substances are grouped into several categories including (i) polyphenols and proanthocyanins, (ii) saponin, (iii) polysaccharides, (iv) organic acids, (v) proteins and polypeptides, (vi) essential oils. Although not fully understood, the mechanism of action for the antiviral activity of the natural compounds is presented. Generally, it is thought to be the prevention of the viral attachment to host cells, either by causing damage on the viral capsids or change of the receptors on the cell membranes. It is recommended that further studies are undertaken not only on the wide-range screening for novel antiviral substances, but also on the mechanism in-depth as well as the exploration for their potential application in controlling virus contamination in foods or food processing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: Biliary atresia (BA) is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atresia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities., Methods: The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extrahepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated α tubulin to identify primary cilia., Results: Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human BA samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peribiliary glands adjacent to extrahepatic ducts in the BA sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared with controls., Conclusions: Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in RRV-infected primary cholangiocytes and the extrahepatic ducts from human patients with BA. This suggests that ciliary abnormalities are part of the pathophysiology of BA.

Researchers need an available and easy-to-use model of the human intestine to better understand human intestinal physiology and pathophysiology of diseases, and to offer an enhanced platform for developing drug therapy. Our work employs human enteroids derived from each of the major intestinal sections to advance understanding of several diarrheal diseases, including those caused by cholera, rotavirus and enterohemorrhagic Escherichia coli. An enteroid bank is being established to facilitate comparison of segmental, developmental, and regulatory differences in transport proteins that can influence therapy efficacy. Basic characterization of major ion transport protein expression, localization and function in the human enteroid model sets the stage to study the effects of enteric infection at the transport level, as well as to monitor potential responses to pharmacological intervention.

Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.

The study was aimed at an analysis of the influence of meglumine acridonacetate--the inductor of endogenous interferon synthesis--on the clinical course of gastroenteritis caused by rotavirus in children. A group of 100 patients aged from 3 months to 14 years were involved on retrospective comparative randomized study. The obtained data showed the advantages of meglumine acridonacetate administration during early terms of treatment of the gastroenteritis caused by rotaviruses. This treatment leads to fast regression of the main clinical symptoms such as fever, diarrhea, and intoxications, which results in reduction of the clinical course duration.

The enaminone 2 was reacted with hydrazonyl halides 3a-d to afford the corresponding pyrazole derivatives (6a-d) which reacted with hydrazine hydrate to afford the new pyrazolo[3,4-d]pyridazine derivatives 7a-d, respectively. In addition, compound 2 was reacted with some primary aromatic amines to afford the corresponding secondary enaminones 10a-c and reacted with sulfapyridine or sulfapyrimidine to afford the corresponding sulfonamide derivatives 12a and 12b. Evaluation of these new compounds against rotavirus Wa strain and adenovirus type 7 showed promising antiviral activity.

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytes in vitro. The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity with Lactobacillus paracasei, Lactobacillus rhamnosus and Bifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S-23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and named L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibited in vitro the meningitis aetiological agent Listeria monocytogenes and human rotavirus infections. B. breve CNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

Novel heterocyclic compounds containing benzimidazole derivatives were synthesized from 2-(1H-benzimidazol-2-yl) acetonitrile (1) and arylhydrazononitrile derivative 2 was obtained via coupling of 1 with 4-methyl phenyldiazonium salt, which was then reacted with hydroxylamine hydrochloride to give amidooxime derivative 3. This product was cyclized into the corresponding oxadiazole derivative 4 upon reflux in acetic anhydride. Compound 4 was refluxed in DMF in the presence of triethylamine to give the corresponding 5-(1H-benzimidazol-2-yl)-2-p-tolyl-2H-1,2,3-triazol-4-amine 6. Treatment of compound 6 with ethyl chloroformate afforded 2,6-dihydro-2-(4-methylphenyl)-1,2,3-triazolo[4",5"-4',5']pyrimido[1,6-a]benzimidazole-5(4H)-one (8). 1,2-bis(2-cyanomethyl-1H-benzimidazol-1-yl)ethane-1,2-dione (10) was synthesized via the condensation reaction of 2-(1H-benzimidazol-2-yl) acetonitrile (1) and diethyloxalate. The reactivity of compound 10 towards some diamine reagents was studied. The in vitro antimicrobial activity of the synthesized compounds was investigated against several pathogenic bacterial strains such as Escherichia coli O157, Salmonella typhimurium, E. coli O119, S. paratyphi, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The results of MIC revealed that compounds 12a-c showed the most effective antimicrobial activity against tested strains. On the other hand, compounds 12a, b exhibited high activity against rotavirus Wa strain while compounds 12b, c exhibited high activity against adenovirus type 7. In silico target prediction, docking and validation of the compounds 12a-c were performed. The dialkylglycine decarboxylase bacterial enzyme was predicted as a potential bacterial target receptor using pharmacophore-based correspondence with previous leads; giving the highest normalized scores and a high correlation docking score with mean inhibition concentrations. A novel binding mechanism was predicted after docking using the MOE software and its validation.

