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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.

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. ispartof: Archives of Virology vol:161 issue:10 pages:2835-2841 ispartof: location:Austria status: published

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., Author summary Viral particles must transport their genome into the cytoplasm or the nucleus of host cells to initiate successful infection. Knowledge of how viruses may pirate host cell signaling cascades or molecules to promote their own replication can facilitate the development of antiviral drugs. Group A rotavirus (RVA) is a major etiological agent of acute gastroenteritis in young children and the young of various mammals. RVA enters cells by a complex multistep process. However, the cellular signaling cascades or molecules that facilitate these processes are incompletely understood. Here, we demonstrate that infection with late-penetration RVA strains results in phosphorylation of PI3K, Akt, and ERK signaling molecules at an early stage of infection, a process mediated by the multistep binding of RVAs outer capsid proteins. Specific inhibitors for PI3K/Akt and MEK/ERK signaling pathways trap the viral particles in late endosome, and acidic replenishment restores and releases them. Moreover, the RVA-induced phosphorylated PI3K, Akt, and ERK directly interact with the subunit E of the V-ATPase proton pump, required for endosomal acidification and RVA uncoating. Understanding how RVA-induced early activation of cellular signaling molecules mediates the V-ATPase-dependent endosomal acidification required for uncoating of viral particles opens up opportunities for targeted interventions against rotavirus entry.

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.

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.

Group A rotaviruses (RVAs) are agents causing severe gastroenteritis in infants and young animals. G9 RVA strains are believed to have originated from pigs. However, this genotype has emerged as the fifth major human RVA genotype worldwide. To better understand the relationship between human and porcine RVA strains, complete RVA genome data are needed. For human RVA strains, the number of complete genome data have grown exponentially. However, there is still a lack of complete genome data on porcine RVA strains. Recently, G9 RVA strains have been identified as the third most important genotype in diarrheic pigs in South Korea in combinations with P[7] and P[23]. This study is the first report on complete genome analyses of 1 G9P[7] and 3 G9P[23] porcine RVA strains, resulting in the following genotype constellation: G9-P[7]/P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. By comparisons of these genotype constellations, it was revealed that the Korean G9P[7] and G9P[23] RVA strains possessed a typical porcine RVA backbone, similar to other known porcine RVA strains. However, detailed phylogenetic analyses revealed the presence of intra-genotype reassortments among porcine RVA strains in South Korea. Thus, our data provide genetic information of G9 RVA strains increasingly detected in both humans and pigs, and will help to establish the role of pigs as a source or reservoir for novel human RVA strains.

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. publisher: Elsevier articletitle: Genetic diversity of the VP7, VP4 and VP6 genes of Korean porcine group C rotaviruses journaltitle: Veterinary Microbiology articlelink: http://dx.doi.org/10.1016/j.vetmic.2014.12.024 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved. ispartof: Veterinary Microbiology vol:176 issue:1 pages:61-9 ispartof: location:Netherlands status: published

Direct interspecies transmissions of group A rotaviruses (RVA) have been reported under natural conditions. However, the pathogenicity of RVA has never been directly compared in homologous and heterologous hosts. The bovine RVA/Cow-tc/KOR/K5/2004/G5P[7] strain, which was shown to possess a typical porcine-like genotype constellation similar to that of the G5P[7] prototype RVA/Pig-tc/USA/OSU/1977/G5P9[7] strain, was examined for its pathogenicity and compared with the porcine G5P[7] RVA/Pig-tc/KOR/K71/2006/G5P[7] strain possessing the same genotype constellation. The bovine K5 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas the porcine K71 strain caused diarrhea and histopathological changes in the small intestine of piglets, but not in calves. Furthermore, the bovine K5 strain showed extra-intestinal tropisms in both piglets and calves, whereas the porcine K71 strain had extra-intestinal tropisms in piglets, but not in calves. Therefore, we performed comparative genomic analysis of the K71 and K5 RVA strains to determine whether specific mutations could be associated with these distinct clinical and pathological phenotypes. Full-length sequencing analyses for the 11 genomic segments for K71 and K5 revealed that these strains were genetically nearly identical to each other. Two nucleotide mutations were found in the 5[prime] untranslated region (UTR) of NSP5 and the 3[prime] UTR of NSP3, and eight amino acid mutations in VP1-VP4 and NSP2. Some of these mutations may be critical molecular determinants for RVA virulence and/or pathogenicity. ispartof: Veterinary Research vol:44 issue:1 ispartof: location:England status: published

