ABSTRACT
A newly identified parvovirus, human bocavirus (HBoV), was found in 21 (8.3%) of 252 nasopharyngeal aspirates from hospitalized children with lower respiratory tract infection in Hunan Province, People's Republic of China. Viral loads were 10(4) to 10(10) copies/mL. Phylogenetic analysis of the VP1 gene showed a single genetic lineage of HBoV worldwide.
Subject(s)
Bocavirus/isolation & purification , Parvoviridae Infections/epidemiology , Parvoviridae Infections/virology , Bocavirus/genetics , Child , Child, Preschool , China/epidemiology , DNA, Viral/isolation & purification , Female , Humans , Infant , Male , Nasopharynx/virology , Phylogeny , Viral LoadABSTRACT
Recently identified interferon-epsilon (IFN-epsilon) belongs to type I interferons. IFN-epsilon is highly and constitutively expressed in the brain, but its biochemical and biological characteristics are poorly understood. In this study, full-length IFN-epsilon cDNA was cloned from human peripheral blood lymphocyte by RT-PCR, and was expressed in Escherichia coli (E. coli). Reverse phase high pressure liquid chromatography was used to purify recombinant human IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About 0.8mg of highly purified rhIFN-epsilon protein was obtained from 100ml of E. coli culture. Functional study of rhIFN-epsilon demonstrated that the antiviral activity of rhIFN-epsilon was 6+/-0.5x10(5)IU/mg, which was lower than that of rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis virus) assay system. As for the activity to promote NK cytotoxicity and antiproliferation activities, rhIFN-epsilon was about 60 times less potent than rhIFN-alpha-2b. However, oligonucleotide microarray analyses revealed dramatic differences in gene expression profiles of cultured human cells treated with IFN-epsilon and IFN-alpha-2b. Particularly, differential regulation of genes related to central nervous system by rhIFN-epsilon suggests a role for IFN-epsilon in maintenance of the structure and function of brain.
Subject(s)
Interferon Type I/genetics , Interferon Type I/isolation & purification , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Base Sequence , Brain/metabolism , Cell Proliferation/drug effects , Cytotoxicity, Immunologic/drug effects , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/genetics , Gene Expression/drug effects , HeLa Cells , Humans , In Vitro Techniques , Interferon Type I/pharmacology , Interferon alpha-2 , Interferon-alpha/pharmacokinetics , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Oligonucleotide Array Sequence Analysis , Recombinant ProteinsABSTRACT
OBJECTIVE: To construct a novel recombinant rhIFN-epsilon155ser, and study its biological activities. METHODS: The whole sequence of rhIFN-epsilon was artificially synthesized and some codons were altered according to the preferred codon using of E.coli. The sequence was cloned into plasmid vector pBV220 to express in E.coli DH5alpha. After purification and re-folding of rhIFN-epsilon155ser inclusion body, the final product was tested for its biological activities, including anti-viral, anti-proliferative and NK cell enhancing activities. At the same time, by using DNA microarray biochips, the gene expression patterns in the rhIFN-epsilon155ser and rhIFN-alpha2b treated cells were compared and analyzed. RESULTS: The re-built rhIFN-epsilon155ser sequence was expressed in E.coli as a form of inclusion body. After purified and re-folded, the rhIFN-epsilon155ser protein reached a purity of above 95%. The rhIFN-epsilon155ser protein had a specific anti-viral activity of about 6 x 10(5) IU/mg in WISH/VSV system. Its anti-proliferative activity and NK cell enhancing activities in vitro seemed to be lower than that of rhIFN-alpha2b. Data obtained from microarray biochips indicated that there were 283 pieces increasing 2 folds and 1489 pieces decreasing 2 folds among totally 22,278 pieces of human genes were found in the rhIFN-epsilon155ser treated cells; more changes in gene expression pattern were detected in the rhIFN-alpha treated cells. CONCLUSION: A novel recombinant rhIFN-epsilon155ser was constructed, which belonged to type 1 interferon. The biological activities of rhIFN-epsilon155ser were compared with rhIFN-alpha2b. The changes of gene expression pattern in the interferon treated cells were detected, analyzed and discussed.
Subject(s)
Antiviral Agents/pharmacology , Cell Proliferation/drug effects , Interferons/pharmacology , Recombinant Proteins/pharmacology , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , HeLa Cells , Humans , Interferons/biosynthesis , Interferons/genetics , K562 Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Microbial Sensitivity Tests , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purificationABSTRACT
OBJECTIVE: To prepare human interferon-k (hIFN-kappa) and study its biological activities. METHODS: Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities. RESULTS: The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity. CONCLUSION: Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.
Subject(s)
Antiviral Agents/pharmacology , Cell Proliferation/drug effects , Interferon Type I/pharmacology , Recombinant Proteins/pharmacology , Animals , Cell Line , Chlorocebus aethiops , Cloning, Molecular , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Humans , Interferon Type I/genetics , Interferon Type I/isolation & purification , K562 Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Microbial Sensitivity Tests , Plasmids/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Vero CellsABSTRACT
<p><b>OBJECTIVE</b>To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation.</p><p><b>METHODS</b>Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program.</p><p><b>RESULTS</b>Analysis of the genomic sequences(nt 25-7,418) showed that the open reading frame contains 6,675 nucleotides in length encoding 2,225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152, 591, 646, 687 and insertion at nt 180-181 in 5?NTR and had mutation at nt 3,889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4,185 (aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5,020-5,025) that caused two amino acids deletion is virus fast growth basis.</p><p><b>CONCLUSION</b>Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.</p>