To assess the presence of three gastroenteritis viruses responsible for human acute gastroenteritis in surface water, a 1-year study was carried out in the city of Beijing, China. A total of 108 urban surface water samples were collected from nine collection sites which were defined with a global positioning system in rivers or lakes from September 2006 to August 2007. The water samples were subjected to virus concentration using an HA electronegative filter, followed by polymerase chain reaction (PCR) for rotavirus (RV) astrovirus (AV), and norovirus (NV). It showed that the number of viruses detected in water samples from different sites was variable, totaling 63 virus strains, with rotavirus (48.1%) verified as the most prevalent detected, followed by astrovirus (AV, 5.6%), and norovirus (NV, 4.6%). RV was also quantified by real-time PCR and the concentration of RV ranged from 0 to 18.27 genome copies·L(-1). And the distributions of RV in surface water were abundant in cold weather (from September to February) while less prevailing in warm weather (from March to August). The high detection rate of RV we encountered in this study provided convincing evidence that RV circulated at a certain frequency in the Beijing population. There was no statistically significant correlation between RV levels and both fecal coliform (R (2) = 0.02) and Enterococcus faecalis (R (2) = 0.02) densities. Our study suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.

Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6-BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6-BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

The human rotavirus G9 strain is the fifth most common rotavirus worldwide. A human rotavirus G9P[8] strain CAU05-202 was isolated from a young child with diarrhea using a cell culture system, and its major gene sequences were determined. Phylogenetic analysis of the VP7 gene revealed that CAU05-202 clustered into genetic lineage III-d and was most closely related to G9 rotaviruses from Turkey (strain GUH13) and Sri Lanka (strain 05SLC056 and 05SLC057). VP4 and NSP4 gene analysis showed that CAU05-202 belongs to the P[8]-3 lineage and genotype B, respectively. In addition, CAU05-202 has a long RNA electropherotype, supported by VP6 gene analysis, which is clearly associated with subgroup II specificity. Analysis of the G9 rotavirus strain CAU05-202 provides information concerning the genetic relationships among global rotavirus G9 strains, suggesting that closely related G9 strains are persistent and widespread in Asian countries.

Some health important enteric viruses are considered to be emerging waterborne pathogens and so the improvement of detection of these viruses in the aquatic environment is one of the most important steps in dealing with these pathogens. Since these viruses may be present in low numbers in water, it is necessary to concentrate water samples before viral detection. Although there are several methods to concentrate viruses in environmental waters, all present some drawbacks and consequently the method should be chosen that, despite its limitations, is adequate to achieve the aim of each study. As the effectiveness of the concentration methods is evaluated by determining the efficiency of viral recovery after concentration, it is important to use a simple and effective approach to evaluate their recovery efficiency. In this work ultracentrifugation, usually used as a secondary step for virus concentration, was evaluated as the main method to concentrate directly viruses in environmental water samples, using the microscopic enumeration of virus-like particles (VLP) as a new approach to estimate the efficiency of recovery. As the flocculation method is currently employed to concentrate viruses in environmental waters, it was also used in this study to assess the efficiency of the ultracentrifugation as the main viral concentration method in environmental waters. The results of this study indicate that ultracentrifugation is an adequate approach to concentrate viruses directly from environmental waters (recovery percentages between 66 and 72% in wastewaters and between 66 and 76% in recreational waters) and that the determination of VLP by epifluorescence microscopy is a simple, fast and cheap alternative approach to determine the recovery efficiency of the viral concentration methods.

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.

Rotavirus is the leading cause of severe acute gastroenteritis among children worldwide. It is well known that breast-feeding and vaccination afford infants protection. Since breast-feeding has drastically decreased in developed countries, efforts have been focused on the potential use of probiotics as preventive agents. In this study, a novel Bifidobacterium longum subsp. infantis strain was isolated from infant feces and selected, based on its capacity to inhibit in vitro rotavirus Wa replication (up to 36.05% infectious foci reduction) and also to protect cells from virus infection (up to 48.50% infectious foci reduction) in both MA-104 and HT-29 cell lines. Furthermore, studies using a BALB/c mouse model have proved that this strain provides preliminary in vivo protection against rotavirus infection. The strain has been deposited in the Spanish Type Culture Collection under the accession number CECT 7210. This novel strain has the main properties required of a probiotic, such as resistance to gastrointestinal juices, biliary salts, NaCl, and low pH, as well as adhesion to intestinal mucus and sensitivity to antibiotics. The food safety status has been confirmed by the absence of undesirable metabolite production and in acute ingestion studies of mice. Overall, these results demonstrate that Bifidobacterium longum subsp. infantis CECT 7210 can be considered a probiotic able to inhibit rotavirus infection.

Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.

Assessment of microbial hazards associated with certain environmental matrices, livelihood strategies, and food handling practices are constrained by time-consuming conventional microbiological techniques that lead to health risk assessments of narrow geographic or time scope, often targeting very few pathogens. Health risk assessment based on one or few indicator organisms underestimates true disease burden due a number of coexisting causative pathogens. Here, we employed molecular techniques in a survey of Cryptosporidium parvum, Giardia lamblia, Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Shigella spp., Vibrio cholera, and Rotavirus A densities in canal water with respect to seasonality and spatial distribution of point-nonpoint pollution sources. Three irrigational canals stretching across nearly a 150-km(2) periurban landscape, traditionally used for agricultural irrigation but function as vital part of municipal wastewater stabilization in recent years, were investigated. Compiled stochastic data (pathogen concentration, susceptible populations) and literature-obtained deterministic data (pathogen dose-response model parameter values) were used in estimating waterborne gastroenteritis burden. Exposure scenarios include swimming or fishing, consuming canal water-irrigated vegetables, and ingesting or inhaling water aerosols while working in canal water-irrigated fields. Estimated annual gastroenteritis burden due individual pathogens among the sampling points was -10.6log(10) to -2.2log(10) DALYs. Aggregated annual gastroenteritis burden due all the target pathogens per sampling point was -3.1log(10) to -1.9log(10) DALYs, far exceeding WHO acceptable limit of -6.0log(10) DALYs. The present approach will facilitate the comprehensive collection of surface water microbiological baseline data and setting of benchmarks for interventions aimed at reducing microbial hazards in similar landscapes worldwide.

Nosocomial transmission of viral and bacterial infections is a major problem worldwide, affecting millions of patients (and causing hundreds of thousands of deaths) per year. Rotavirus infections affect most children worldwide at least once before age five. We present here deterministic and stochastic models for the transmission of rotavirus in a pediatric hospital ward and draw on published data to compare the efficacy of several possible control measures in reducing the number of infections during a 90-day outbreak, including cohorting, changes in healthcare worker-patient ratio, improving compliance with preventive hygiene measures, and vaccination. Although recently approved vaccines have potential to curtail most nosocomial rotavirus transmission in the future, even short-term improvement in preventive hygiene compliance following contact with symptomatic patients may significantly limit transmission as well, and remains an important control measure, especially where resources are limited.

Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.

Two human G12 rotaviruses, CAU 195 and CAU 214, were isolated from South Korea using cell culture and characterized on the basis of sequence divergence in the VP7, VP4, and NSP4 genes. Phylogenetic analysis of the VP7 gene sequences indicated that these strains clustered into lineage III and were most closely related to G12 rotaviruses isolated in the United States. The VP4 and NSP4 gene sequences showed that two strains belonged to the P[6]-Ia lineage and genotype [B]. This finding provides information that can be used to evaluate G12 strains and aid in the development of effective vaccines in the future.

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 10(7) wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny.

The effects of free chlorine disinfection of tap water and wastewater effluents on the infectivity, gene integrity and surface antigens of rotaviruses were evaluated by a bench-scale chlorine disinfection experiments. Plaque assays, integrated cell culture-quantitative RT-PCR (ICC-RT-qPCR), RT-qPCR, and enzyme-linked immunosorbent assays (ELISA), respectively, were used to assess the influence of the disinfectant on virus infectivity as well as genetic and antigenic integrity of simian rotavirus SA11 as a surrogate for human rotaviruses. The ICC-RT-qPCR was able to detect rotaviruses survival from chlorine disinfection at chlorine dose up to 20 mg/L (60 min contact), which suggested a required chlorine dose of 5 folds (from 1 to 5 mg/L) higher than that indicated by the plaque assay to achieve 1.8 log10 reductions in tap water with 60 min exposing. The VP7 gene was more resistant than the infectivity and existed at chlorine dose up to 20 mg/L (60 min contact), while the antigencity was undetectable with chlorine dose more than 5 mg/L (60 min contact). The water quality also impacted the inactivation efficiencies, and rotaviruses have a relatively higher resistant in secondary effluents than in the tap water under the same chlorine disinfection treatments. This study indicated that rotaviruses have a higher infectivity, gene and antigencity resistance to chlorine than that previously indicated by plaque assay only, which seemed to underestimate the resistance of rotaviruses to chlorine and the risk of rotaviruses in environments. Present results also suggested that re-evaluation of resistance of other waterborne viruses after disinfections by more sensitive infectivity detection method (such as ICC-RT-qPCR) may be necessary, to determine the adequate disinfectant doses required for the inactivation of waterborne viruses.

