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Rotavirus infects intestinal epithelial cells and is the leading cause of gastroenteritis in infants worldwide. Upon viral infection, intestinal cells produce type I and type III interferons (IFNs) to alert the tissue and promote an antiviral state. These two types of IFN bind to different receptors but induce similar pathways that stimulate the activation of interferon-stimulated genes (ISGs) to combat viral infection. In this work, we studied the spread of a fluorescent wild-type (WT) SA11 rotavirus in human colorectal cancer cells lacking specific interferon receptors and compared it to that of an NSP1 mutant rotavirus that cannot interfere with the host intrinsic innate immune response. We could show that the WT rotavirus efficiently blocks the production of type I IFNs but that type III IFNs are still produced, whereas the NSP1 mutant rotavirus allows the production of both. Interestingly, while both exogenously added type I and type III IFNs could efficiently protect cells against rotavirus infection, endogenous type III IFNs were found to be key to limit infection of human intestinal cells by rotavirus. By using a fluorescent reporter cell line to highlight the cells mounting an antiviral program, we could show that paracrine signaling driven by type III IFNs efficiently controls the spread of both WT and NSP1 mutant rotavirus. Our results strongly suggest that NSP1 efficiently blocks the type I IFN-mediated antiviral response; however, its restriction of the type III IFN-mediated one is not sufficient to prevent type III IFNs from partially inhibiting viral spread in intestinal epithelial cells. Additionally, our findings further highlight the importance of type III IFNs in controlling rotavirus infection, which could be exploited as antiviral therapeutic measures. IMPORTANCE Rotavirus is one of the most common causes of gastroenteritis worldwide. In developing countries, rotavirus infections lead to more than 200,000 deaths in infants and children. The intestinal epithelial cells lining the gastrointestinal tract combat rotavirus infection by two key antiviral compounds known as type I and III interferons. However, rotavirus has developed countermeasures to block the antiviral actions of the interferons. In this work, we evaluated the arms race between rotavirus and type I and III interferons. We determined that although rotavirus could block the induction of type I interferons, it was unable to block type III interferons. The ability of infected cells to produce and release type III interferons leads to the protection of the noninfected neighboring cells and the clearance of rotavirus infection from the epithelium. This suggests that type III interferons are key antiviral agents and could be used to help control rotavirus infections in children.

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Drinking water supplies in the developing world often serve as a biosphere for various organisms. Viral gastroenteritis is a neglected area of research in Pakistan, there are no data for the prevalence of enteric viruses in drinking water of the largest city of Karachi. The present study aimed to provide a survey of the existence of enteric viruses: human adenovirus (HAdV), human enteroviruses (hEV), and genotype A rotavirus (GARV) in tap water. Using a simple PCR approach, we detected 20%, 43%, and 23% of HAdV, hEV, and GARV in tap water samples, respectively. We have also shown an overall quality deficit of tap water at the pumping station and consumer tap. We have found no sample free from bacterial contaminations. The ranges for a total number of the heterotrophic plate count and coliform were found 8.7 × 10 2 -4.5 × 10 6  CFU/mL and 210 to uncountable coliforms/100 mL, respectively. Moreover, we assessed the efficiency of small-scale water treatment methods for the removal of viruses.

Rotavirus is the most common cause of severe diarrhea among infants and young children and is responsible for more than 200,000 pediatric deaths per year. There is currently no pharmacological treatment for rotavirus infection in clinical activity. Although cholesterol synthesis has been proven to play a key role in the infections of multiple viruses, little is known about the relationship between cholesterol biosynthesis and rotavirus replication. The models of rotavirus infected two cell lines and a human small intestinal organoid were used. We investigated the effects of cholesterol biosynthesis, including inhibition, enhancement, and their combinations on rotavirus replication on these models. The knockdown of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was built by small hairpin RNAs in Caco2 cells. In all these models, inhibition of cholesterol synthesis by statins or HMGCR knockdown had a significant inhibitory effect on rotavirus replication. The result was further confirmed by the other inhibitors: 6-fluoromevalonate, Zaragozic acid A and U18666A, in the cholesterol biosynthesis pathway. Conversely, enhancement of cholesterol production increased rotavirus replication, suggesting that cholesterol homeostasis is relevant for rotavirus replication. The effects of all these compounds toward rotavirus were further confirmed with a clinical rotavirus isolate. We concluded that rotavirus replication is dependent on cholesterol biosynthesis. To be specific, inhibition of cholesterol synthesis can downregulate rotavirus replication; on the contrary, rotavirus replication is upregulated. Statin treatment is potentially an effective novel clinical anti-rotavirus strategy.

Interferon (IFN) family cytokines stimulate genes (interferon-stimulated genes [ISGs]) that are integral to antiviral host defense. Type I IFNs act systemically, whereas type III IFNs act preferentially at epithelial barriers. Among barrier cells, intestinal epithelial cells (IECs) are particularly dependent on type III IFN for the control and clearance of virus infection, but the physiological basis of this selective IFN response is not well understood. Here, we confirm that type III IFN treatment elicits robust and uniform ISG expression in neonatal mouse IECs and inhibits the replication of IEC-tropic rotavirus. In contrast, type I IFN elicits a marginal ISG response in neonatal mouse IECs and does not inhibit rotavirus replication. In vitro treatment of IEC organoids with type III IFN results in ISG expression that mirrors the in vivo type III IFN response. However, IEC organoids have increased expression of the type I IFN receptor relative to neonate IECs, and the response of IEC organoids to type I IFN is strikingly increased in magnitude and scope relative to type III IFN. The expanded type I IFN-specific response includes proapoptotic genes and potentiates toxicity triggered by tumor necrosis factor alpha (TNF-α). The ISGs stimulated in common by type I and III IFNs have strong interferon-stimulated response element (ISRE) promoter motifs, whereas the expanded set of type I IFN-specific ISGs, including proapoptotic genes, have weak ISRE motifs. Thus, the preferential responsiveness of IECs to type III IFN in vivo enables selective ISG expression during infection that confers antiviral protection but minimizes disruption of intestinal homeostasis. IMPORTANCE Enteric viral infections are a major cause of gastroenteritis worldwide and have the potential to trigger or exacerbate intestinal inflammatory diseases. Prior studies have identified specialized innate immune responses that are active in the intestinal epithelium following viral infection, but our understanding of the benefits of such an epithelium-specific response is incomplete. Here, we show that the intestinal epithelial antiviral response is programmed to enable protection while minimizing epithelial cytotoxicity that can often accompany an inflammatory response. Our findings offer new insight into the benefits of a tailored innate immune response at the intestinal barrier and suggest how dysregulation of this response could promote inflammatory disease., (Copyright © 2020 American Society for Microbiology.)

