Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both G6P[5] WC3 and mono-reassortant G4P[5] strains recognized the αGal HBGA. The infectivity of both P[5]-bearing strains was significantly reduced in αGal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by coincubation with the α2,6-linked SA-specific Sambucus nigra lectin, but not by the α2,3-linked specific sialidase or by Maackia amurensis lectin. Free NeuAc and the αGal trisaccharide also prevented the infectivity of both strains. This indicated that both P[5]-bearing strains utilize α2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack α2,6-linked SA or αGal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight-junction proteins bound directly to the VP5* domain can be used by the P[5]-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P[5]-bearing strains have potential for cross-species transmission.IMPORTANCE Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P[5] VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P[5] vaccine strains recognize both α2,6-linked SA and αGal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P[5]-bearing strains. However, we observed that the P[5]-bearing WC3 and G4P[5] RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P[5] RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.

Schisandrin C (Sch C) is one of the main components of Schisandra chinensis (Schisandra). Since the olden times, Schisandra has been used as a traditional herbal medicine in Asia. Recent studies have shown that Schisandra is effective against irritable bowel syndrome (IBS) in an animal model and affects IBS through the 5-HT3A pathway in the IBS rat model. However, there lacks fundamental research on the interaction of specific components of Schisandra with the 5-HT3A receptor for the treatment of IBS. We hypothesized that a component of Schisandra binds to the 5-HT3A receptor and identified Sch C via a screening work using two electrode-voltage clamps (TEVC). Thus, we aimed to elucidate the neuropharmacological actions between Sch C and the 5-HT3A receptor at molecular and cellular levels. Co-treatment of Sch C with 5-HT inhibited I5-HT in a reversible, concentrate-dependent, like-competition, and voltage-independent manner, and IC50 values of Sch C. Besides, the main binding positions of Sch C were identified through 3D modeling and point mutation were V225A and V288Y on 5-HT3A receptor. Thus, we suggest the potential of Sch C in treating IBS in a manner that suppresses excessive neuronal serotonin signaling in the synapse of sensory neurons and enterochromaffin (EC) cells. In conclusion, the results demonstrate the mechanism of interaction between Sch C and 5-HT3A receptor and reveal Sch C as a novel antagonist.

Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor.IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

The biosynthesis of host lipids and/or lipid droplets (LDs) has been studied extensively as a putative therapeutic target in diverse viral infections. However, directly targeting the LD lipolytic catabolism in virus-infected cells has not been widely investigated. Here, we show the linkage of the LD-associated lipase activation to the breakdown of LDs for the generation of free fatty acids (FFAs) at the late stage of diverse RNA viral infections, which represents a broad-spectrum antiviral target. Dysfunction of membrane transporter systems due to virus-induced cell injury results in intracellular malnutrition at the late stage of infection, thereby making the virus more dependent on the FFAs generated from LD storage for viral morphogenesis and as a source of energy. The replication of SARS-CoV-2 and influenza A virus (IAV), which is suppressed by the treatment with LD-associated lipases inhibitors, is rescued by supplementation with FFAs. The administration of lipase inhibitors, either individually or in a combination with virus-targeting drugs, protects mice from lethal IAV infection and mitigates severe lung lesions in SARS-CoV-2-infected hamsters. Moreover, the lipase inhibitors significantly reduce proinflammatory cytokine levels in the lungs of SARS-CoV-2- and IAV-challenged animals, a cause of a cytokine storm important for the critical infection or mortality of COVID-19 and IAV patients. In conclusion, the results reveal that lipase-mediated intracellular LD lipolysis is commonly exploited to facilitate RNA virus replication and furthermore suggest that pharmacological inhibitors of LD-associated lipases could be used to curb current COVID-19- and future pandemic outbreaks of potentially troublesome RNA virus infection in humans.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.

The receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6'-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensislectin andSambucus nigralectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

Tight junctions (TJs) are a major barrier and also an important portal of entry for different pathogens. Porcine sapovirus (PSaV) induces early disruption of the TJ integrity of polarized LLC-PK cells, allowing it to bind to the buried occludin co-receptors hidden beneath the TJs on the basolateral surface. However, the signaling pathways involved in the PSaV-induced TJ dissociation are not yet known. Here, we found that the RhoA/ROCK/MLC signaling pathway was activated in polarized LLC-PK cells during the early infection of PSaV Cowden strain in the presence of bile acid. Specific inhibitors of RhoA, ROCK, and MLC restored PSaV-induced reduction of transepithelial resistance, increase of paracellular flux, intracellular translocation of occludin, and lateral membrane lipid diffusion. Moreover, each inhibitor significantly reduced PSaV replication, as evidenced by a reduction in viral protein synthesis, genome copy number, and progeny viruses. The PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, known to dissociate TJs, were not activated during early PSaV infection. Among the above signaling pathways, the RhoA/ROCK/MLC signaling pathway was only activated by PSaV in the absence of bile acid, and specific inhibitors of this signaling pathway restored early TJ dissociation. Our findings demonstrate that PSaV binding to cell surface receptors activates the RhoA/ROCK/MLC signaling pathway, which in turn disrupts TJ integrity via the contraction of the actomyosin ring. Our study contributes to understanding how PSaV enters the cells and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.IMPORTANCEPorcine sapovirus (PSaV), one of the most important enteric pathogens, is known to disrupt tight junction (TJ) integrity to expose its buried co-receptor occludin in polarized LLC-PK cells. However, the cellular signaling pathways that facilitate TJ dissociation are not yet completely understood. Here, we demonstrate that early infection of PSaV in polarized LLC-PK cells in either the presence or absence of bile acids activates the RhoA/ROCK/MLC signaling pathway, whose inhibitors reverse the early PSaV infection-induced early dissociation of TJs and reduce PSaV replication. However, early PSaV infection did not activate the PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, which are also known to dissociate TJs. This study provides a better understanding of the mechanism involved in early PSaV infection-induced disruption of TJs, which is important for controlling or preventing PSaV and other calicivirus infections.

Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis-which have proviral and antiviral effects, respectively-exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

Intestinal epithelial tight junctions (TJ) are a major barrier restricting the entry of various harmful factors including pathogens; however, they also represent an important entry portal for pathogens. Although the rotavirus-induced early disruption of TJ integrity and targeting of TJ proteins as coreceptors are well-defined, the precise molecular mechanisms involved remain unknown. In the present study, infection of polarized MDCK cells with the species A rotavirus (RVA) strains human DS-1 and bovine NCDV induced a redistribution of TJ proteins into the cytoplasm, a reversible decrease in transepithelial resistance, and an increase in paracellular permeability. RhoA/ROCK/MLC signaling was identified as activated at an early stage of infection, while inhibition of this pathway prevented the rotavirus-induced early disruption of TJ integrity and alteration of TJ protein distribution. Activation of pMYPT, PKC, or MLCK, which are known to participate in TJ dissociation, was not observed in MDCK cells infected with either rotavirus strain. Our data demonstrated that binding of RVA virions or cogent VP8* proteins to cellular receptors activates RhoA/ROCK/MLC signaling, which alters TJ protein distribution and disrupts TJ integrity via contraction of the perijunctional actomyosin ring, facilitating virion access to coreceptors and entry into cells.

Cyclooxygenases (COXs)/prostaglandin E2 (PGE2) signaling pathways are known to modulate a variety of homeostatic processes and are involved in various pathophysiological conditions. COXs/PGE2 signaling pathways have also been demonstrated to have proviral or antiviral effects, which appeared different even in the same virus family. A porcine sapovirus Cowden strain, a member of genus Sapovirus within the Caliciviridae family, induces strong COX-2/PGE2 but transient COX-1/PGE2 signaling to enhance virus replication. However, whether infections of other viruses in the different genera activate COXs/PGE2 signaling, and thus affect the replication of viruses, remains unknown. In the present study, infections of cells with the feline calicivirus (FCV) F9 strain in the genus Vesivirus and murine norovirus (MNV) CW-1 strain in the genus Norovirus only activated the COX-2/PGE2 signaling in a time-dependent manner. Treatment with pharmacological inhibitors or transfection of small interfering RNAs (siRNAs) against COX-2 enzyme significantly reduced the production of PGE2 as well as FCV and MNV replications. The inhibitory effects of these pharmacological inhibitors against COX-2 enzyme on the replication of both viruses were restored by the addition of PGE2. Silencing of COX-1 via siRNAs and inhibition of COX-1 via an inhibitor also decrease the production of PGE2 and replication of both viruses, which can be attributed to the inhibition COX-1/PGE2 signaling pathway. These data indicate that the COX-2/PGE2 signaling pathway has proviral effects for the replication of FCV and MNV, and pharmacological inhibitors against these enzymes serve as potential therapeutic candidates for treating FCV and MNV infections.

Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection.