OBJECTIVETo prepare the armored RNA containing M gene of influenza H3N2.

METHODSThe vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked.

RESULTSAR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture.

CONCLUSIONIt showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.

Influenza A Virus, H3N2 Subtype ; genetics ; Plasmids ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; standards ; Viral Matrix Proteins ; genetics

Animals ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Viral Matrix Proteins ; immunology ; Viral Vaccines

According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.
Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genes, Viral ; Genetic Vectors ; Genome, Viral ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; SARS Virus ; genetics ; Temperature ; Time ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

To establish method suitable to assay HCMVpp65 of the blood donors in blood bank and to supply safe blood to the patients, the immunocytochemical techniques were used, (6 - 8) x 10(6)/ml cells were counted, 50 x 10(3) cells were detected by light microscope, The results showed that 10 positive samples in 103 samples were found, positive rate was 9.71%, among 10 positive samples, 2 samples were still positive in the second detecting. In conclusion, this method is simple, quick and effective, suitable to detect HCMVpp65 of the blood donors in the blood bank.
Blood Banks ; Blood Donors ; Female ; Humans ; Immunohistochemistry ; Male ; Phosphoproteins ; blood ; Viral Matrix Proteins ; blood

Rabies virus (RABV) causes a neurological disease in warm-blooded animals that is nearly always fatal. In this study, we analyzed the matrix (M) genes in 10 Korean street RABV strains isolated from two Provinces during 2011–2013. The M genes in these 10 Korean strains were highly conserved during 1999–2013. Phylogenetic analysis revealed they were closely related to the M genes of RABVs isolated in northeastern China. Specific amino acid substitutions were identified in the KRVB1206, KRVF1301, and BV9901PJ strains. However, functional domains, including those involved in virus production and pathogenicity, were conserved in all 10 strains.
Amino Acid Substitution ; Animals ; China ; Korea* ; Phylogeny ; Rabies virus* ; Rabies* ; Viral Matrix Proteins ; Virulence

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.
Cell Line ; Coronavirus ; genetics ; pathogenicity ; Genes, Viral ; Humans ; Interferons ; antagonists & inhibitors ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

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 explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus.

METHODSA total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed.

RESULTSThe 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates.

CONCLUSIONSignificant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.

China ; epidemiology ; Evolution, Molecular ; Genes, Viral ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza B virus ; genetics ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Reassortant Viruses ; genetics ; Viral Core Proteins ; genetics ; Viral Matrix Proteins ; genetics ; Viral Nonstructural Proteins ; genetics

OBJECTIVE: To determine whether U(251) cells are permissive for human cytomegalovirus (HCMV), and to investigate the characteristics of temporal expression of proteins IE1 and pp65. METHODS: U(251) cells were infected with HCMV, and then the cells were observed under the transmission electronic microscope, and the viral nucleic acid was detected by PCR, and the expression levels of IE1 and pp65 were analyzed by immunohistochemical assay with anti-IE1 monoclonal antibody and anti-pp65 monoclonal antibody at various time spost infection. RESULTS: Morphological changes of the infected cells appeared under the transmission electron microscope. The viral nucleic acid was detected successfully by PCR. The expression of IE1 was detected firstly at 4h post infection, and reached a peak within 14h, and then decreased. The incoming pp65 was detected at 1h, the low expression levels of pp65 were detected firstly at 4h, and they could remain relatively constant through 96 h, but the maximum expression occurred at 120 h. CONCLUSION Human glioma U(251) cells are permissive for HCMV, the temporal cascade of HCMV gene expression can be observed in the infected U(251) cells, but it is delayed obviously in the human fibroblast.
Cell Line, Tumor ; Cytomegalovirus ; Cytomegalovirus Infections ; Glioma ; metabolism ; virology ; Humans ; Immediate-Early Proteins ; metabolism ; Phosphoproteins ; metabolism ; Viral Matrix Proteins ; metabolism

BACKGROUNDEpstein-Barr virus (EBV) associated malignancies with a Type II latency gene expression pattern, such as Hodgkin's disease, and nasopharyngeal carcinoma (NPC), frequently express the EBV antigen latent membrane protein 2A (LMP2A). We expected to establish a highly expressing LMP2A yeast cell strain and get the high quality LMP2A protein, which was used for detection, analysis and characterization of its antibodies in various patients' sera of EBV associated malignancies.

METHODSThe plasmid pPICZalphaA-LMP2A containing the full length of LMP2A cDNA was constructed and transformed to Pichia pastoris GS115 to express LMP2A protein. After fermentation and purification, the LMP2A protein was used as an antigen to detect anti-LMP2A antibodies (Abs) in the sera of patients with EBV-associated malignancies in enzyme linked immunosorbent assay (ELISA) or Western-blot.

RESULTSLMP2A was expressed successfully with an expected molecular weight of approximately 54 kD and Abs to LMP2A were strikingly specific to NPC. Two-thirds or more sera from NPC patients were positive for anti-LMP2A immunoglobulin G (IgG) Abs. The antibodies were absent from the sera of other EBV-associated diseases except a small fraction of the gastric carcinoma. Comparing anti-viral capsid Ags (VCA) IgA and LMP2A IgA titers in the sera from 76 NPC patients, only 55% were positive for anti-LMP2A IgA Abs while 70% were positive for anti-VCA IgA. However, we found that 3 sera negative for VCA IgA were positive for LMP2A IgA.

CONCLUSIONThe results suggested the potential significance of LMP2A specific Abs for the diagnosis of EBV-associated malignancies, especially NPC.

Antibodies, Viral ; blood ; Capsid Proteins ; immunology ; Epstein-Barr Virus Infections ; complications ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Nasopharyngeal Neoplasms ; diagnosis ; immunology ; virology ; Viral Matrix Proteins ; immunology ; isolation & purification