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 10 3 to 10 7 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 10 3 to 10 7 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 10 3 to 10 7 GC/L, 75% of drainage sediment with viral concentrations ranging from 10 3 to 10 7 GC/L, 50% of Nile water with viral concentrations ranging from 10 3 to 10 7 GC/L, and 45% of Nile sediment with viral concentrations ranging from 10 3 to 10 7 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 10 3 to 10 7 GC/L, 75% of activated sludge with viral concentrations ranging from 10 3 to 10 7 GC/L, 58% of drainage water with viral concentrations ranging from 10 3 to 10 7 GC/L, 50% of drainage sediment with viral concentrations ranging from 10 3 to 10 7 GC/L, and 45% of Nile water with viral concentrations ranging from 10 3 to 10 7 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

Live oral rotavirus vaccines have been developed by serial passaging in cell culture and found to be safe in infants. However, mechanisms for the adaptation and attenuation of rotavirus vaccines are not fully understood. We prepared a human rotavirus vaccine strain, CDC-9 (G1P[8]), which when grown in MA104 cells to passage 11 or 12 (P11/P12) had no nucleotide or amino acid sequence changes from the original virus in stool. Upon adaptation and passages in Vero cells, the strain underwent five amino acid changes at P28 and one additional change at P44/P45 in the VP4 gene. We performed virologic, immunological, and pathogenic characterization of wild-type CDC-9 virus at P11/P12 and its two mutants at P28 or P44/P45 using in vitro and in vivo model systems. We found that mutants CDC-9 P28 and P44 induced upregulated expression of immunomodulatory cytokines. On the other hand, the two mutant viruses induced lower STAT1 phosphorylation and grew to 2-log-higher titers than wild-type virus in human Caco-2 cells and simian Vero cells. In neonatal rats, CDC-9 P45 showed reduced rotavirus shedding in fecal specimens and did not induce diarrhea compared to wild-type virus and modulated cytokine responses comparably to Rotarix infection. These findings indicate that mutant CDC-9 is attenuated and safe. Our study is the first to provide insight into the possible mechanisms of human rotavirus adaptation and attenuation and supports ongoing efforts to develop CDC-9 as a new generation of rotavirus vaccine for live oral or parenteral administration. IMPORTANCE Mechanisms for in vitro adaptation and in vivo attenuation of human rotavirus vaccines are not known. The present study is the first to comprehensively compare the in vitro growth characteristics, virulence, and host response of a wild-type and an attenuated human rotavirus strain, CDC-9, in Caco-2 cells and neonatal rats. Our study identifies critical sequence changes in the genome that render human rotavirus adapted to growth to high levels in Vero cells and attenuated and safe in neonatal rats; thus, the study supports clinical development of CDC-9 for oral or parenteral vaccination in children., (Copyright © 2020 American Society for Microbiology.)

The availability of drinking water is one of the main determinants of quality of life, disease prevention and the promotion of health. Viruses are important agents of waterborne diseases and have been described as important markers of human faecal contamination. This study aimed to investigate viruses' presence as an indicator of drinking water quality in low-income communities in the Manguinhos area, Rio de Janeiro, Brazil. Three hundred and four drinking water samples (2L/each) were collected along the drinking water distribution-to-consumption pathway in households, as well as healthcare and school units. Water samples were collected both directly from the water supply prior to distribution and after storage in tanks and filtration units. Using qPCR, viruses were detected 50 times in 45 water samples (15%), 19 of these being human adenovirus, 17 rotavirus A and 14 norovirus GII. Viral loads recovered ranged from 5E+10 to 8.7E+10 6 genome copies/Liter. Co-detection was observed in five household water samples and there was no difference regarding virus detection across sampling sites. Precarious and inadequate environmental conditions characterized by the lack of local infrastructure regarding basic sanitation and waste collection in the territory, as well as negligent hygiene habits, could explain viral detection in drinking water in regions with a water supply system.

RNA viruses form a dynamic distribution of mutant swarms (termed "quasispecies") due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences. IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated., (Copyright © 2020 Kadoya et al.)

Bivalve mollusks as filter-feeders concentrate in their digestive tissue microorganisms likely present in the harvesting water, thus becoming risky food especially if consumed raw or poorly cooked. To eliminate bacteria and viruses eventually accumulated, they must undergo a depuration process which efficacy on viruses is on debate. To better clarify the worth of the depuration process on virus elimination from mussels, in this study we investigated rotavirus kinetics of accumulation and depuration in Mytilus galloprovincialis experimentally contaminated. Depuration process was monitored for 9 days and virus residual presence and infectivity were evaluated by real time quantitative polymerase chain reaction, cell culture and electron microscopy at days 1, 2, 3, 5, 7, 9 of depuration. Variables like presence of ozone and of microalgae feeding were also analyzed as possible depuration enhancers. Results showed a two-phase virus removal kinetic with a high decrease in the first 24 h of depuration and 5 days necessary to completely remove rotavirus.

Coxsackievirus B3 (CVB3) and rotavirus (RV) are pathogens of some chronic human diseases. The aim of this study was to determine in vitro antiviral activity of some cyanobacteria against RV and CVB3 infections. Five cyanobacteria were collected from Egypt, identified, and analyzed biochemically. Then, the inhibition of the cytopathic effect of RV and CVB3 viruses by cyanobacterial extracts was examined. Methanol extract of the cyanobacterial isolates showed high antiviral activity against CVB3 with Therapeutic index (TI) of 50.0, 30.0, 27.6, 16.6, and 20.0 for Leptolyngbya boryana, Arthrospira platensis, Nostoc punctiforme, Oscillatoria sp., and Leptolyngbya sp., respectively. The extracts reduced CVB3 titers comparing to 50% tissue culture infectious doses (TCID 50 ) with values 3.25-5.75 log 10 of TCID 50 . Moreover, extracts of A. platensis, and Oscillatoria sp. exhibited high antiviral activity against RV with TI values of 45 and 42.5, respectively, and a reduction in virus titers by 5.75 log 10 and 5.5 log 10 of TCID 50 , respectively. Extracts of L. boryana, Leptolyngbya sp., and N. punctiforme had a moderate to low antiviral activity against RV with TI ranging between 2.8 and 7, respectively, and a reduction in virus titers between 0.5 log 10 and 1.5 log 10 of TCID 50 , respectively. This study concluded that extracts of five cyanobacterial isolates possess a potent antiviral effect against CVB3 and RV, making them promising sources of new safe antiviral drugs.

We modeled Group A Rotavirus (RVA) and Norovirus genogroup II (GII NoV) transport experiments in standardized (crystal quartz sand and deionized water with adjusted pH and ionic strength) and natural soil matrix-water systems (MWS). On the one hand, in the standardized MWS, Rotavirus and Norovirus showed very similar breakthrough curves (BTCs), showing a removal rate of 2 and 1.7 log 10 , respectively. From the numerical modeling of the experiment, transport parameters of the same order of magnitude were obtained for both viruses. On the other hand, in the natural MWS, the two viruses show very different BTCs. The Norovirus transport model showed significant changes; BTC showed a removal rate of 4 log 10 , while Rotavirus showed a removal rate of 2.6 log 10 similar to the 2 log 10 observed on the standardized MWS. One possible explanation for this differential behavior is the difference in the isoelectric point value of these two viruses and the increase of the ionic strength on the natural MWS.

Background: Rotavirus infection is one of the most common gastroenteritis in the world, and a million cases are registered to enter hospital every year. Promyelocytic leukemia proteins (PMLs) are IFN-up-regulated proteins, and one of their critical functions is working as antiviral proteins. Recently, promyelocytic leukemia isoform II (PML-II) has been depicted as an isoform responsible for the antiviral function., Methods: Rotavirus prevalence determination was achieved by PCR and Rapid Adeno/Rota Virus test, while the relative expression assay was carried out by real-time PCR technique. Blood and stool samples were collected from 34 children under five years admitted to the hospital with acute gastroenteritis showing signs of dehydration. RNA samples were extracted from blood specimens and converted to cDNA to be used in gene expression analysis of PML, PML-II, and IFN-γ in rotavirus positive or negative samples., Results: Rapid Adeno/Rota Virus Antigen Combo Test and PCR assay could detect the virus in stool samples in 45% and 17.6% of cases, respectively. PML in positive samples decreased to 104fold less than the level in negative ones. The same trend was noticed in the level of IFN-γ and PML-II expression as their expression reduced to 104 or 13fold in rotavirus-infected samples compared to the control, respectively., Conclusion: Altogether, our data showed that the gene expression of PML, PML-II, and type II IFN considerably diminished in rotavirus-infected samples compared to the negative control.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines., Competing Interests: The authors declare no conflict of interest.

Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as "swollen" endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of "budding" occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication. IMPORTANCE In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes., (Copyright © 2019 American Society for Microbiology.)

Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4. IMPORTANCE Rotavirus, a member of the family Reoviridae , is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4., (Copyright © 2019 American Society for Microbiology.)

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles., Competing Interests: YG, JM, DT, HH, AP, MM, CW, JR, DS, SL, AG, CA No competing interests declared, (© 2019, Garcés Suárez et al.)

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 °C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Several microbial modulatory concepts, such as certain probiotics and prebiotics, confer protection against gastrointestinal infections, among which is acute diarrhea caused by the rotavirus (RV). Other microbiota modulators, such as postbiotics, produced during fermentation, might also have the potential to counteract RV infection. In light of this, a fermented milk, made by using Bifidobacterium breve C50 (BbC50) and Streptococcus thermophilus 065 (St065) with a prebiotic mixture-short chain galactooligosaccharides/long chain fructooligosaccharides (scGOS/lcFOS 9:1)-with potential to impact the intestinal microbiota composition was tested. An RV infected rat model was used to evaluate the amelioration of the infectious process and the improvement of the immune response induced by the fermented milk with prebiotic mixture. The dietary intervention caused a reduction in the clinical symptoms of diarrhea, such as severity and incidence. Furthermore, a modulation of the immune response was observed, which might enhance the reduction of the associated diarrhea. In addition, the fermented milk with prebiotic mixture was able to bind the virus and reduce its clearance. In conclusion, the postbiotic components in the fermented milk in combination with the prebiotics used here showed protective properties against RV infection.

Background: In 2016, the Government of India introduced the oral rotavirus vaccine (ROTAVAC, Bharat Biotech, India) in 4 states of India as part of the Universal Immunization Programme, and expanded to 5 more states in 2017. We report four years of data on rotavirus gastroenteritis in hospitalized children < 5 years of age prior to vaccine introduction., Methods: Children from 7 sites in southern and northern India hospitalized for diarrhoea were recruited between July 2012 and June 2016. Stool samples were screened for rotavirus using enzyme immunoassay (EIA). The EIA positive samples were genotyped by reverse-transcription polymerase chain reaction., Results: Of the 5834 samples from the 7 sites, 2069 (35.5%) were positive for rotavirus by EIA. Genotyping was performed for 2010 (97.1%) samples. G1P[8](56.3%), G2P[4](9.1%), G9P[4](7.6%), G9P[8](4.2%), and G12P[6](3.7%) were the common genotypes in southern India and G1P[8](36%), G9P[4](11.4%), G2P[4](11.2%), G12P[6](8.4%), and G3P[8](5.9%) in northern India., Conclusions: The study highlights the high prevalence of rotavirus gastroenteritis in India and the diversity of rotavirus genotypes across different geographical regions. Pre- vaccine surveillance data is necessary to evaluate the potential change in admission rates for gastroenteritis and circulating rotavirus genotypes after vaccine introduction, thus assessing impact.

