Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Whole Genomic Analysis of Human G12P[6] and G12P[8] Rotavirus Strains that Have Emerged in Myanmar

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.     

Genome Sequence of an Unusual Human G10P[8] Rotavirus Detected in Vietnam

A rare human G10P[8] rotavirus with a reassortment between bovine and human viruses was detected from a patient with acute gastroenteritis in Vietnam. Genetic analysis using complete coding sequences of all segments showed a genomic constellation of this virus of G10-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Its VP7 region was genetically related to that of a bovine rotavirus derived from Australia (strain VICG10.01), whereas all other genes were identical to those of a human rotavirus derived from Australia (strain Victoria/CK00047). These results indicate a possibility that the reassortment of the rotavirus was caused by immune escape in Australia and the rotavirus was carried to Vietnam. Additionally, this finding will help further understanding the evolution of rotaviruses circulating in Vietnam.     

Structural Basis of Glycan Recognition in Globally Predominant Human P[8] Rotavirus

Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3 ?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.     

The Functional Characterization of Bat and Human P[3] Rotavirus VP8*s

Virologica Sinica - P[3] rotavirus (RV) has been identified in many species, including human, simian, dog, and bat. Several glycans, including sialic acid, histo-blood group antigens (HBGAs) are...     

Emergence and Characterization of Unusual DS-1-Like G1P[8] Rotavirus Strains in Children with Diarrhea in Thailand

The emergence and rapid spread of unusual DS-1-like G1P[8] rotaviruses in Japan have been recently reported. During rotavirus surveillance in Thailand, three DS-1-like G1P[8] strains (RVA/Human-wt/THA/PCB-180/2013/G1P[8], RVA/Human-wt/THA/SKT-109/2013/G1P[8], and RVA/Human-wt/THA/SSKT-41/2013/G1P[8]) were identified in stool specimens from hospitalized children with severe diarrhea. In this study, we sequenced and characterized the complete genomes of strains PCB-180, SKT-109, and SSKT-41. On whole genomic analysis, all three strains exhibited a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation is shared with Japanese DS-1-like G1P[8] strains. Phylogenetic analysis revealed that the G/P genes of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P[8] strains. On the other hand, the non-G/P genes of the three strains were assumed to have originated from human DS-1-like strains. Thus, strains PCB-180, SKT-109, and SSKT-41 appeared to be derived through reassortment event(s) between Wa-like G1P[8] and DS-1-like human rotaviruses. Furthermore, strains PCB-180, SKT-109, and SSKT-41 were found to have the 11-segment genome almost indistinguishable from one another in their nucleotide sequences and phylogenetic lineages, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of the three strains were closely related to those of Japanese DS-1-like G1P[8] strains. Therefore, DS-1-like G1P[8] strains that have emerged in Thailand and Japan were assumed to have originated from a recent common ancestor. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like G1P[8] strains that have emerged in an area other than Japan. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] rotaviruses.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

In Vitro and in Vivo Evaluation of the Efficacy of Bovine Colostrum against Human Rotavirus Infection

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6–7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.     

Full Genome Characterization of Novel DS-1-Like G8P[8] Rotavirus Strains that Have Emerged in Thailand: Reassortment of Bovine and Human Rotavirus Gene Segments in Emerging DS-1-Like Intergenogroup Reassortant Strains

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotavirus strains have been recently reported in Asia, Australia, and Europe. During rotavirus surveillance in Thailand in 2013–2014, novel DS-1-like intergenogroup reassortant strains having G8P[8] genotypes (i.e., strains KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, SWL-12, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55) were identified in stool samples from hospitalized children with severe diarrhea. In this study, we determined and characterized the complete genomes of these 12 strains (seven strains, KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, and SWL-12, found in 2013 (2013 strains), and five, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55, in 2014 (2014 strains)). On full genomic analysis, all 12 strains showed a unique genotype constellation comprising a mixture of genogroup 1 and 2 genes: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. With the exception of the G genotype, the unique genotype constellation of the 12 strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) was found to be shared with DS-1-like intergenogroup reassortant strains. On phylogenetic analysis, six of the 11 genes of the 2013 strains (VP4, VP2, VP3, NSP1, NSP3, and NSP5) appeared to have originated from DS-1-like intergenogroup reassortant strains, while the remaining four (VP7, VP6, VP1, and NSP2) and one (NSP4) gene appeared to be of bovine and human origin, respectively. Thus, the 2013 strains appeared to be reassortant strains as to DS-1-like intergenogroup reassortant, bovine, bovine-like human, and/or human rotaviruses. On the other hand, five of the 11 genes of the 2014 strains (VP4, VP2, VP3, NSP1, and NSP3) appeared to have originated from DS-1-like intergenogroup reassortant strains, while three (VP7, VP1, and NSP2) and one (NSP4) were assumed to be of bovine and human origin, respectively. Notably, the remaining two genes, VP6 and NSP5, of the 2014 strains appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses. Thus, the 2014 strains were assumed to be multiple reassortment strains as to DS-1-like intergenogroup reassortant, bovine, bovine-like human, human, and/or locally circulating DS-1-like G2P[4] human rotaviruses. Overall, the great genomic diversity among the DS-1-like intergenogroup reassortant strains seemed to have been generated through additional reassortment events involving animal and human strains. Moreover, all the 11 genes of three of the 2014 strains, NP-130, PCB-656, and SSL-55, were very closely related to those of Vietnamese DS-1-like G8P[8] strains that emerged in 2014–2015, indicating the derivation of these DS-1-like G8P[8] strains from a common ancestor. To our knowledge, this is the first report on full genome-based characterization of DS-1-like G8P[8] strains that have emerged in Thailand. Our observations will add to our growing understanding of the evolutionary patterns of emerging DS-1-like intergenogroup reassortant strains.     

In Vitro Thymidine Labelling In Human and Porcine Growth Plates

Abstract. An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. the patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.     

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix?. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix? vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.     

In Vitro and In Vivo Isolation of a Novel Rearranged Porcine Circovirus Type 2

Here, we present the first report of a novel rearranged porcine circovirus type 2 (PCV2) strain named BIV, isolated from both in vitro and in vivo sources. The complete circular genome of BIV is 896 nucleotides in length. The data will help us to update current knowledge of the replication of PCV2 viruses in cell culture and of their molecular evolution, as well as their diagnosis.     

Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens

Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Le^b and H type 1 antigens, while P[6] RV weakly bound the Le^b antigen. These findings may facilitate further research into RV host specificity and vaccine development.     

G9型人A组轮状病毒LL52696与LL52727株的主要基因及其编码蛋白特征的分析

人A组轮状病毒(Human rotavirus,HRV)是引起世界范围婴幼儿重症腹泻的最主要病原,也是导致发展中国家婴幼儿死亡的主要病因之一[1-3],世界卫生组织统计每年大约引起611 000婴儿和儿童死亡,特别是发展中国家[4].HRV感染广泛,且改善营养状况和卫生条件对HRV发病危险影响不大,因此在发达国家和发展中国家HRV感染率接近.HRV引起的腹泻至今无特效药,发展疫苗对控制HRV感染的作用就显得特别突出.     

Radiation-Induced DNA Methylation Disorders: In Vitro and In Vivo Studies

Biology Bulletin - Phenomenological aspects and mechanisms of DNA methylation disorders (changes in the total level of DNA methylation and in genome repetitive elements, locus-specific methylation...     

The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.     

The Molecular Chaperone Calnexin Interacts with the NSP4 Enterotoxin of Rotavirus In Vivo and In Vitro

Calnexin is an endoplasmic reticulum (ER)-associated molecular chaperone proposed to promote folding and assembly of glycoproteins that traverse the secretory pathway in eukaryotic cells. In this study we examined if calnexin interacts with the ER-associated luminal (VP7) and transmembrane (NSP4) proteins of rotavirus. Only glycosylated NSP4 interacted with calnexin and did so in a time-dependent manner (half-life, 20 min). In vitro translation experiments programmed with gene 10 of rhesus rotavirus confirmed that calnexin recognizes only glycosylated NSP4. Castanospermine (a glucosidase I and II inhibitor) experiments established that calnexin associates only with partly deglucosylated (di- or monoglucosylated) NSP4. Furthermore, enzymatic removal of the remaining glucose residues on the N-linked glycan units was essential to disengage the NSP4-calnexin complex. Novel experiments with castanospermine revealed that glucose trimming and the calnexin-NSP4 interaction were not critical for the assembly of infectious virus.     

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.