G9 group A rotaviruses (RVAs) are considered important pathogens in pigs and humans, and pigs are hypothesized to be a potential host reservoir for human. However, intestinal and extra-intestinal pathogenicity and viremia of porcine G9 RVAs has remained largely unreported. In this study, colostrum-deprived piglets were orally infected with a porcine G9P[23] or G9P[7] strain. Histopathologically, both strains induced characteristic small intestinal lesions. Degeneration and necrosis of parenchymal cells were observed in the extra-intestinal tissues, but most predominantly in the mesenteric lymph nodes (MLNs). RVA antigen was continuously detected in the small intestinal mucosa and MLNs, but only transiently in cells of the liver, lung, and choroid plexus. Viral RNA levels were much higher in the feces and the MLNs compared to other tissues. The onset of viremia occurred at day post infection (DPI) 1 with the amount of viral RNA reaching its peak at DPI 3 or 5, before decreasing significantly at DPI 7 and remaining detectable until DPI 14. Our data suggest that porcine G9 RVAs have a strong small intestinal tropism, are highly virulent for piglets, have the ability to escape the small intestine, spread systemically via viremia, and replicate in extra-intestinal tissues. In addition, MLNs might act as a secondary site for viral amplification and the portal of systemic entry. These results add to our understanding of the pathogenesis of human G9 RVAs, and the validity of the pig model for use with both human and pig G9 RVAs in further studies.

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.

Rotaviruses are important human and animal pathogens with high impact on public health and livestock industry. There is little evidence about the cross-species pathogenicity and extra-intestinal infections of animal and human reassortant rotaviruses, particularly based on all 11 genotyping data. In this study, the bovine triple reassortant KJ56-1 strain harboring two bovine-like genome segments, eight porcine-like genome segments, and one human-like genome segment was used to evaluate the cross-species pathogenicity in its parent species, calves and piglets, and to determine its abilities of causing viremia and extra-intestinal tropisms in piglets. The KJ56-1 strain isolated from a calf diarrhea fecal sample replicated without causing diarrhea and severe intestinal pathology in calves. However, piglets inoculated with this strain showed persistent severe diarrhea and marked intestinal pathology. By SYBR Green real-time RT-PCR, viral RNA was detected in the sera, mesenteric lymph node, lung, liver, choroid plexus, and cerebrospinal fluid in the experimental piglets. An immunofluorescence assay confirmed viral replication in these extra-intestinal organs and tissues. These results indicated that the bovine triple reassortant KJ56-1 strain was virulent to piglets but not to calves. Our data also demonstrated that the reassortant rotaviruses had the ability to spread to the bloodstream from the gut, enter and amplify in the mesenteric lymph node, and disseminate to the extra-intestinal organs and tissues.

Group A rotaviruses (GARVs) cause severe acute gastroenteritis in children and young animals. Although zoonotic infections with bovine-like G6 and G8 GARVs have been reported in many countries, there is little evidence for reassortment between bovine GARVs and GARVs from heterologous species. The finding of bovine GARVs with the G6 and G8 genotypes in combination with the typical porcine P[7] prompted us to characterize all 11 genes of 30 bovine GARVs isolated from clinically infected calves. By the comparison of the full-length ORF of VP7 and NSP1–5, and the partial VP1–4 and VP6 nucleotide sequences between the 30 Korean and other known strains, three different genome constellations were found. Twenty seven strains showed the G8-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotypes, a single strain possessed the G6-P[7]-I2-R2-C1-M2-A1-N2-T1-E2-H1 genotype constellation and 2 strains the G6-P[7]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotype constellation. The complete genome of a single reference strains for each of these three genotype constellations (KJ25, KJ9-1 and KJ19-2) was determined and analyzed. A detailed phylogenetic analysis revealed a complicated picture, with several reassortments among bovine-like, porcine-like and human-like GARV strains, resulting in several different reassortant strains successfully infecting cattle.