Background: Rоtaviruses are amоng the leading causes of severe diarrhea in children all over the Wоrld. Vaccination is considered to be the mоst effective way to cоntrоl the disease. Currently available vaccines for prevention of rоtavirus infection are based on live attenuated rotavirus strains human оr animal origin., Objectives: The aim of this investigation was to study the biological and genetic properties of an actual epidemic human rotavirus A (RVA) strain Wa G1P[8] genotype., Methods: RVA Wa reproduction in a monolayer continuous cell lines, purification and concentration of RVA antigen, PAAG electrophoresis and Western-Blot, electrophoresis of viral genomic RNA segments, sequencing., Results: Human RVA G1P[8] Wa strain biological and molecular genetic properties were assessed in the process of the adaptation to MARC145 continuous cell line. Cell cultured RVA antigen was purified, concentrated and then characterized by the method of PAAG electrophoresis and immunoblot. To verify RVA Wa genome identity, electrophoresis of viral genomic RNA segments was performed. The lack of accumulation of changes in the RVA Wa genome during adaptation to various cell cultures and during serial passages was demonstrated by sequencing fragments of the viral genome., Conclusions: RVA Wa strain is stable, it possesses high biological activity: it has been successfully adapted to the MARC145 cell line and RVA Wa virus titer after the adaptation reached 7,5-7,7 lg TCID50/ml. The identity of the cultivated RVA to the original strain Wa G1P[8] was confirmed., Competing Interests: The authors declare no conflict of interest.

Rotaviruses (RVs), a leading cause of severe diarrhea in young children and many mammalian species, have evolved multiple strategies to counteract the host innate immunity, specifically interferon (IFN) signaling through RV non-structural protein 1 (NSP1). However, whether RV structural components also subvert antiviral response remains under-studied. Here, we found that MAVS, critical for the host RNA sensing pathway upstream of IFN induction, is degraded by the RV RNA methyl- and guanylyl-transferase (VP3) in a host-range-restricted manner. Mechanistically, VP3 localizes to the mitochondria and mediates the phosphorylation of a previously unidentified SPLTSS motif within the MAVS proline-rich region, leading to its proteasomal degradation and blockade of IFN-λ production in RV-infected intestinal epithelial cells. Importantly, VP3 inhibition of MAVS activity contributes to enhanced RV replication and to viral pathogenesis in vivo . Collectively, our findings establish RV VP3 as a viral antagonist of MAVS function in mammals and uncover a novel pathogen-mediated inhibitory mechanism of MAVS signaling., Competing Interests: SD, SZ, LR, NF, YS, XG, BL, RF, HG No competing interests declared, (© 2018, Ding et al.)

A stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. Because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. As subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. First, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain RVA/pig-tc/BEL/RV277/1977/G1P[7] and different other rotavirus genotypes (fecal samples containing G5P[7], G5P[13], G9P[23], G4P[6]). Next, the TGEV Purdue strain infected both ileocytes and colonocytes whereas the Miller strain only infected ileocytes. Last, the PEDV CV777 Vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. The infectivity of the CV777 Vero adapted strain was higher when the cells were cultured without fetal bovine serum and the CV777 fecal suspension only infected the ileocytes cultured without fetal bovine serum. In conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses.

Rotavirus (RV) and diarrheagenic are waterborne pathogens commonly causing diarrhea in children below five years old worldwide. Our study is a first step toward a loads-concentrations-risk modeling and scenario analysis framework. We analyzed current and future human RV and indicator (EC) emissions from sanitation facilities to surface waters in Uganda using two process-based models. Emissions were estimated for the baseline year 2015 and for three scenarios in 2030 using population, excretion rates, sanitation types, and wastewater treatment. The first model is a downscaled GloWPa-Rota H1 version, producing emissions at a 1-km resolution. The second model is newly developed for Kampala and adds emissions from pit latrines and septic tanks excluded in the first model. The scenarios Business as Usual, Industrious, and Low Emissions reflect government prospects in sanitation coverage and wastewater treatment. For the first model, 6.14 × 10 RV particles d and 1.31 × 10 EC colony-forming units (CFU) d are emitted to surface waters in 2015. The RV emissions are expected to increase in 2030 by 75% for Business as Usual and 212% for Industrious and decrease by 58% in Low Emissions. Emissions from the second model are higher for Kampala than in the first model, at 3.74 × 10 vs. 5.95 × 10 RV particles d and 8.18 × 10 vs. 1.75 × 10 EC CFU d in 2015, most of which come from the onsite-not-contained category. Simulated emissions for Kampala show the importance of including onsite sanitation in our modeling. Our study is replicable in other locations and helps identify key emission sources, their hotspots, and the importance of wastewater treatment. The scenarios can guide future sanitation safety planning., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

In Uruguay, groundwater is frequently used for agricultural activities, as well as for human consumption in urban and rural areas. As in many countries worldwide, drinking water microbiological quality is evaluated only according to bacteriological standards and virological analyses are not mentioned in the legislation. In this work, the incidence of human viral (Rotavirus A, Norovirus GII, and human Adenovirus) and bacterial (total and thermotolerant coliform and Pseudomonas aeruginosa) contamination in groundwater in the Salto district, Uruguay, as well as the possible correlation between these groups of microorganisms, was studied. From a total of 134 groundwater samples, 42 (32.1%) were positive for Rotavirus, only 1 (0.7%) for both Rotavirus and Adenovirus, and 96 (72.6%) samples were positive for bacterial indicators. Results also show that Rotavirus presence was not associated with changes in chemical composition of the aquifer water. Bacteriological indicators were not adequate to predict the presence of viruses in individual groundwater samples (well scale), but a deeper spatial-temporal analysis showed that they are promising candidates to assess the viral contamination degree at aquifer scale, since from the number of wells with bacterial contamination the number of wells with viral contamination could be estimated.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

