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Molecular detection of genotype 3 porcine hepatitis E virus in aborted fetuses and their sows
Hosmillo, Myra,Jeong, Young-Ju,Kim, Hyun-Jeong,Park, Jun-Gyu,Nayak, Mukti Kant,Alfajaro, Mia Madel,Collantes, Therese Marie,Park, Su-Jin,Ikuta, Kazuyoshi,Yunoki, Mikihiro,Kang, Mun-Il,Park, Sang-Ik,Ch Springer-Verlag 2010 Archives of virology Vol.155 No.7
Porcine sapovirus replication is restricted by the type I interferon response in cell culture
Hosmillo, Myra,Sorgeloos, Fré,dé,ric,Hiraide, Rintaro,Lu, Jia,Goodfellow, Ian,Cho, Kyoung-Oh Society for General Microbiology 2015 The Journal of general virology Vol.96 No.1
<P><I>Porcine sapovirus</I> (PSaV) of the family <I>Caliciviridae</I>, is the only member of the genus <I>Sapovirus</I> with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-β resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2′,5′-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100–150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host–cell interactions.</P>
Sapovirus Translation Requires an Interaction between VPg and the Cap Binding Protein eIF4E
Hosmillo, Myra,Chaudhry, Yasmin,Kim, Deok-Song,Goodfellow, Ian,Cho, Kyoung-Oh American Society for Microbiology 2014 Journal of virology Vol.88 No.21
<P>Sapoviruses of the <I>Caliciviridae</I> 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 <I>Sapovirus</I> 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 <I>Vesivirus</I> 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.</P><P><B>IMPORTANCE</B> Sapoviruses, which are members of the <I>Caliciviridae</I> 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.</P>
Kim, Deok-Song,Hosmillo, Myra,Alfajaro, Mia Madel,Kim, Ji-Yun,Park, Jun-Gyu,Son, Kyu-Yeol,Ryu, Eun-Hye,Sorgeloos, Frederic,Kwon, Hyung-Jun,Park, Su-Jin,Lee, Woo Song,Cho, Duck,Kwon, Joseph,Choi, Jong- Public Library of Science 2014 PLoS pathogens Vol.10 No.6
<▼1><P>Sapovirus, a member of the <I>Caliciviridae</I> 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 <I>Sapovirus</I> 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 <I>Vibrio cholerae</I> 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 <I>Maackia amurensis</I> lectin (MAL), both specific for α2,3-linked sialic acid, or <I>Sambucus nigra</I> 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-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits <I>O</I>-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or <I>N</I>-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 <I>O</I>-linked glycosylation.</P></▼1><▼2><P><B>Author Summary</B></P><P>Although enteropathogenic sapoviruses and noroviruses are leading causes of acute gastroenteritis in both humans and animals, the study of viral pathogenesis and immunity of these ubiquitous pathogens has been hampered due to the lack of a fully permissive cell culture system. Porcine sapovirus Cowden strain provides a suitable system that can be used to identify the molecular mechanisms of viral pathogenesis. Previous studies have shown that carbohydrates and glycolipids play important roles in the attachment of members of the <I>Caliciviridae</I>; histo-blood group antigens (HBGAs) are used by <I>Norovirus</I> genogroups I to IV, as well as members of the <I>Lagovirus</I>, and <I>Recovirus</I> genera, whereas terminal sialic acid is recognized as a receptor for feline calicivirus and murine norovirus. To date, however, the role of carbohydrates in the life cycle of sapoviruses has remained largely unknown. We found that porcine sapovirus binds to susceptible host cells through both α2,3- and α2,6-linked terminal sialic acids which are attached to <I>O</I>-linked glycoproteins. These efforts, findings and insights will significantly contribute to a better understanding of the sapovirus life cycle.</P></▼2>
Detection and molecular characterization of porcine group C rotaviruses in South Korea
Jeong, Y.J.,Park, S.I.,Hosmillo, M.,Shin, D.J.,Chun, Y.H.,Kim, H.J.,Kwon, H.J.,Kang, S.Y.,Woo, S.K.,Park, S.J.,Kim, G.Y.,Kang, M.I.,Cho, K.O. Elsevier Scientific Pub. Co 2009 Veterinary microbiology Vol.138 No.3
Group C rotaviruses (GCRVs) cause acute diarrhea in humans and animals worldwide and the evidence for a possible zoonotic role of GCRVs has been recently provided. However, there is little evidence of porcine GCRV infections or of their genetic diversity in South Korea. We examined 137 diarrheic fecal specimens from 55 farms collected from six provinces. RT-PCR utilizing primer pairs specific for the GCRV VP6 gene detected GCRV-positive reactions in 36 (26.2%) diarrheic fecal samples. Of these, 17 samples (12.4%) tested positive for porcine GCRVs alone and 19 samples (13.8%) were also positive for other pathogens. Other enteric pathogens except for GCRV were detected in 64 feces samples (46.7%) and no enteric pathogens were evident in 37 feces samples (27.0%). Phylogenetic and sequence homology analyses of GCRV partial VP6 gene between 23 Korean and other known porcine GCRVs demonstrated that Korean strains belonged to the porcine lineage. Furthermore, one Korean porcine strain shared the highest nucleotide (89.7-89.0%) and deduced amino acid sequence (92.9-93.9%) identities with bovine GCRV strains and was placed in the bovine GCRV lineage indicative of bovine origin. In conclusion, porcine GCRV infections are widespread in piglets with diarrhea in South Korea. The infecting porcine GCRVs mostly belong to the porcine lineage with the exception of one bovine-like GCRV, which possibly originated from bovine GCRV due to interspecies transmission.
Pathogenicity characterization of a bovine triple reassortant rotavirus in calves and piglets
Kim, H.J.,Park, J.G.,Alfajaro, M.M.,Kim, D.S.,Hosmillo, M.,Son, K.Y.,Lee, J.H.,Bae, Y.C.,Park, S.I.,Kang, M.I.,Cho, K.O. Elsevier Scientific Pub. Co 2012 Veterinary microbiology Vol.159 No.1
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.
Chun, Young-Hyun,Jeong, Young-Ju,Park, Sang-Ik,Hosmillo, Myra,Shin, Dong-Jun,Kwon, Hyung-Jun,Kang, Shien-Young,Woo, Sang-Kyu,Kang, Mun-Il,Cho, Kyoung-Oh AAVLD 2010 Journal of veterinary diagnostic investigation Vol.22 No.1
<P>Although the widespread occurrence of porcine group C rotaviruses (GCRV) is assumed, precise prevalence remains largely unknown because of the absence of reliable, specific, and rapid diagnostic methods. To detect and quantify porcine GCRV, the authors evaluated and optimized SYBR Green and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) assays and applied them to 108 piglet fecal samples. Using serially diluted standard RNA transcripts of porcine GCRV VP6 gene, both SYBR Green and TaqMan real-time RT-PCR assays detected as few as 1 x 10(1) genome copies/microl (correlation coefficiency >0.99), whereas conventional RT-PCR detected 1.0 x 10(3) copies/microl. In addition, the conventional assay detected porcine GCRV in 24% (26/108) of fecal samples, whereas the detection rates of both SYBR Green and TaqMan assays were 72% (78 of 108) and 64% (70 of 108), respectively. The current study indicated that both real-time RT-PCR assays were reliable, specific, and rapid methods for the detection of porcine GCRV in porcine fecal samples.</P>