ML241 Antagonizes ERK 1/2 Activation and Inhibits Rotavirus Proliferation.

Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

Suppression of classical nuclear import pathway by importazole and ivermectin inhibits rotavirus replication.

BACKGROUND: Rotavirus is the foremost cause of acute gastroenteritis among infants in resource-poor countries, causing severe morbidity and mortality. The currently available rotavirus vaccines are effective in reducing severity of the disease but not the infection rates, thus antivirals as an adjunct therapy are needed to reduce the morbidity in children. Viruses rely on host cellular machinery for nearly every step of the replication cycle. Therefore, targeting host factors that are indispensable for virus replication could be a promising strategy. OBJECTIVES: To assess the therapeutic potential of ivermectin and importazole against rotaviruses. METHODS: Antirotaviral activity of importazole and ivermectin was measured against various rotavirus strains (RV-SA11, RV-Wa, RV-A5-13, RV-EW) in vitro and in vivo by quantifying viral protein expression by western blot, analysing viroplasm formation by confocal microscopy, and measuring virus yield by plaque assay. RESULTS: Importin-ß1 and Ran were found to be induced during rotavirus infection. Knocking down importin-ß1 severely impaired rotavirus replication, suggesting a critical role for importin-ß1 in the rotavirus life cycle. In vitro studies revealed that treatment of ivermectin and importazole resulted in reduced synthesis of viral proteins, diminished production of infectious virus particles, and decrease in viroplasm-positive cells. Mechanistic study proved that both drugs perform antirotavirus activity by inhibiting the function of importin-ß1. In vivo investigations in mice also confirmed the antirotavirus potential of importazole and ivermectin at non-toxic doses. Treatments of rotavirus-infected mice with either drug resulted in diminished shedding of viral particles in the stool sample, reduced expression of viral protein in the small intestine and restoration of damaged intestinal villi comapared to untreated infected mice. CONCLUSIONS: The study highlights the potential of importazole and ivermectin as antirotavirus therapeutics.

Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

VP4 Is a Determinant of Alpha-Defensin Modulation of Rotaviral Infection.

Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.

Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses.

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.

Rotavirus A infection in pre- and post-vaccine period: Risk factors, genotypes distribution by vaccination status and age of children in Nampula Province, Northern Mozambique (2015-2019).

Mozambique introduced the monovalent rotavirus vaccine (Rotarix®, GSK Biologicals, Rixensart, Belgium) in September 2015. Previous analysis, showed that Nampula province continues reporting a high frequency of Rotavirus A (RVA) infection and the emergence of G9P[6], G9P[4] and G3P[4] genotypes. This analysis aimed to determine the RVA frequency; risk factors; genotype distribution by vaccination status and age between pre- and post-vaccine periods in children under-five years old with diarrhea in Nampula. A cross-sectional, hospital-based surveillance study was conducted in the Hospital Central de Nampula in Mozambique. Socio-demographic and clinical data were collected to assess factors related to RVA infection in both periods. Stool specimens were screened to detect RVA by ELISA, and positive samples were genotyped. Between 2015 (pre-vaccine period) and 2016-2019 (post-vaccine period), 614 stool specimens were collected and tested for RVA in which 34.9% (67/192) were positive in pre-vaccine period and 21.8% (92/422) in post-vaccine (p = 0.001). In the post-vaccine period, age, year, and contact with different animal species (chicken, duck, or multiple animals) were associated with RVA infection. RVA infection was higher in children partially vaccinated (40.7%, 11/27) followed by the fully vaccinated (29.3%, 56/191) and the unvaccinated (15.3%, 21/137) (p = 0.002). G1P[8] and G9P[4] were common in vaccinated children less than 12 months. The present analysis showed that RVA infection reduced slightly in the post-vaccine period, with a high proportion of infection and genotype diversity in children, under 12 months of age, vaccinated. Further research on factors associated with RVA infection on vaccinated compared to unvaccinated children and vaccination optimization should be done.

Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis.

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.

18ß-Glycyrrhetinic acid inhibits the apoptosis of cells infected with rotavirus SA11 via the Fas/FasL pathway.

CONTEXT: 18ß-Glycyrrhetinic acid (18ß-GA), a pentacyclic triterpenoid saponin metabolite of glycyrrhizin, exhibits several biological activities. OBJECTIVE: We investigated the effects of 18ß-GA on MA104 cells infected with rotavirus (RV) and its potential mechanism of action. MATERIALS AND METHODS: Cell Counting Kit-8 was used to assess tissue culture infective dose 50 (TCID50) and 50% cellular cytotoxicity (CC50) concentration. MA104 cells infected with RV SA11 were treated with 18ß-GA (1, 2, 4, and 8 µg/mL, respectively). Cytopathic effects were observed. The virus inhibition rate, concentration for 50% of maximal effect (EC50), and selection index (SI) were calculated. Cell cycle, cell apoptosis, and mRNA and protein expression related to the Fas/FasL pathway were detected. RESULTS: TCID50 of RV SA11 was 10-4.47/100 µL; the CC50 of 18ß-GA on MA104 cells was 86.92 µg/mL. 18ß-GA showed significant antiviral activity; EC50 was 3.14 µg/mL, and SI was 27.68. The ratio of MA104 cells infected with RV SA11 in the G0/G1 phase and the G2/M phase decreased and increased, respectively, after 18ß-GA treatment. 18ß-GA significantly induced apoptosis in the infected cells. Furthermore, after 18ß-GA treatment, the mRNA and protein expression levels of Fas, FasL, caspase 3, and Bcl-2 decreased, whereas the expression levels of Bax increased. DISCUSSION AND CONCLUSIONS: The study demonstrates that 18ß-GA may be a promising candidate for the treatment of RV SA11 infection and provides theoretical support for the clinical development of glycyrrhizic acid compounds for the treatment of RV infection.

Bovine Lactoferricin Induces Intestinal Epithelial Cell Activation through Phosphorylation of FAK and Paxillin and Prevents Rotavirus Infection.

We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.

Advances in the Development of Antiviral Compounds for Rotavirus Infections.

Group A rotaviruses (RVAs) are the major cause of severe acute gastroenteritis (AGE) in children under 5 years of age, annually resulting in nearly 130,000 deaths worldwide. Social conditions in developing countries that contribute to decreased oral rehydration and vaccine efficacy and the lack of approved antiviral drugs position RVA as a global health concern. In this minireview, we present an update in the field of antiviral compounds, mainly in relation to the latest findings in RVA virion structure and the viral replication cycle. In turn, we attempt to provide a perspective on the possible treatments for RVA-associated AGE, with special focus on novel approaches, such as those representing broad-spectrum therapeutic options. In this context, the modulation of host factors, lipid droplets, and the viral polymerase, which is highly conserved among AGE-causing viruses, are analyzed as possible drug targets.

The anti-rotavirus effect of baicalin via the gluconeogenesis-related p-JNK-PDK1-AKT-SIK2 signaling pathway.

Rotavirus (RV) infection is a leading cause of severe, dehydrating gastroenteritis in children < 5 years of age, and by now, the prevention and treatment of RV are still the major public health problems due to a lack of specific clinical drugs. Thus, the aims of this study are to explore the anti-RV effect of baicalin and its influence on glucose metabolism. Here, we demonstrated for the first time that baicalin had an anti-RV attachment effect with the strongest effect at a concentration of 100 µM, and also inhibited the replication of RV at concentrations of 100, 125, 150, 175, and 200 µM. Moreover, baicalin helped to overcome the weight loss and reduced the diarrhea rate and score with the best therapeutic effect at a concentration of 0.3 mg/g in RV-infected neonatal mice. Interestingly, baicalin decreased glucose consumption in RV-infected Caco-2 cells with the optimal concentration of 125 µM. Next, metabolomic analysis indicated that there were 68 differentially expressed metabolites, including an increase in pyruvic acid, asparagine, histidine and serine, and a decrease in dihydroxyacetone phosphate, which suggested that the underlying signaling pathway was gluconeogenesis. Further studies demonstrated that baicalin inhibited gluconeogenesis via improving glucose 6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxylase (PEPCK). Moreover, baicalin upregulated the potential gluconeogenesis proteins named salt inducible kinase 2, pyruvate dehydrogenase kinase 1, AKT serine/threonine kinase 1 and down-regulated phosphorylated c-Jun NH2-terminal kinase, which are associated with G-6-Pase and PEPCK expressions. Therefore, baicalin improved the gluconeogenesis disruption caused by RV.