We aimed to determine the effects of Lactobacillus strains against rotaviral infections. Rotaviruses are the major causative agent of acute gastroenteritis in infants and children worldwide. However, to date, no specific antiviral drugs for the treatment of rotavirus infection have been developed. We identified 263 Lactobacillus strains from 35 samples of the traditional Korean fermented vegetable food kimchi. Among them, Lactobacillus plantarum LRCC5310, more specifically the exopolysaccharides produced by these cells, were shown to have an antiviral effect against human rotavirus Wa strain in vitro. In vivo, the oral administration of exopolysaccharides for 2 d before and 5 d after mouse infection with the murine rotavirus epidemic diarrhea of infant mice strain led to a decrease in the duration of diarrhea and viral shedding and prevented the destruction of enteric epithelium integrity in the infected mice. We demonstrated here that the exopolysaccharides extracted from L. plantarum LRCC5310 can be used for the effective control of rotavirus infection., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.

It is well known that Korean red ginseng has various biological activities. However, there is little knowledge about the antiviral activity of Korean red ginseng and its ginsenosides. In this study, we addressed whether oral administration of ginsenoside-Rb2 and -Rg3 is able to protect against rotavirus (RV) infection. The protective effect of ginsenosides against RV infection was examined using an in vivo experiment model in which newborn mice (10-day-old) were inoculated perorally (p.o.) with 1.5 × 10 6 plaque-forming units/mouse of RV strain SA11. When various dosages of ginsenoside-Rb2 (25-250 mg/kg) were administered 3days, 2 days, or 1 day before virus challenge, treatment with this ginsenoside at the dosage of 75 mg/kg 3days before virus infection most effectively reduced RV-induced diarrhea. In addition, consecutive administration of ginsenoside-Rb2 (75 mg/kg) at 3 days, 2 days, and 1 day before virus infection was more effective than single administration on day -3. The consecutive administration of ginsenoside-Rb2 also reduced virus titers in the bowels of RV-infected mice. In an experiment to compare the protective activity between ginsenoside-Rb2 and its two hydrolytic products (20(S)- and 20(R)-ginsenoside-Rg3), 20(S)-ginsenoside-Rg3, but not 20(R)-ginsenoside-Rg3, prevented RV infection. These results suggest that ginsenoside-Rb2 and its hydrolytic product, 20(S)-ginsenoside-Rg3, are promising candidates as an antiviral agent to protect against RV infection.

Aim: To analyse group A rotavirus (RVA) environmental contamination in waters used for calves' consumption and to assess viral viability in dairy farm water sources., Methods and Results: We analysed 202 samples of water used for calves' consumption and RVA was detected by RT-qPCR in 35·1% (95% CI: 28·9-42·0%). A marked pattern of seasonality was observed with higher frequency of detection in colder than warmer months (P = 0·002). There was no association between viral load and season or between the number of milking cows in the herd and the detection of RVA in the farm. The viability of the RVA particles detected was confirmed by isolation of RVA in cell culture from 5 of 10 water samples. Furthermore, an RVA waterborne outbreak of neonatal calf diarrhoea was described., Conclusions: We demonstrate that RVA is frequent in dairy farm waters, and that the virus is infectious and capable of generating a diarrhoea outbreak., Significance and Impact of the Study: Neonatal diarrhoea syndrome leads to economic losses to the livestock industry worldwide. To determine transmission routes is essential to take action in this regard and reduce the impact that this syndrome has for the livestock production. The results obtained in this work alert the dairy industry and highlight that mitigation strategies are crucial to improve the microbiological quality of this water., (© 2018 The Society for Applied Microbiology.)

Cathepsins, endosomal acid proteases, are transported from the trans-Golgi network to late endosomes by the mannose-6-phosphate receptor (M6PR). We have previously demonstrated that some rotavirus strains, like UK, Wa, WI61, DS-1, and YM, require the cation-dependent (CD-) M6PR and cathepsins to enter from late endosomes to the cytoplasm in MA104 cells, while other strains, like the simian strain RRV, which enter cells from maturing endosomes, do not. However, the role of other trans-Golgi network-late endosome transporters, such as the cation-independent (CI-) M6PR and sortillin-1, has not been evaluated. In this work, we found that several rotavirus strains that require the CD-M6PR for cell entry are also dependent on CI-M6PR and sortilin-1. Furthermore, we showed that the infectivity of all these rotavirus strains also requires cathepsins to enter not only MA104 cells, but also human intestinal Caco-2 cells. This study identifies sortilin-1 as a novel cell factor necessary for the infectivity of a virus; in addition, our results strongly suggest that cathepsins could be common cell factors needed for the infectivity of most rotavirus strains., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Diarrheal diseases (DD) have distinct etiological profiles in immune-deficient and immune-competent patients. This study compares detection rates, genotype distribution and viral loads of different enteric viral agents in HIV-1 seropositive (n = 200) and HIV-1 seronegative (n = 125) children hospitalized with DD in Rio de Janeiro, Brazil. Except for group A rotavirus (RVA), which were detected through enzyme immunoassay, the other enteric viruses (norovirus [NoV], astrovirus [HAstV], adenovirus [HAdV] and bocavirus [HBoV]) were detected through PCR or RT-PCR. A quantitative PCR was performed for RVA, NoV, HAstV, HAdV and HBoV. Infections with NoV (19% vs. 9.6%; p<0.001), HBoV (14% vs. 7.2%; p = 0.042) and HAdV (30.5% vs. 14.4%; p<0.001) were significantly more frequent among HIV-1 seropositive children. RVA was significantly less frequent among HIV-1 seropositive patients (6.5% vs. 20%; p<0.001). Similarly, frequency of infection with HAstV was lower among HIV-1 seropositive children (5.5% vs. 12.8%; p = 0.018). Among HIV-1 seropositive children 33 (16.5%) had co-infections, including three enteric viruses, such as NoV, HBoV and HAdV (n = 2) and NoV, HAstV and HAdV (n = 2). The frequency of infection with more than one virus was 17 (13.6%) in the HIV-1 negative group, triple infection (NoV + HAstV + HBoV) being observed in only one patient. The median viral load of HAstV in feces was significantly higher among HIV-1 positive children compared to HIV-1 negative children. Concerning children infected with RVA, NoV, HBoV and HAdV, no statistically significant differences were observed in the medians of viral loads in feces, comparing HIV-1 seropositive and HIV-1 seronegative children. Similar detection rates were observed for RVA, HAstV and HAdV, whilst NoV and HBoV were significantly more prevalent among children with CD4+ T lymphocyte count below 200 cells/mm3. Enteric viruses should be considered an important cause of DD in HIV-1 seropositive children, along with pathogens more classically associated with intestinal infections in immunocompromised hosts.

