Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

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.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

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 sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II.

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-ß-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.

Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells.

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.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

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.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

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.

Glycan-specificity of four neuraminidase-sensitive animal rotavirus strains.

Group A rotaviruses (RVAs) are divided into neuraminidase (NA)-sensitive and NA-insensitive strains depending upon their binding affinity to the VP8* domain in the terminal sialic acids (SAs) of cell surface carbohydrates. Although NA-sensitive strains are known to use terminal SAs as an attachment factor, the exact nature of this attachment factor is largely unknown. Here we show that the specific linkage of SA-containing glycan to glycoprotein or glycolipid is an attachment factor used by NA-sensitive porcine G9P[7] PRG9121 and G9P[23] PRG942, bovine G6P[1] NCDV, and canine G3P[3] strains. Infectivity of porcine G9P[7] and G9P[23] strains was markedly blocked by α2,3-linkage and α2,6-linkage inhibitors, indicating that these strains bind to both α2,3- and α2,6-linked SAs. However, the infectivity of bovine G6P[1] and canine G3P[3] strains was significantly reduced by α2,6-linkage inhibitor but not by α2,3-linkage blockers, demonstrating a predilection of these strains for α2,6-linked SAs. The infectivity of four NA-sensitive strains was equally reduced by inhibitors of lipid membrane and N-linked glycoprotein but not by an inhibitor of O-linked glycoprotein, indicating that these strains utilize both glycolipid and N-linked glycoprotein. Our study demonstrates that four NA-sensitive animal strains could have a strain-dependent binding preference toward α2,6-linked SAs (P[1] NCDV and P[3] CU-1 strains) or both α2,3- and α2,6-linked SAs (P[7] PRG9121 and P[23] PRG942 strains) to the glycolipid and N-linked glycoprotein.

Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production.

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. IMPORTANCE: Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E2 (PGE2) pathway. Targeting of COX-1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2-specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We further demonstrate that the production of PGE2 provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be a new target for treatment of sapovirus infection.

Occurrence and molecular characterization of Sapelovirus A in diarrhea and non-diarrhea feces of different age group pigs in one Korean pig farm.

To determine the occurrence and genetic diversity of Sapelovirus A (SV-A) in diarrhea and non-diarrhea feces of Korean pigs, 110 specimens from different age groups of pigs in the same farm were analyzed by RT-nested PCR. SV-As were detected in 60% of both diarrhea and non-diarrhea specimens regardless of age groups with primer pairs for 2C region, in which all diarrhea samples were co-infected by other enteric pathogens. Phylogenetical analysis of partial VP1 region showed that our strains and several other Korean strains belonged to cluster I, distinct from some strains reported in Korea and other countries. These data indicate that genetically distinct SV-As are frequently detected in Korean pigs irrespective of diarrhea and age.

Pathogenesis of Korean SapelovirusA in piglets and chicks.

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.

Whole genomic characterization of Korean porcine G8P[7] reassortant rotaviruses.

This study analyzed eleven genomic segments of three Korean porcine G8P[7] group A rotavirus (RVA) strains. Phylogenetically, these strains contained two bovine-like and nine porcine-like genomic segments. Eight genes (VP1, VP2, VP6 and NSP1-NSP5) of strains 156-1 and 42-1 and seven genes (VP1, VP2, VP6 and NSP2-NSP5) of strain C-1 clustered closely with porcine and porcine-like animal strains and distantly from typical human Wa-like strains. The VP3-M2 genotype of these strains clustered closely with bovine-like strains, but distantly with typical human DS-1-like strains. These data indicate that multiple reassortments involving porcine and bovine RVA strains in Korea must have occurred.

Porcine Sapelovirus Uses α2,3-Linked Sialic Acid on GD1a Ganglioside as a Receptor.

UNLABELLED: 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. IMPORTANCE: The porcine sapelovirus (PSV) is known to cause enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. However, the receptor(s) that the PSV utilizes to enter host cells remains largely unknown. Using a variety of approaches, we showed that α2,3-linked terminal sialic acid (SA) on the cell surface GD1a ganglioside could be used for PSV binding and infection as a receptor. On the other hand, histo-blood group antigens also present in the cell surface carbohydrates could not be utilized as PSV receptors for binding and infection. These findings should contribute to the understanding of the sapelovirus life cycle and to the development of affordable, useful and efficient drugs for anti-sapelovirus therapy.

Genetic diversity of the VP7, VP4 and VP6 genes of Korean porcine group C rotaviruses.

Porcine group C rotaviruses (RVCs) are considered important pathogens due to their economic impact on pig industry and may also cross the host species barrier toward humans. Unlike RVA, however, genetic and phylogenetic data on RVCs from pigs and other host species are scarce. In the present study, full-length ORF sequences of 26 VP7, 9 VP4 and 9 VP6 genes of Korean porcine RVC strains were compared with those of other known RVC strains by phylogenetic analyses and pairwise identity frequency graphs. Applying the established 85% nucleotide identity cut-off value for RVC VP7 classification, the 26 Korean porcine RVC strains belonged to the G1, G3, G6 and G7 genotypes. Although more complete RVC VP4 sequences are warranted before a definitive cut-off value could be determined, a provisional 83% nucleotide cut-off value proposed for RVC VP4 classification resulted in 7 P-genotypes, 5 of which possessed porcine RVC strains. A 90% nucleotide cut-off value for VP6 divided RVC strains into 7 I-genotypes, 5 of which had porcine RVC strains. G/P/I-genotype comparisons suggested the occurrence of rather frequent reassortment events among Korean porcine RVC strains, and strong geographical differences in the distribution of RVC G-genotypes worldwide. Our data indicate that a large genetic diversity exists among porcine RVC strains. For the final genotype determination of each gene segment, more intensified epidemiological studies on animal and human RVC strains throughout the world are needed.

