Gene Expression Regulation, Viral ; Proteomics ; methods ; trends ; Viral Proteins ; analysis ; genetics ; Viruses ; genetics ; growth & development ; metabolism

Rubella virus (RV), one of pathogens in TORCH syndrome, can lead to anisotropy of the fetus, and therefore it is of great significance to screen RV infection among women at child-bearing age. At the present, the screening of RV infection is mainly based on the ELISA method, the specificity of RV antigens is very important for ELISA tests. The antigenicity of RV is closely associated with its structural proteins, including capsid protein C, and envelope glycoproteins E1 and E2, which are important surface antigens of RV. This paper reviews the advances in the studies on the structure features, immunogenicity and recombinant proteins of the three structural proteins.
Antibodies, Viral ; analysis ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins ; immunology ; Rubella virus ; genetics ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology

OBJECTIVETo purify recombinant nuclear protein of Hantaan virus.

METHODSThe recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein is a fusion protein with GST and existed in inclusion bodies. The inclusion bodies were processed through denaturation and renaturation and were purified with Glutathione Sepharose 4B affinity chromatography column. Then the purified fusion protein and nuclear protein were examined by sandwich ELISA and Western blot.

RESULTSThe expressed fusion protein was separated from the mixture proteins by the first affinity chromatography. GST was cut from the purified fusion protein with thrombin. The nuclear protein was separated from GST by the second affinity chromatography. The crossed peak represents nuclear protein and the elute peak represents GST. The purified fusion protein and nuclear protein were single band by SDS-PAGE. Both of them had available antigen activity.

CONCLUSIONSPurification of nuclear protein of Hantaan virus with Glutathione Sepharose 4B affinity chromatography is an effective method.

Blotting, Western ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Hantaan virus ; Nuclear Proteins ; analysis ; isolation & purification ; Plasmids ; Recombinant Fusion Proteins ; analysis ; isolation & purification ; Viral Proteins ; analysis ; isolation & purification

BACKGROUNDTo clarify the features of gene variation among epidemic strains of human rotavirus NSP4 in China.

METHODSSP4 genes from 27 epidemic strains of human rotavirus isolated in different area of China in recent years were amplified with RT-PCR, the resulted cDNAs were cloned and sequenced. The sequences of full length cDNAs were compared with 10 rotavirus NSP4 sequences available in the GenBank using the Clustal x 1.8 TreeView32 and DNA Star softwares. The G serotype of VP7 was analysed by PCR.

RESULTSThe homology of the amino acid among the 27 rotavirus strains isolated in China was 81.7%-99.4%. Based on the variation of amino acid sequence, the virus strains can be divided into two groups, represented by Wa and KUN with the homology of 92.0%-99.4% and 92.0%-98.9% within each group, respectively. The diversity between the two groups were 16.6%-21.0%. The Wa group could further be separated into three subgroups, according to the diversity between those strains and the characterization in the highly variable domain. The association between VP7 serotype and NSP4 genotype was not strong.

CONCLUSIONSThe NSP4 gene of human rotavirus epidemic strains in China can be divided into Wa and KUN two groups, Wa group is the main group and contains three subgroups possessing characteristic amino acid sites. Samples isolated in the same year but not in the same area shared higher homology.

Antigens, Viral ; Capsid Proteins ; genetics ; DNA, Complementary ; analysis ; DNA, Viral ; analysis ; Diarrhea ; virology ; Genetic Variation ; Genotype ; Glycoproteins ; genetics ; Humans ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Toxins, Biological ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo investigate the evolution features of whole-genome of influenza virus H3N2 prevalent in Qingdao from year 2007 to 2011.

METHODSThe RNA of 58 strains of influenza virus H3N2 prevalent in Qingdao between 2007 and 2011 was extracted and all segments amplified by RT-PCR. The sequence was then detected and assembled by software Sequencer. A total of 589 strains of influenza virus H3N2 with more than 300 amino acid recorded by GenBank were selected. The phylogeny and molecular features of all gene segments were analyzed by software Mega 5.0, referred by the heavy chain of hemagglutinin (HA1).

RESULTSHemagglutinin (HA) genes of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 formed a single trunk of phylogenetic tree. Every prevalent strain originated in last season. The analysis of the evolution of whole genome found that reassortment virus strains were prevalent between year 2009 and 2010, but between 2010 and 2011 there were two series of prevalent strains, which showed complicated reassortment. Compared with the vaccine strains, the variant amino acids of protein of virus HA1 between year 2007 and 2011 were 8, 6, 6, 8 and 11, involving 13 antigenic sites. The sequence analysis of M2 protein showed that the isolated influenza virus H3N2 mutated in amino acid site 31, from serine to asparagine (S31N). HA1 gene of influenza virus H3N2 isolated in Qingdao between 2007 and 2011 shared the similar phylogenetic tree with the globally prevalent strain. The comparison of the sequence and the analysis of the antigenicity found co-infection between H3N2 and A/H1N1 in the strain A/Qingdao/F521/2011.

CONCLUSIONThe evolution features of all segments of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 were complicated.

China ; Evolution, Molecular ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Phylogeny ; RNA, Viral ; Reassortant Viruses ; genetics ; Sequence Analysis ; Viral Matrix Proteins ; genetics

OBJECTIVETo perform variation and phylogenetics analysis on the SARS-CoV genome sequence (PUMC01) isolated in the Peking Union Medical College Hospital.