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Objective: Diarrheal diseases are a leading cause of morbidity and mortality worldwide, but the etiology of diarrhea and its relation to nutritional outcomes in resource-limited settings is poorly defined. We sought to determine the etiology of community-acquired diarrhea in Tanzanian infants and to assess the association with anthropometrics and novel intestinal biomarkers., Methods: A convenience sample of infants in a trial of zinc and/or multivitamin supplementation in Tanzania was selected. Subjects were enrolled at age 6 weeks and studied for 18 months. Stool samples were obtained from children with acute diarrhea. A novel, polymerase chain reaction-based TaqMan array was used to screen stool for 15 enteropathogens. A subset of subjects had serum gastrointestinal biomarkers measured., Results: One hundred twenty-three subjects with diarrhea were enrolled. The mean ± SD age at stool sample collection was 12.4 ± 3.9 months. Thirty-five enteropathogens were identified in 34 (27.6%) subjects: 11 rotavirus, 9 Cryptosporidium spp, 7 Shigella spp, 3 Campylobacter jejuni/coli, 3 heat stable-enterotoxigenic Escherichia coli, and 2 enteropathogenic E coli. Subjects with any identified enteropathogen had significantly lower weight-for-length z scores (-0.55 ± 1.10 vs 0.03 ± 1.30, P = 0.03) at the final clinic visit than those without an identified pathogen. Fifty of the 123 subjects (40.7%) had serum analyzed for antibodies to lipopolysaccharide (LPS) and flagellin. Subjects with any identified enteropathogen had lower immunoglobulin (IgA) antibodies to LPS (0.75 ± 0.27 vs 1.13 ± 0.77, P = 0.01) and flagellin (0.52 ± 0.16 vs 0.73 ± 0.47, P = 0.02) than those without an identified pathogen., Conclusions: This quantitative polymerase chain reaction method may allow identification of enteropathogens that place children at higher risk for suboptimal growth. IgA anti-LPS and flagellin antibodies hold promise as emerging intestinal biomarkers., Competing Interests: The authors report no conflicts of interest.

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Environmental approach has proven to be a useful tool for epidemiological studies demonstrating through environmental studies the diversity of viruses circulating in a given population. The aim of this study was to perform a phylogenetic characterization of the group A rotavirus (RVA) glycoprotein (G)- and protease-sensitive (P)-genotypes obtained from sewage samples (n = 116) collected in six cities of Uruguay during March 2011 to April 2013. A worldwide standardized semi-nested multiplex RT-PCR (SNM RT-PCR) protocol directed against VP4 and VP7 genes were conducted for RVA detection and consensual DNA fragments were submitted to nucleotide sequencing. P and/or G genotype was successfully determined by phylogenetic analysis in 61% (n = 37) of the positive samples obtained by SNM RT-PCR (n = 61). The RVA genotypes were as follow: G1 (n = 2), G2 (n = 14), G3 (n = 5), G12 (n = 2), P[4] (n = 4), P[8] (n = 16), and P[3] (n = 2). Interestingly, through phylogenetic analysis, emerging, and uncommon human genotypes could be detected. Results obtained from the comparison of RVA genotypes detected in the current study and Uruguayan RVA strains previously described for contemporary clinical pediatric cases showed that monitoring sewage may be a good screening option for a rapid and economical overview of the circulating genotypes in the surrounding human population and a useful approximation to study RVA epidemiology in a future vaccine monitoring program. The present study represents the first report in Uruguay that describes the phylogenetic diversity of RVA from urban sewage samples.

This study determined whether a hand sanitizing wipe can reduce virus transmission in households, and could reduce the probability of infection by rhinovirus and rotavirus. Bacteriophage MS-2 (a marker virus) was used to assess viral transmission in five households having at least two children of ages 2-18. Hands of one female adult were inoculated with ~10(8) PFU MS-2 bacteriophages in each home, and after 8 h, hands of all family members and select fomites were sampled to determine baseline contamination without intervention. This sequence was repeated with the intervention, where all family members were instructed to use a quaternary ammonium compound-based sanitizing wipe at least once per day. A significant reduction of virus after the intervention occurred on inoculated hands (95.3%; p = 0.0039), all fomites combined (74.5%; p < 0.005), and non-inoculated hands and fomites combined (73.5%; p < 0.005). However, viral reduction on non-inoculated hands was not significant, likely due to small sample size. Using rhinovirus and rotavirus as models it was estimated that infection risk was reduced by ~30 to 89% with the use of sanitizing wipes once per day depending on the starting concentration of these viruses on hands of susceptible individuals. Therefore, using a hand sanitizing wipe can significantly reduce viral transmission and risk of illness in homes. Previous studies have shown other hand hygiene interventions, such as alcohol-based hand sanitizers, are even more effective for reducing risk of illness in homes; however the sanitizing wipe used in this study is appropriate to use for microbial reduction.