Background: During rotavirus replication cycle, electron-dense cytoplasmic inclusions named viroplasms are formed, and two non-structural proteins, NSP2 and NSP5, have been shown to localize in these membrane-free structures. In these inclusions, replication of dsRNA and packaging of pre-virion particles occur. Despite the importance of viroplasms in the replication cycle of rotavirus, the information regarding their formation, and the possible sites of their nucleation during the early stages of infection is scarce. Here, we analyzed the formation of viroplasms after infection of MA104 cells with the rotavirus strain RRV, using different multiplicities of infection (MOI), and different times post-infection. The possibility that viroplasms formation is nucleated by the entering viral particles was investigated using fluorescently labeled purified rotavirus particles., Results: The immunofluorescent detection of viroplasms, using antibodies specific to NSP2 showed that both the number and size of viroplasms increased during infection, and depend on the MOI used. Small-size viroplasms predominated independently of the MOI or time post-infection, although at MOI's of 2.5 and 10 the proportion of larger viroplasms increased. Purified RRV particles were successfully labeled with the Cy5 mono reactive dye, without decrease in virus infectivity, and the labeled viruses were clearly observed by confocal microscope. PAGE gel analysis showed that most viral proteins were labeled; including the intermediate capsid protein VP6. Only 2 out of 117 Cy5-labeled virus particles colocalized with newly formed viroplasms at 4 hours post-infection., Conclusions: The results presented in this work suggest that during rotavirus infection the number and size of viroplasm increases in an MOI-dependent manner. The Cy5 in vitro labeled virus particles were not found to colocalize with newly formed viroplasms, suggesting that they are not involved in viroplasm nucleation.

Following virus infection, one of the cellular responses to limit the virus spread is induction of apoptosis. In the present study, we report role of rotavirus nonstructural protein 1 (NSP1) in regulating apoptosis by activating prosurvival pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB (nuclear factor kappaB) during early hours of infections (2 to 8 hpi). The NSP1 mutant strain A5-16 induces weak and transient activation of Akt (protein kinase B) and p65 NF-kappaB compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-kappaB promoter activity or Akt phosphorylation after A5-16 infection could be complemented in cells transfected with plasmid expressing NSP1 after infection with the rotavirus A5-16 strain. In cells either infected with A5-13 or transfected with pcD-NSP1, coimmunoprecipitation of NSP1 with phosphoinositide 3-kinase (PI3K) was observed, indicating that strong activation of PI3K/Akt could be due to its interaction with NSP1. In addition, after infection with same multiplicity of infection, A5-16 showed reduced number of viral particles compared to the A5-13 strain at the end of the replication cycle. A lower growth rate could be due to weak induction of PI3K/Akt and NF-kappaB, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-kappaB inhibitors. This effect was interferon independent; however, it was partly due to significantly higher caspase-3 activity, poly-ADP ribose polymerase (PARP) cleavage, and apoptosis during earlier stages of infection with the NSP1 mutant. Thus, our data suggest that NSP1 positively supports rotavirus growth by suppression of premature apoptosis for improved virus growth after infection.

Rotaviruses exist widely in water environments and are the major cause to the gastroenteritis in children. To overcome the limitations associated with the current methods for detecting rotaviruses in environmental samples, such as long duration with the traditional cell culture-based plaque assay, inability to detect infectivity with RT-PCR-based molecular methods and lower sensitivity with ELISA tests, we developed an integrated cell culture and reverse transcription quantitative PCR (ICC-RT-qPCR) assay to detect infectious rotaviruses based on detection of viral RNA during replication in cells. The cell culturing step before qPCR allows the infectious rotaviruses to replicate and be detected because they are the only ones that can infect cells and produce RNA. The results showed that as low as 0.2 PFU/ml rotaviruses were detected by ICC-RT-qPCR after 2 days of incubation. With samples, the copy numbers of VP7 gene of rotaviruses linearly correlated (with a coefficient (R(2)) of 0.9575) with initial virus concentrations ranging from 0.2 to 200 PFU/ml. In parallel comparing tests, the ICC-RT-qPCR exhibited higher sensitivity than both the plaque assay and the RT-qPCR when applied to field samples. ICC-RT-qPCR detected infectious rotavirus in 42% (10/24) of secondary effluents, while only 21% (5/24) and 12% (3/24) of samples were positive with either the plaque counting or the RT-qPCR method, respectively. Concentrations of rotaviruses in secondary effluent samples were determined to be 1-30 PFU/l. The results demonstrated that the developed ICC-RT-qPCR method reduced test duration and improved sensitivity towards infectious rotavirus and therefore can be an effective and quantitative tool for detecting infectious rotaviruses in water environments., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