METHODSThe cDNA library of SARS-CoV (PUMC01 isolate) was constructed by means of random-priming strategy. Random selected plasmid was sequenced and the genome sequence of SARS-CoV-PUMC01 was assembled by conventional methods (The Genebank Accession No. of SARS-CoV-PUMC01 is AY350750). The variation and phylogenetics analysis were performed by comparing the PUMC01 sequence with other SARS-CoV isolates.

RESULTSTen variation sites were found by comparing PUMC01 isolate with Tor2 and Urbani isolates. In phylogenetic analysis of 18 SARS-CoV isolates, two classes were observed and there is different differential time between these two classes and the different isolates in each class.

CONCLUSIONSThe evidence of phylogenetic analysis of different SARS-CoV isolates from different region is instructive for understanding the clinical relations between the different isolates and the transmission chain of SARS-CoV.

Amino Acid Sequence ; Base Sequence ; China ; DNA, Viral ; genetics ; Genetic Variation ; Genome, Viral ; Molecular Sequence Data ; Phylogeny ; SARS Virus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Viral Proteins ; genetics

BACKGROUNDThere is a higher rate of cytomegalovirus (CMV) reactivation in patients with multiple myeloma after an autologous stem cell transplantation, but no attention has been given thus far to a possible pathogenetic interplay between CMV and multiple myeloma. CMV can infect many kinds of cells, and CMV infection has been shown to inhibit apoptotic responses in several cell systems. In this study the authors investigated the alterations in apoptosis in the multiple myeloma cell line KM3 after infection with CMV and proposed a possible mechanism.

METHODSKM3 cells were infected with 100, 10, or 1 TCID50 of CMV and then cultured in serum-free RPMI 1640. An RT-PCR-based assay was used to detect mRNA expression of CMV-IE, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and IL-6 in CMV-infected and mock-infected cells. Flow cytometry was used to detect apoptotic cells. CMV particles and apoptotic cells were also examined with an electron microscope.

RESULTSCMV-infected KM3 cells clearly expressed immediate early (IE) antigen mRNA when compared to uninfected cells, and there were fewer apoptotic cells among cells treated with 100 or 10 TCID50 of CMV after culturing in serum-free RPMI 1640. CMV particles were observable in infected cells under an electron microscope. Expression of IL-6 mRNA increased after infection.

CONCLUSIONCMV can infect the multiple myeloma cell line KM3, inhibit the apoptotic response in these cells after apoptosis induction in serum-free culture, and increase the expression of IL-6 mRNA.

Apoptosis ; physiology ; Cell Line, Tumor ; Cytomegalovirus ; physiology ; Flow Cytometry ; Humans ; Immediate-Early Proteins ; analysis ; Interleukin-6 ; genetics ; physiology ; Multiple Myeloma ; pathology ; RNA, Messenger ; analysis ; Viral Proteins ; analysis

BACKGROUNDSeveral coronaviruses establish persistent infections in vitro and in vivo, however it is unknown whether persistence is a feature of the severe acute respiratory syndrome coronavirus (SARS-CoV) life cycle. This study was conducted to investigate viral persistence.

METHODSWe inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID50 and passaged the remaining cells every 4 to 8 days for a total of 11 passages. Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays. The presence of viral particles in passage 11 cells was assessed by electron microscopy. Changes in viral genomic sequences during persistent infection were examined by DNA sequencing.

RESULTSCytopathic effect was extensive after initial inoculation but diminished with serial passages. Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells. Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40% of cells at passages 2-11. Electron microscopy confirmed the presence of viral particles in passage 11 cells. Sequence analysis at passage 11 revealed fixed mutations in the spike (S) gene and ORFs 7a-8b but not in the nucleocapsid (N) gene.

CONCLUSIONSSARS-CoV can establish a persistent infection in vitro. The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release. Persistence is associated with selected mutations in the SARS-CoV genome. This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.

Animals ; Antigens, Viral ; analysis ; Cercopithecus aethiops ; Microscopy, Electron ; Nucleocapsid Proteins ; analysis ; SARS Virus ; genetics ; growth & development ; Vero Cells

With the development of bio-technological drugs, drug immunogenicity evaluation has become key factor of clarifying safety and efficacy of these drugs. It has become the focus to establish a stable and reliable evaluation system. Due to the advantages such as continuous real-time monitoring, surface plasmon resonance (SPR) technology has been widely used in bio-technological drugs immunogenicity assessments. Our study applied this technology to detect anti-drug antibody (ADA) of a recombinant human anti-rabies monoclonal antibody NM57 in the sera of 48 volunteers admitted in phase I clinical trials. This method could satisfy the basic requirements of detection of ADA.
Antibodies, Anti-Idiotypic ; analysis ; blood ; immunology ; Antibodies, Monoclonal ; analysis ; blood ; immunology ; Antibodies, Viral ; analysis ; blood ; immunology ; Humans ; Rabies virus ; immunology ; Recombinant Proteins ; analysis ; blood ; immunology ; Surface Plasmon Resonance

OBJECTIVETo study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.

METHODSPersistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.

RESULTSIn the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.

CONCLUSIONSThe virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Carcinoma, Hepatocellular ; virology ; Defective Viruses ; physiology ; Encephalitis Virus, Japanese ; chemistry ; genetics ; physiology ; Humans ; Membrane Glycoproteins ; analysis ; Mutation ; RNA Helicases ; Serine Endopeptidases ; Tumor Cells, Cultured ; Viral Envelope Proteins ; analysis ; Viral Nonstructural Proteins ; analysis