Unlabelled: The genetic diversity of rotavirus A (RVA) strains is facilitated in part by genetic reassortment. Although this process of genome segment exchange has been reported frequently among mammalian RVAs, it remained unknown if mammalian RVAs also could package genome segments from avian RVA strains. We generated a simian RVA strain SA11 reassortant containing the VP4 gene of chicken RVA strain 02V0002G3. To achieve this, we transfected BSR5/T7 cells with a T7 polymerase-driven VP4-encoding plasmid, infected the cells with a temperature-sensitive SA11 VP4 mutant, and selected the recombinant virus by increasing the temperature. The reassortant virus could be stably passaged and exhibited cytopathic effects in MA-104 cells, but it replicated less efficiently than both parental viruses. Our results show that avian and mammalian rotaviruses can exchange genome segments, resulting in replication-competent reassortants with new genomic and antigenic features., Importance: This study shows that rotaviruses of mammals can package genome segments from rotaviruses of birds. The genetic diversity of rotaviruses could be broadened by this process, which might be important for their antigenic variability. The reverse genetics system applied in the study could be useful for targeted generation and subsequent characterization of distinct rotavirus reassortant strains., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A total of 136 rotavirus positive samples from diarrhoeic animals of different species were submitted for isolation and cultural propagation of rotavirus on MA-104 cells. The samples were collected from animals with diarrhoea, between 1980 and 2010, originating from herds or farms located in several parts of Germany. Rotaviruses of species A were isolated from 102 faecal samples in cultures of MA-104 cells under the following conditions: pre-treatment of virus with trypsin, incorporation of trypsin into culture medium, use of roller cultures, and centrifugation of the samples on the cells. The cell culture adapted viruses produced a cytopathic effect, accompanied by the release of cells from the glass surface of the cultivation vessels. After 10 passages the virus isolates yielded titres between 10(5.5) and 10(7.5)ml(-1) TCID50. Isolation and serial propagation of the virus in MA-104 cells was confirmed by immunofluorescence assay, transmission electron microscopy, and polyacrylamide-gel electrophoresis of viral dsRNA. Eight (5.9%) of the electrophoretic profiles were characteristic of species B or D rotaviruses, which were not replicated in MA-104 cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We wished to select a cold-adapted genotype G1P[8] ZTR-68 rotavirus (China southwest strain) in MA104 cells for possible use as a live vaccine. ZTR-68 was recovered originally from children with diarrhea. The virus was cultivated at 37 degrees C at the first passage. Then, the cultivation temperature was decreased stepwise by 3 degrees C per eight passages. In total, the virus was passaged 32 times, and cultivation was terminated at 28 degrees C. Biological characteristics of the virus were analyzed during serial passages. There was no difference between the migration patterns of genomic dsRNA segments according to polyacrylamide gel electrophoresis of original and cold-adapted viruses. Infectious and red cell-agglutination titers of cold-adapted virus were lower than those of the parent virus. Also, the virus formed small-size plaques with irregular shapes at 31 degrees C and 28 degrees C. These results suggested that a genetically stable attenuated virus can be obtained through serial cold-adapted passages. Thus, an alternative strategy is provided by cold-adaption for development of attenuated live rotavirus vaccines.

Rotavirus (RV) contains 11 double-stranded RNA segments that encode for twelve structural and nonstructural proteins. The separation and isolation of viral RNA is a necessary precursor for many experimental techniques and can be useful for rapid RV RNA typing and sequencing of different rotavirus strains. The segmented genome enables RV to recombine easily. These recombinant viruses are essential for many purposes, including generation of potential vaccine strains. Rotavirus gene 10 expresses the viral enterotoxin, NSP4, which has been the focus of several studies due to the influence of NSP4 on rotavirus replication, morphogenesis, and pathogenesis. This unit will describe the isolation and separation of viral RNAs, the production characterization of recombinant RV in culture, and the expression and isolation of NSP4 in mammalian and insect cells., (Copyright © 2005 John Wiley & Sons, Inc. All rights reserved.)

Urine from urine-diversion toilets (UDTs) is routinely used as fertilizer for urban agriculture in Ouagadougou, Burkina Faso. Because urine from UDTs can be accidentally spoiled by feces, we determined whether virulent enteric viruses could persist in urine that is used for agricultural purposes and pose a threat to human health. Urine samples (N = 60) were first collected from 42 UDTs during the months of January and February 2012 in Ouagadougou and screened negative for the presence of norovirus (NoV) and group A rotavirus (RV). Composite urine from five collection sites was used to determine whether spiked murine norovirus (MNV) and group A bovine rotavirus (boRVA) could remain infectious at 15, 25, and 42 °C over an incubation period of 42 days in phosphate buffered saline (control) and urine. For both viruses, infectivity was determined by plaque assay and the presence of viral genome was evaluated by real-time RT-PCR. A decrease in the infectious titer was observed in composite urines that were experimentally seeded with MNV and boRVA. The decrease in the infectious titer was greater for MNV than for boRVA. Given that MNV was more labile to urine than boRVA was, MNV and boRVA genomes were still detectable after the 42 and 49 days incubation period for MNV and boRVA, respectively. Our data using substitutes of human NoV and RV suggested that there is a virucidal activity of urine against RVs and NoVs, given that the effect was lesser for RV. In spite of disappointing results for boRVA, the use of urine as fertilizer is still promising provided that future safety studies are extended to other enteric viruses.