In Europe, rotavirus gastroenteritis peaks in late winter or early spring suggesting a role for weather factors in transmission of the virus. In this study, multivariate regression models adapted for time-series data were used to investigate effects of temperature, humidity and rainfall on reported rotavirus infections and the infection-rate parameter, a derived measure of infection transmission that takes into account population immunity, in England, Wales, Scotland and The Netherlands. Delayed effects of weather were investigated by introducing lagged weather terms into the model. Meta-regression was used to pool together country-specific estimates. There was a 13 per cent (95% confidence interval (CI), 11-15%) decrease in reported infections per 1 degrees C increase in temperature above a threshold of 5 degrees C and a 4 per cent (95% CI, 3-5%) decrease in the infection-rate parameter per 1 degrees C increase in temperature across the whole temperature range. The effect of temperature was immediate for the infection-rate parameter but delayed by up to four weeks for reported infections. There was no overall effect of humidity or rainfall. There is a direct and simple relationship between cold weather and rotavirus transmission in Great Britain and The Netherlands. The more complex and delayed temperature effect on disease incidence is likely to be mediated through the effects of weather on transmission.

Group A rotaviruses (RVs) infect the young of numerous animal species and cause acute gastroenteritis. Cultivation of animal and human RVs in cells requires proteolytic activation of the viral attachment protein using trypsin. Continuous cell lines, such as rhesus monkey kidney cells, as well as primary monkey kidney cells, are routinely used for the growth and characterization of RVs. Isolation and cultivation of human RVs from clinical fecal specimens is difficult and adaptation to growth in vitro requires multiple rounds of passage in primary cells. Following growth, RV stocks can be purified by centrifugation, if required, and quantified using plaque assay or fluorescence focus assay. This unit describes easily applicable procedures for the culturing, storage, and quantification of RVs.

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-alpha/beta and IFN-gamma receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-alpha/beta receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-gamma receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

The final assembly of rotavirus particles takes place in the endoplasmic reticulum (ER). In this work, we evaluated by RNA interference the relevance to rotavirus assembly and infectivity of grp78, protein disulfide isomerase (PDI), grp94, calnexin, calreticulin, and ERp57, members of the two ER folding systems described herein. Silencing the expression of grp94 and Erp57 had no effect on rotavirus infectivity, while knocking down the expression of any of the other four chaperons caused a reduction in the yield of infectious virus of about 50%. In grp78-silenced cells, the maturation of the oligosaccharide chains of NSP4 was retarded. In cells with reduced levels of calnexin, the oxidative folding of VP7 was impaired and the trimming of NSP4 was accelerated, and in calreticulin-silenced cells, the formation of disulfide bonds of VP7 was also accelerated. The knockdown of PDI impaired the formation and/or rearrangement of the VP7 disulfide bonds. All these conditions also affected the correct assembly of virus particles, since compared with virions from control cells, they showed an altered susceptibility to EGTA and heat treatments, a decreased specific infectivity, and a diminished reactivity to VP7 with monoclonal antibody M60, which recognizes only this protein when its disulfide bonds have been correctly formed. In the case of grp78-silenced cells, the virus produced bound less efficiently to MA104 cells than virus obtained from control cells. All these results suggest that these chaperones are involved in the quality control of rotavirus morphogenesis. The complexity of the steps of rotavirus assembly that occur in the ER provide a useful model for studying the organization and operation of the complex network of chaperones involved in maintaining the quality control of this organelle.

Survival of rotavirus in fresh fruit juices of papaya (Caraca papaya L.), honeydew melon (Cucumis melo L.), and pineapple (Ananas comosus [L.] Merr.) was studied. Clarified juices were prepared from pulps of ripe fruits and sterilized by ultrafiltration. One milliliter of juice from each fruit was inoculated with 20 microl of 1 x 10(6) PFU of SA11 rotavirus and sampled immediately (0-h exposure) and 1 and 3 h later at 28 degrees C. Mean viral titers in juices of papaya (pH 5.1) and honeydew melon (pH 6.3) at 1 and 3 h were not significantly different from titers at 0-h exposure. Mean viral titers in juices from pineapples with ripening color indices of 3 (pH 3.6) and 6 (pH 3.7) at 1-h exposure (color index 3: 4.0 +/- 1.7 x 10(4); color index 6: 2.3 +/- 0.3 x 10(5)) and 3-h exposure (color index 3: 1.1 +/- 0.4 x 10(4); color index 6:1.3 +/- 0.6 x 10(5)) were significantly lower than titers at 0-h exposure (color index 3: 5.7 +/- 2.9 x 10(5); color index 6: 7.4 +/- 1.3 x 10(5)). Virus titers in pineapple juices of color index 3 were significantly lower than titers of the virus in juices of index 6. In cell culture medium (pH 7.4), SA11 titer remained stable over 3 h at 28 degrees C. However, at pH 3.6, the virus titer was reduced to a level not significantly different from that of the virus in pineapple juice of color index 6 (pH 3.7). In conclusion, papaya and honeydew melon juices, in contrast to pineapple juice, have the potential to transmit rotavirus. Inactivation of SA11 virus in pineapple juice can be possibly attributed to low pH and constituent(s) in the juice.

Mucosal epithelial cells are infected by a wide variety of pathogens and determining their response to infection is critical for understanding disease pathogenesis. A protocol was developed for culturing primary epithelial cells from fetal bovine intestine and the cultured cells were evaluated for susceptibility to an enteric viral infection. Immunohistochemical staining for cytokeratin confirmed that 60-75% of cultured cells were epithelial cells. Furthermore, following infection with bovine rotavirus (BRV) over 80% of cells in the ileal and jejunal cultures contained viral protein at 16 h post-infection. The intestinal epithelial cell cultures also contained fibroblasts so a jejunal fibroblast culture was established and infected with BRV. Viral protein was detected in jejunal fibroblasts but viral-induced cytopathology was delayed in fibroblast cultures when compared to epithelial cell cultures. This study describes an effective protocol for culturing bovine epithelial cells from fetal intestine and confirmed that the epithelial cells were susceptible to BRV infection. Ileal and jejunal cultures displayed limited growth following continuous passage but early passage epithelial cells provide competent target cells for studying host cell responses to an enteric viral pathogen.

Current approaches to the prevention of severe rotavirus diarrhea and deaths in children have all been through the use of live oral vaccines. To develop a safe and effective inactivated rotavirus vaccine (IRV), a new simple, rapid and robust method for the inactivation is critical and essential because chemical inactivation commonly used for a number of killed vaccines has been a challenge and problematic for rotavirus. We have examined an array of thermal conditions and demonstrated that purified YK-1 rotavirus in diluent buffer can be completely inactivated by heat treatment, as evidenced by the lack of virus growth in two successive passages in cell culture. Unlike chemical treatment that often causes physical and biochemical damages to viruses, thermally inactivated rotavirus particles maintained their structural, biochemical and antigenic integrity. A two-dose intramuscular administration of thermally inactivated YK-1 rotavirus without adjuvant resulted in high titers of total and neutralizing antibody in serum of mice. Adjuvant Al(OH)(3) further led to enhanced antibody titers and also dramatically lowered the amount of antigens in the vaccine formulation. Our results demonstrate the potential of heat inactivation as a novel approach to the manufacture of a safe and efficacious parenteral rotavirus vaccine, which should serve as an important addition to and back up for live oral rotavirus vaccine in children.

Alkaline versus neutral cultivation of bovine rotavirus strain 101 in the continuous cell cultures resulted in a significant acceleration of a reproduction cycle with a very significant cytopathic effect.

The structure and function of the intestinal epithelium is briefly described, with the principal mechanisms involved in diarrhea. Human enteric viruses and probiotics are presented. We then review how probiotic bacteria could interfere with virus-induced pathology, we present our own view and describe specific interactions that would be valuable targets for future studies.

