Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8] / 中国实验动物学报

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Monitoring and molecular characterization of group Drotavirus in brazilian poultry farms / Monitoramento e caracterização molecular de rotavírus do grupo D em criações comerciais brasileiras

Rotaviruses are etiological agents of diarrhea both in humans and in several animal species. Data on avian Group D rotaviruses (RVD) are scarce, especially in Brazil. We detected RVD in 4 pools of intestinal contents of broilers, layer and broiler breeders out of a total of 111 pools from 8 Brazilian states, representing an occurrence of 3.6%, by a specific RVD RT-PCR targeting the VP6 gene. Phylogenetic tree confirmed that the Brazilian strains belong to group D and 3 of the sequences were identical in terms of amino acid whereas one showed 99.5% identity with the others. The sequences described in this study are similar to other sequences previously detected in Brazil, confirming the conserved nature of the VP6 protein.

Rotavírus são agentes etiológicos de diarreia tanto em humanos como em várias espécies animais. Dados sobre rotavírus do grupo D (RVD) em aves são escassos, especialmente no Brasil. Nós detectamos RVD em 4 pools de conteúdo intestinal de frango de corte, poedeiras e matrizes de um total de 111 pools originários de 8 estados brasileiros, representando uma ocorrência de 3,6% a partir de uma RT-PCR específica para RVD, tendo como alvo o gene VP6. A árvore filogenética confirmou que as amostras brasileiras pertencem ao grupo D e três das sequências obtidas foram idênticas em termos de aminoácidos enquanto uma apresentou 99,5% de identidade com as demais. As sequências aqui definidas são semelhantes a outras sequências previamente definidas no Brasil, confirmando a natureza conservada da proteína VP6.

Changes in Anti-Group A Rotavirus Antibody Seroprevalence and Levels in the Western Gyeongnam Province of Korea Over 16 Years

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.

Antibody Production of Baculovirus-expressed VP6 from Porcine Group C Rotavirus

The emerging pathogen, group C rotavirus (RVC) has been reported to cause acute diarrhea. But there was the limitation on the detection and monitoring for the absence of rapid sensitive diagnosis system. For the molecular biology study and diagnostic system development, we could detect porcine RVC by reverse transcriptase PCR (RT-PCR) analyses from 60 diarrheal disease porcine stool samples. VP6 full length RT-PCR product (CA-2 RVC, 1352 bp) was cloned and compared the nucleotide and deduced amino acid sequences with those of previously reported other porcine, human, and bovine rotavirus group A, B and C strains. Analyses data showed >82% homology on the nucleotide sequences and >90% homology on the deduced amino acid sequences with other RVCs. Recombinant baculovirus was prepared with cloned PCR product corresponding to VP6 coding sequence (CDS) (position 22~1206) into BaculoDirect(TM) C-term linear DNA, and used for the transfection of insect cells. The polyclonal antibody was produced from mice with purified recombinant VP6 and confirmed with western blot. Both of VP6 antigen and antibody, are useful for the development of rapid diagnostic system against RVC.

Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection / 대한소아소화기영양학회지

PURPOSE: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. METHODS: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. RESULTS: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. CONCLUSION: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.

Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection / 대한소아소화기영양학회지

PURPOSE: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. METHODS: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. RESULTS: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. CONCLUSION: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells / 中国病毒学

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Cloing and High Level Expression of VP6 Gene From Group A Human Rotavirus in E.coli / 中国生物工程杂志

The structural protein VP6 of rotavirus form the middle layer of the triple-layered viral capsid, playing a key role in the organization of the virion. The gene of structural protein 6 of rotavirus strain TB-Chen isolated from a clinic sample was amplified using PCR from the reverse transcription product of RV genome RNA, using pET as expression vector, a recombinant plasmid pET-VP6 containing coding sequence of VP6 protein was constructed. The results showed that the VP6 was highly efficiently expressed in E. coli BL21(DE3) cells which were transformed with the recombinant plasmid pET-VP6.The expressed VP6 protein possessed 27.4% of total cells protein, with an approximately 45kDa of molecular weight, and could be recognized by guinea pig anti-SA11 antibody on Western blot. The results obtained provide important basis for further study on structure and function of the VP6 protein.