The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms.

This study reports the prevalence of duck hepatitis A virus (DHAV) types 1 and 3 on Korean duck farms. By RT-nested PCR assays specific for DHAV-1 or DHAV-3, DHAV-1 was detected in 9 of 157 liver samples (5.7 %) from 2 of 30 farms (6.7 %), and DHAV-3 was positive in 104 of 157 liver samples (66.2 %) from 23 of 30 farms (76.7 %). Dual infections with DHAV-1 and DHAV-3 were detected in 23 of 157 samples (14.6 %) from 5 of 30 farms (16.7 %). The data indicate that DHAV-3 infections are prevalent and that DHAV-1 reemerged in Korea, resulting in dual infections on several farms. Our data will help to establish a vaccination policy against DHAV-1 and DHAV-3 in Korea.

Full-length genomic analysis of Korean porcine Sapelovirus strains.

Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, is associated with diarrhea, pneumonia, severe neurological disorders, and reproductive failure in pigs. However, the structural features of the complete PSV genome remain largely unknown. To analyze the structural features of PSV genomes, the full-length nucleotide sequences of three Korean PSV strains were determined and analyzed using bioinformatic techniques in comparison with other known PSV strains. The Korean PSV genomes ranged from 7,542 to 7,566 nucleotides excluding the 3' poly(A) tail, and showed the typical picornavirus genome organization; 5'untranslated region (UTR)-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'UTR. Three distinct cis-active RNA elements, the internal ribosome entry site (IRES) in the 5'UTR, a cis-replication element (CRE) in the 2C coding region and 3'UTR were identified and their structures were predicted. Interestingly, the structural features of the CRE and 3'UTR were different between PSV strains. The availability of these first complete genome sequences for PSV strains will facilitate future investigations of the molecular pathogenesis and evolutionary characteristics of PSV.

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

UNLABELLED: Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. IMPORTANCE: Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation.

Both α2,3- and α2,6-linked sialic acids on O-linked glycoproteins act as functional receptors for porcine Sapovirus.

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-ß-D-galactopyranosyl-ß-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

Comparison of pathogenicities and nucleotide changes between porcine and bovine reassortant rotavirus strains possessing the same genotype constellation in piglets and calves.

Although reassortment is one of the most important characteristics of group A rotavirus (RVA) evolution, the host range restriction and/or virulence of reassortant RVAs remain largely unknown. The porcine 174-1 strain isolated from a diarrheic piglet was identified as a reassortant strain, harboring the same genotype constellation as the previously characterized bovine strain KJ56-1. Owing to its same genotype constellation, the pathogenicity of the porcine strain 174-1 in piglets and calves was examined for comparison with that of the bovine reassortant KJ56-1 strain, whose pathogenicity has already been demonstrated in piglets and calves. The porcine 174-1 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas KJ56-1 had been reported to be virulent only in piglets, but not in calves. Therefore, full genomic sequences of 174-1 and KJ56-1 strains were analyzed to determine whether specific mutations might be associated with clinical and pathological phenotypes. Sequence alignment between the 174-1 and KJ56-1 strains detected one nucleotide substitution at the 3' untranslated region of the NSP3 gene and 16 amino acid substitutions at the VP7, VP4, VP1, VP3, NSP1 and NSP4 genes. These mutations may be critical molecular determinants for different virulence and/or pathogenicity of each strain. This study presents new insights into the host range restriction and/or virulence of RVAs.

Anti-rotavirus effects by combination therapy of stevioside and Sophora flavescens extract.

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

Effects of crosslinked dextran in hydroxylpropyl methylcellulose on soft tissue augmentation in rats.

This study compared crosslinked dextrans in hydroxylpropyl methycellulose (DiHMs, pH 5 or 7) with polymethylmethacrylate in bovine collagen (PMMA) and hyaluronic acid (HA) fillers on soft tissue augmentation and safety in rats. HA tended to maintain its size throughout the experimental period but was moveable and friable because of a lack of thick fibroconnective tissue formation. Although, PMMA induced moderately thick fibroconnective tissue formation, its size was decreased markedly from 3-week postimplantation (PI) and became the smallest at 24-month PI. DiHM (pH 7) elicited strong fibrous encapsulation around the filler. Its size decreased slowly but was still considerably maintained at 24-month PI. In contrast, the rate of the DiHM (pH 5) size decrease was slower than that of PMMA, faster DiHM (pH 7), but comparable to HA. Immunohistochemically, types I and III collagens were deposited inside and outside DiHMs (pH 5 and 7). DiHMs (pH 5 and 7), PMMA, and HA showed no adverse reactions. These results suggest that DiHM (pH 7) assures efficacy and safety and is a good candidate for soft tissue augmentation in both humans and animals.