Background: Biliary atresia is a disease of newborns that results in obliteration of the biliary tree. Infection of mice with rhesus rotavirus (RRV) results in a cholangiopathy mirroring human disease. The Mitogen Associated Protein Kinase (MAPK) signaling pathway can be activated by viral binding to cell-surface receptors. We hypothesized that RRV infection of cholangiocytes results in activation of MAPK signaling., Methods: Extrahepatic bile ducts from BALB/c pups or immortalized cholangiocytes subjected to RRV infection or control were analyzed, using Western blots, for phosphorylated members of the MAPK family: p38, ERK 1/2, JNK 1/2, and downstream transcription factors. Inhibitors of the MAPK were used to downregulate activity. Viral replication and cytolysis in cholangiocytes were evaluated post-MAPK inhibition., Results: Phosphorylation of all MAPK increased in RRV-infected mice and cholangiocytes. Several downstream transcription factors had increased activity in vitro. Inhibition of p38 and ERK 1/2 resulted in decreased viral replication. ERK 1/2 inhibition decreased cytolysis without affecting viral entry or binding., Conclusions: RRV infection of cholangiocytes resulted in increased MAPK signaling. Inhibition of p38 and ERK 1/2 influenced the ability of rotavirus to replicate. These novel findings provide insight into the signaling cascade involved in RRV-induced cholangiocyte injury.

Storage of water that was deliberately contaminated with enteric viruses in polyethylene terephthalate (PET) bottles led to a rapid decrease of the apparent viral load, thereby hampering the development of samples for a collaborative evaluation of viral detection methods for bottled water. To determine if this decrease was due to spontaneous inactivation or to adhesion, an elution protocol was developed and combined with a rapid and sensitive real-time reverse transcription-PCR-based method to quantify adsorbed norovirus (NV), hepatitis A virus (HAV), and rotavirus (RV) on bottle walls. The NV retention on PET bottle walls after 20 and 62 days reached an average level of 85% and 95% of the recovered inoculum, respectively. HAV and RV also showed adsorption onto PET bottles, reaching 90% and 80%, respectively, after 20 days of storage. NV and RV attachment was demonstrated to be dependent on the presence of autochthonous flora, whereas HAV adsorption was independent of it. Application of the elution and viral detection protocol to 294 commercially available water bottles obtained from 25 different countries did not give any positive result, thereby providing further evidence that the sources used for this product are free from enteric viruses and support for the theory that bottled water is not a vehicle for viral diseases.

In this study, a 15-mer phage display peptide library was employed to pan against human rotavirus immobilized on solid phase. 4 different peptides were selected and could bind with rotavirus particles specifically. Plaque reduction neutralization test and MTT analysis results indicated that 3 of the peptides can inhibit rotavirus infecting in vitro. A peptide which sequence is QSNPIHIITNTRNHP showed the best efficiency--93% neutralization infectivity. Two other peptides, A and B, showed 40% and 50% neutralization infectivity respectively. Amino sequence analysis results indicate the 3 peptides containing 2 conserved motifs: SNPIHII and NIP. No putative trypsin hydrolysis site was found in C peptide, however, 4 and 3 potential sites were found in A and B peptides respectively. Using trypsin inhibitor, both A and B peptides showed the similar antiviral effect as that of C peptide. It suggests that the intactness of the 2 conserved motifs play an important role in counteracting virus infection. According to the results of this study, peptide C is hopeful to be exploited as an antiviral peptide drug.

Rotavirus is the leading cause of severe dehydrating diarrhoea in infants and young children worldwide. This virus infects mature enterocytes in the small intestine, and induces structural and functional damage. In the present study, we have identified a new mechanism by which rotavirus impairs a brush border-associated intestinal protein. We show that infection of enterocyte-like Caco-2/TC7 cells by rhesus monkey rotavirus (RRV) impairs the biosynthesis of dipeptidyl peptidase IV (DPP IV), an important hydrolase in the digestion of dietary proline-rich proteins. We show that the enzyme activity of DPP IV was reduced, and that rearrangements of the protein occurred at the apical domain of the RRV-infected cells. Using pulse-chase experiments and cell surface immunoprecipitation, we have demonstrated that RRV infection did not affect the stability or apical targeting of DPP IV, but did induce a dramatic decrease in its biosynthesis. Using quantitative RT-PCR, we showed that RRV had no effect on the level of expression of DPP IV mRNA, suggesting that the observed decrease in the biosynthesis of the protein is related to an effect of the virus at the translational level.

Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.

A total of 3,101 fecal specimens were collected during an 8-year survey for rotavirus infection in São Paulo, Brazil. Group A rotavirus was detected in 774 (25.0%) specimens. Of these, 431 strains (55.7%) were analyzed for G and P types by reverse transcription-polymerase chain reaction; G1 was the predominant serotype (68.2%), followed by G9 (17.2%), G4 (6.3%), G2 (1.2%), G3 (0.7%), mixed infection (1.8%), and untypeable (4.6%). Both rotavirus G and P types could be established in 332 strains (77.0%). We identified the 4 most common strains worldwide: P[8]G1 (66.6%), P[4]G2 (1.0%), P[8]G3 (0.6%), and P[8]G4 (7.2%). Among the single G9 strains detected, VP4 genotyping showed that P[8]G9 was the most prevalent, followed by P[4]G9 and P[6]G9. The emergence and high frequency of rotavirus G9 in São Paulo, Brazil, and other parts of the world will affect the development and evaluation of future vaccines.