Viral gastroenteritis is a serious viral infection which affects a large number of individuals around the world, most of them being children. The infection may occur due to different viruses, for example, coxsackievirus, adenovirus, and rotavirus. There is no available cure for such infections, and the treatment mainly depends on hospitalization and administration of nutritional supports. A new antiviral agent against gastroenteritis viral infection will be a breakthrough in healthcare. Pyrrole and pyrrolopyrimidine derivatives are well known for their biological activity as antibacterial, antifungal, and anticancer agents. These compounds also proved to possess antiviral activity. Here, we synthesized novel pyrrole and pyrrolopyrimidine compounds and examined their antiviral activity. We synthesized several new pyrrole, pyrrolo[2,3-d]pyrimidine, and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. The characterization of all synthesized compounds was based on microanalysis and spectral data. Moreover, we determined the non-toxic doses of these compounds on BGM, Hep-2, and MA-104 cells. We tested all the synthesized compounds for their antiviral activities against coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain. Several compounds exhibited significant activities as antiviral agents., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: Because rotavirus gastroenteritis is associated with high morbidity and mortality especially in developing countries, it is necessary to develop antirotavirus drugs for the treatment of rotavirus infection. Previous studies have demonstrated that cyclosporin A (CsA) has antiviral properties against rotavirus. Its effect has not yet been evaluated against rotavirus diarrheal disease. The aim of this study was to assess the anti-rotavirus efficacy of CsA in neonatal mice after induction of rotavirus diarrhea., Methods: Suckling mice were inoculated with murine rotavirus. On the onset of diarrhea, mice were given different concentrations of CsA. To evaluate the effects of CsA on reduction of rotavirus diarrhea, diarrhea score, fecal virus shedding, and pathological lesion change in the small intestine, messenger RNA (mRNA) expression levels in the small intestine and spleen of mice were measured for type I interferon (IFN-α and IFN-β), inflammation-related cytokines (interleukin [IL]-8, IL-10, IFN-γ, and tumor necrosis factor-α), and inflammatory signaling pathways (p38, c-Jun N-terminal kinase, activator protein-1, and nuclear factor-kappa B)., Results: Among virus-inoculated and CsA-treated groups, a dose of 5 mg · kg⁻¹ · day⁻¹ of CsA inhibited diarrhea and improved fecal virus shedding and intestinal lesion changes. IFN-β mRNA expression was significantly increased in rotavirus-induced diarrhea mice treated with 5 mg · kg⁻¹ · day⁻¹ of CsA, whereas the mRNA expression levels of inflammation-related cytokines (IL-8, IL-10, IFN-γ, and tumor necrosis factor-α) and inflammatory signaling pathways (p38, c-Jun N-terminal kinase, activator protein-1, and nuclear factor-kappa B) were markedly decreased. Antiviral effects of CsA were dose dependent., Conclusions: CsA can inhibit rotavirus infection in neonatal mice through its antiviral properties. The mechanism for this may be through CsA suppression of inflammation by viral inhibition in animal models.

This report from the Australian Rotavirus Surveillance Program, together with collaborating laboratories Australia-wide, describes the rotavirus genotypes responsible for the hospitalisation of children with acute gastroenteritis during the period 1 January to 31 December 2013. During the survey period, 1,035 faecal samples were referred for rotavirus G and P genotype analysis. Of these 828 were confirmed as rotavirus positive. A total of 503 specimens were collected from children under 5 years of age, while 325 were from older children and adults. Genotype analysis of the 828 rotavirus samples collected from both children and adults revealed that G12P[8] was the dominant genotype in this reporting period, identified in 33% of strains nationally. Genotype G3P[8] was the second most common strain nationally, representing 31% of samples, followed by genotype G2P[4] (14%). This represents the first report where G12P[8] strains are the major cause of disease in this population. The genotype distribution was slightly altered when the analysis was restricted to samples collected from children under 5 years of age, with G3P[8] being the dominant genotype (39.2%) followed by G12P[8] as the second most common genotype (31%). Fluctuations in genotype distribution were also observed based on the vaccine type in use. Genotype G12P[8] was more common in states and territories using RotaTeq, while G3P[8] was more common in the locations using Rotarix. This survey highlights the yearly fluctuations in rotavirus genotypes observed since vaccine introduction, with changes in dominant genotypes an annual event. The emergence of G12P[8] as the dominant genotype further illustrates the ongoing changes in the wild type rotavirus population evident in the Australian population since vaccine introduction., (This work is copyright. You may download, display, print and reproduce the whole or part of this work in unaltered form for your own personal use or, if you are part of an organisation, for internal use within your organisation, but only if you or your organisation do not use the reproduction for any commercial purpose and retain this copyright notice and all disclaimer notices as part of that reproduction. Apart from rights to use as permitted by the Copyright Act 1968 or allowed by this copyright notice, all other rights are reserved and you are not allowed to reproduce the whole or any part of this work in any way (electronic or otherwise) without first being given the specific written permission from the Commonwealth to do so. Requests and inquiries concerning reproduction and rights are to be sent to the Online, Services and External Relations Branch, Department of Health, GPO Box 9848, Canberra ACT 2601, or by email to copyright@health.gov.au.)

The numbers of coliforms, Escherichia coli, F-RNA coliphages, bovine enteric calicivirus (BEC) and rotavirus (RV) and presence of non-O157 shiga toxigenic E. coli (STEC) were determined on commercial vacuum packaged beef subprimals at the retail level from swabs obtained from the entire surfaces of 150 cuts that originated from federally and provincially registered plants. The prevalence and log mean numbers of E. coli were higher in provincially registered plants than in federally registered plants; 64% vs 20%, respectively, and -0.3 vs -1.22 log cfu/100 cm(2), respectively. In contrast, the prevalence and mean log numbers of F-RNA coliphages were lower for the provincially registered plants than for the federally registered plants; 31% vs 68% and -0.86 vs -0.13 log cfu/100 cm(2), respectively. One E. coli sample tested positive for stx2 and eae. F-RNA coliphages associated with human origin (GII/GIII) were detected in 12% and 30% of samples that originated from provincially and federally registered plants, respectively. RV RNA was detected in 4% of samples while BEC RNA was not detected. Although the infectivity of RV is unknown, the presence of viable F-RNA coliphages suggests that consumers could potentially be at risk when consuming undercooked meat that is contaminated with RV., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

The production of neutralizing immunoglobulin A (IgA) in edible fruits as a means of oral passive immunization is a promising strategy for the inexpensive treatment of mucosal diseases. This approach is based on the assumption that the edible status remains unaltered in the immunoglobulin-expressing fruit, and therefore extensive purification is not required for mucosal delivery. However, unintended effects associated with IgA expression such as toxic secondary metabolites and protein allergens cannot be dismissed a priori and need to be investigated. This paper describes a collection of independent transgenic tomato lines expressing a neutralizing human IgA against rotavirus, a mucosal pathogen producing severe diarrhea episodes. This collection was used to evaluate possible unintended effects associated with recombinant IgA expression. A comparative analysis of protein and secondary metabolite profiles using wild type lines and other commercial varieties failed to find unsafe features significantly associated with IgA expression. Preliminary, the data indicate that formulations derived from IgA tomatoes are as safe for consumption as equivalent formulations derived from wild type tomatoes.

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.)