Antiviral activity of the sea cucumber tegument extract (Pattalus mollis) on human rotavirus A (RVA).

The antiviral effect against RVA in cell culture was evaluated by using an aqueous extract of Patallus mollis sea cucumber, applying the titration methodology. This technique is used to measures the ability of the extract dilutions to inhibit the cytopathic effect (CPE) of the virus, expressed as percentage of inhibition (IP). The mean extract cytotoxic concentration (CC50) used in the antiviral assay was 27,042.10 µg/mL and the PI of the antiviral activity extract was greater than 99.9% for each concentration. To determine the viral action mode, the cells were previously treated with the extracts in different stages during the viral infection cycle. The result analysis suggests that the extract inhibits 99% of the virus during the absorption and viral inactivation phase. These results show the P. mollis extract has a remarkable antiviral effect against the RVA in cell culture. So that, it is crucial to investigate its action mechanisms.

Mutations in the VP2 gene of rotavirus associated with benzimidazole sensitivity.

Rotavirus species A (RVA) is the etiological agent of acute gastroenteritis in young individuals of various animal species, including humans. Vaccination has helped to reduce the impact of these viruses on humans and some species of domestic mammals, but they do not confer complete immunity, so antirotavirus agents are another important control option. In this study, millimolar concentrations of benzimidazole inhibited the replication of the Rhesus rotavirus (RRV) strain of RVA. Two mutants partially resistant to the inhibitory effect of benzimidazole were independently selected, and their genomes and those of their parental strains were fully sequenced. Most (7/11) mutations occurred in the gene that encodes the VP2 protein, and similarly most of the missense mutations (5/9), including the only one shared by the two mutants (G2,414 → R[G/A], D800 N), occurred in the VP2 gene. Our results identify the VP2 gene as the primary target affected by benzimidazole.

Biosynthesis of silver nanoparticles using isolated Bacillus subtilis: characterization, antimicrobial activity, cytotoxicity, and their performance as antimicrobial agent for textile materials.

Silver nanoparticles (AgNPs) have unique properties and a large range of applications. Biosynthesis of stable AgNPs using the extracellular filtrate of Bacillus subtilis was proved by the characteristic surface plasmon resonance at about 420-430 nm. They were polycrystalline with spherical, hexagonal, and irregular shapes and they were negatively charged (-40 mV) with an average diameter of 20 nm. FTIR spectrum confirmed the presence of protein molecules coating AgNPs. The optimum conditions for the synthesis of tested AgNPs were 1:6 filtrate dilution, 1 mM AgNO3, pH 7, 30 °C , 48 h contact time under static and illuminating conditions. The synthesized AgNPs showed antibacterial activities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Salmonella typhi, antifungal activity against Candida albicans and antiviral activity against rotavirus. Also, they showed potent cytotoxic effects on lung and hepatic carcinoma human cell lines. Meanwhile, the acute toxicity study against mice showed no significant changes in hematological, biochemical, and histological parameters of AgNPs treated mice. They also showed mild hepatoprotective effect in thioacetamide (TAA) - induced hepatic fibrosis in rats. AgNPs treated textiles fabrics showed high antimicrobial activities against different pathogenic microorganisms as well as UV protection adequacy.