A novel multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) that can detect porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (GAR) was developed. The 3 viruses (PEDV, TGEV, and porcine GAR) are major agents in viral enteric diseases of piglets. As the clinical signs of these diseases are similar, including watery diarrhea, differential detection is required for etiologic diagnosis. A mixture of 3 pairs of published primers was used for amplification of viral nucleic acids, yielding 3 different amplicons with sizes of 859 bp, 651 bp, and 309 bp for TGEV, PEDV, and porcine GAR, respectively. A total of 157 specimens (78 fecal and 79 intestinal samples) from piglets with acute gastroenteritis were collected in Korea between January 2004 and May 2005. They were tested for the presence of 3 viruses by multiplex RT-PCR. Coinfections with PEDV and porcine GAR were identified in 16 farms (43.2%). PEDV, porcine GAR, and TGEV infection were 26.3%, 13.2%, and 2.7% respectively. The relative sensitivity and specificity of multiplex RT-PCR were evaluated, with results suggesting that this assay is equal in quality to conventional single-agent RT-PCR assays (sensitivity:100%, 92.9%, 100% for TGEV, PEDV, GARs; specificity: 100% for all 3 viruses). This multiplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets.

A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analysis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have molecular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron microscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein extract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contribution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection.

Rotaviral diarrheal illness is one of the most common infectious diseases in children worldwide, but our understanding of its pathophysiology is limited. This study examines whether the enhanced net chloride secretion during rotavirus infection in young rabbits may occur as a result of hypersecretion in crypt cells that would exceed the substantial Cl(-) reabsorption observed in villi. By using a rapid filtration technique, we evaluated transport of (36)Cl and D-(14)C glucose across brush border membrane (BBM) vesicles purified from villus tip and crypt cells isolated in parallel from the entire small intestine. Rotavirus infection impaired SGLT1-mediated Na(+)-D-glucose symport activity in both villus and crypt cell BBM, hence contributing to the massive water loss along the cryptvillus axis. In the same BBM preparations, rotavirus failed to stimulate the Cl(-) transport activities (Cl(-)/H(+) symport, Cl(-)/anion exchange and voltage-activated Cl(-) conductance) at the crypt level, but not at the villus level, questioning, therefore, the origin of net chloride secretion. We propose that the chloride carrier might function in both normal (absorption) and reversed (secretion) modes in villi, depending on the direction of the chloride electrochemical gradient resulting from rotavirus infection, agreeing with our results that rotavirus accelerated both Cl(-) influx and Cl(-) efflux rates across villi BBM.

The rotaviruses, members of the family Reoviridae, are icosahedral triple-layered viruses with genomes consisting of 11 segments of double-stranded (ds)RNA. A characteristic feature of rotavirus-infected cells is the formation of large cytoplasmic inclusion bodies, termed viroplasms. These dynamic and highly organized structures serve as viral factories that direct the packaging and replication of the viral genome into early capsid assembly intermediates. Migration of the intermediates to the endoplasmic reticulum (ER) initiates a budding process that culminates in final capsid assembly. Recent information on the development and organization of viroplasms, the structure and function of its components, and interactive pathways linking RNA synthesis and capsid assembly provide new insight into how these microenvironments serve to interface the replication and morphogenetic processes of the virus.

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.

Although rotavirus infections are generally considered to be confined to the intestine, recent reports suggest that extraintestinal disease occurs. We studied whether rotavirus infection was associated with antigenemia during a major outbreak of gastroenteritis in the Kingston metropolitan area, during July-August 2003. Rotavirus antigen was identified in 30 of 70 acute-phase serum samples (including from 2 deceased individuals) but in only 1 of 53 control samples. Serum antigen levels were inversely associated with time since symptom onset and were directly associated with antigen levels in stool (P = .02). Serum antigen levels were significantly elevated during primary infections (acute-phase serum immunoglobulin G [IgG] titers, <25), compared with those in subsequent infections (acute-phase serum IgG titers, > or = 25) (P = .02). Antigenemia was common in this outbreak and might provide a mechanism to help explain rare but well-documented reports of findings of extraintestinal rotavirus. In situations in which stool samples are not readily available (i.e., patients with severe dehydration or those recently recovered or deceased), serum testing by enzyme immunoassay offers a new and practical diagnostic tool.