Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Deokc Song Kim,Myra Hosmillo,Mia Madel Alfajaro,Ji Yun Kim,Jun Gyu Park,Kyu Yeol Son,Eun Hye Ryu,Frederic Sorgeloos,Hyung Jun Kwon,Su Jin Park,Woo Song Lee,Duck Cho,Joseph Kwon,Jong Soon Choi,Mun Il K 대한수의학회 2014 대한수의학회 학술대회발표집 Vol.2014 No.-

Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

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>

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.

Glycan-specificity of four neuraminidase-sensitive animal rotavirus strains

Kim, Ji-Yun,Kim, Deok-Song,Seo, Ja-Young,Park, Jun-Gyu,Alfajaro, Mia Madel,Soliman, Mahmoud,Baek, Yeong-Bin,Cho, Eun-Hyo,Kwon, Hyung-Jun,Park, Su-Jin,Kang, Mun-Il,Cho, Kyoung-Oh Elsevier Scientific Pub. Co 2017 Veterinary microbiology Vol.207 No.-

<P><B>Abstract</B></P> <P>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 <I>N</I>-linked glycoprotein but not by an inhibitor of <I>O</I>-linked glycoprotein, indicating that these strains utilize both glycolipid and <I>N</I>-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 <I>N</I>-linked glycoprotein.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Porcine P[7] and P[23] strains use α2,3- and α2,6-linked SAs as receptors. </LI> <LI> Bovine P[1] and canine P[3] strains bind α2,6-linked SAs as receptors. </LI> <LI> These strains use SAs on glycolipid and N-linked glycoprotein as receptors. </LI> </UL> </P>

Different virulence of porcine and porcine-like bovine rotavirus strains with genetically nearly identical genomes in piglets and calves

Park, Jun-Gyu,Kim, Hyun-Jeong,Matthijnssens, Jelle,Alfajaro, Mia Madel,Kim, Deok-Song,Son, Kyu-Yeol,Kwon, Hyoung-Jun,Hosmillo, Myra,Ryu, Eun-Hye,Kim, Ji-Yun,Cena, Rohani B,Lee, Ju-Hwan,Kang, Mun-Il,Pa BioMed Central 2013 Veterinary research Vol.44 No.-

<P>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′ untranslated region (UTR) of NSP5 and the 3′ 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.</P>

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

Jeong, Y.J.,Matthijnssens, J.,Kim, D.S.,Kim, J.Y.,Alfajaro, M.M.,Park, J.G.,Hosmillo, M.,Son, K.Y.,Soliman, M.,Baek, Y.B.,Kwon, J.,Choi, J.S.,Kang, M.I.,Cho, K.O. Elsevier Scientific Pub. Co 2015 Veterinary microbiology Vol.176 No.1

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.

Different Virulence of Porcine and Porcine-Like Bovine Rotavirus Strains with Genetically Nearly Identical Genomes in Piglets Andcalves

Jun Gyu Park,Hyun Jeong Kim,Jelle Matthijnssens,Mia Madel Alfajaro,Deok Song Kim,Kyu Yeol Son,Hyoung Jun Kwon,Myra Hosmillo,Eun Hye Ryu,Ji Yun Kim,Rohani B. Cena,Ju Hwan Lee,Mun Il Kang,Sang Ik Park,K 대한수의학회 2014 대한수의학회 학술대회발표집 Vol.2014 No.-

Molecular epidemiology of Korean porcine sapeloviruses

Son, Kyu-Yeol,Kim, Deok-Song,Matthijnssens, Jelle,Kwon, Hyoung-Jun,Park, Jun-Gyu,Hosmillo, Myra,Alfajaro, Mia Madel,Ryu, Eun-Hye,Kim, Ji-Yun,Kang, Mun-Il,Cho, Kyoung-Oh Springer-Verlag 2014 Archives of virology Vol.159 No.5

<P>To evaluate the prevalence and genetic diversity of porcine sapeloviruses (PSVs) in Korea, a total of 100 diarrhea fecal samples from pigs were analyzed by RT-PCR and nested PCR assays with primer pairs specific for the VP1 gene. Overall, 34 % of the diarrhea samples tested positive for PSV, and a high proportion of infections occurred along with a variety of other enteric viruses and bacteria. Genomic and phylogenetic analysis of the VP1 genes revealed pronounced genetic diversities between PSVs from Korean and elsewhere. Our results indicate that PSV infections are very common in Korean pigs with diarrhea. The infecting strains are genetically diverse.</P>

Intestinal and extra-intestinal pathogenicity of a bovine reassortant rotavirus in calves and piglets

Kim, H.J.,Park, J.G.,Matthijnssens, J.,Lee, J.H.,Bae, Y.C.,Alfajaro, M.M.,Park, S.I.,Kang, M.I.,Cho, K.O. Elsevier Scientific Pub. Co 2011 Veterinary microbiology Vol.152 No.3

Despite the impact of bovine group A rotaviruses (GARVs) as economically important and zoonotic pathogens, there is a scarcity of data on cross-species pathogenicity and extra-intestinal spread of bovine reassortant GARVs. During the course of characterizing the genotypes of all 11 genomic segments of bovine GARVs isolated from diarrheic calves in South Korea, a unique G6P[7] reassortant GARV strain (KJ9-1) was isolated. The strain harbors five bovine-like gene segments (VP7: G6; VP6: I2; VP1: R2; VP3: M2; NSP2: N2, and NSP4: E2), five porcine-like gene segments (VP4: P[7]; NSP1: A1; NSP3: T1, and NSP5: H1), and one human-like gene segment (VP2: C2). To investigate if this reassortant strain possessed cross-species pathogenicity in calves and piglets, and could induce viremia and extra-intestinal spread in calves, colostrum-deprived calves and piglets were experimentally inoculated with the KJ9-1 strain. The KJ9-1 strain caused severe diarrhea in experimentally infected calves with extensive intestinal villous atrophy, but replicated without causing clinical symptoms in experimentally infected piglets. By SYBR Green real-time RT-PCR, viral RNA was detected in sera of the calves at post-inoculation day (PID) 1, reaching a peak at PID3, and then rapidly decreasing from PID4. In addition, viral RNA was detected in the mesenteric lymph node, lungs, liver, choroid plexus, and cerebrospinal fluid. An immunofluorescence assay confirmed viral replication in the extra-intestinal organs and tissues of virus-inoculated calves. The data indicates that the homologous/heterologous origin of the NSP4 gene segment (E2 genotype), may play a key role in the ability to cause diarrhea in calves and piglets.

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

Park, J.G.,Kim, D.S.,Matthijnssens, J.,Kwon, H.J.,Zeller, M.,Alfajaro, M.M.,Son, K.Y.,Hosmillo, M.,Ryu, E.H.,Kim, J.Y.,Lee, J.H.,Park, S.J.,Kang, M.I.,Kwon, J.,Choi, J.S.,Cho, K.O. Elsevier Scientific Pub. Co 2014 Veterinary microbiology Vol.172 No.1

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.