Valeriana jatamansi Jones Inhibits Rotavirus-Induced Diarrhea via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway.

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1ß (IL-1ß), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

In-vitro inhibitory effect of maternal breastmilk components on rotavirus vaccine replication and association with infant seroconversion to live oral rotavirus vaccine.

BACKGROUND: Despite contributing to a significant reduction in rotavirus associated diarrhoea in highly burdened low- and middle-income countries, live attenuated, oral rotavirus vaccines have lower immunogenicity and efficacy in these settings in comparison to more developed countries. Breastmilk has been implicated among factors contributing to this lowered oral vaccine efficacy. We conducted in-vitro experiments to investigate the inhibitory effects of maternal antibody and other non-antibody components in breastmilk on rotavirus vaccine strain (Rotarix) multiplication in MA104 cell culture system and assessed associations with in-vivo vaccine seroconversion in vaccinated infants. METHODS: Breastmilk samples were collected from mothers before routine rotavirus vaccination of their infant at 6 weeks of age. For each sample, whole breastmilk, purified IgA, purified IgG and IgG and IgA depleted breastmilk samples were prepared as exposure preparations. A 96 well microtitre plate was set up for each sample including a control in which only MA104 cells were grown as well as a virus control with MA104 cells and virus only. The outcome of interest was 50% inhibition dilution of each of the exposure preparations calculated as the titer at which 50% of virus dilution was achieved. Samples from 30 women were tested and correlated to vaccine seroconversion status of the infant. HIV status was also correlated to antiviral breastmilk proteins. RESULTS: The mean 50% inhibitory dilution titer when whole breastmilk was added to virus infected MA104 cells was 14.3 (95% CI: 7.1, 22.7). Incubation with purified IgG resulted in a mean 50% inhibitory dilution of 5 (95%CI -1.6, 11.6). Incubating with purified IgA resulted in a mean 50% inhibitory dilution of 6.5 (95% CI -0.7, 13.7) and IgG and IgA depleted breastmilk did not yield any inhibition with a titer of 1.06 (95%CI 0.9, 1.2). Higher milk IgA levels contributed to a failure of infants to seroconvert. HIV was also not associated with any antiviral breastmilk proteins. DISCUSSION AND CONCLUSION: Whole breastmilk and breastmilk purified IgG and IgA fractions showed inhibitory activity against the rotavirus vaccine Rotarix™ whilst IgA and IgG depleted breastmilk with non-antibody breastmilk fraction failed to show any inhibition activity in-vitro. These findings suggest that IgA and IgG may have functional inhibitory properties and indicates a possible mechanism of how mothers in rotavirus endemic areas with high titres of IgA and IgG may inhibit viral multiplication in the infant gut and would potentially contribute to the failure of their infants to serocovert. There was not association of HIV with either lactoferrin, lactadherin or tenascin-C concentrations.

Genipin inhibits rotavirus-induced diarrhea by suppressing viral replication and regulating inflammatory responses.

Rotavirus is the leading cause of acute gastroenteritis among young children worldwide. However, agents specifically designed to treat rotavirus infection have not been developed yet. In this study, the anti-rotavirus and anti-inflammatory effects of genipin, a chemical compound found in the fruit of Gardenia jasminoides, were evaluated. Genipin had an antiviral effect against the human rotavirus Wa and SA-11 strains in vitro, and it inhibited two distinct stages of the viral replication cycle: attachment and penetration (early stage) in pre-treatment and assembly and release (late stage) in post-treatment. Additionally, genipin downregulated nitric oxide synthase and pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 cells and rotavirus-infected Caco-2 cells. Oral administration of genipin before and after viral infection with the murine rotavirus epidemic diarrhea of infant mice strain led to a reduced duration of diarrhea and faecal viral shedding and to decreased destruction of the enteric epithelium. Genipin could have potential as a natural compound with preventive and therapeutic effects against infection and colitis caused by rotavirus.

Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity.

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.