To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; Cross Protection ; Female ; Genetic Vectors ; biosynthesis ; genetics ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; administration & dosage ; genetics ; immunology ; Viral Core Proteins ; administration & dosage ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology

Peg-interferon and ribavirin has been the standard therapy of chronic hepatitis C for the past 15 years in Korea. However, the treatment paradigm is changing. Direct acting agents (DAAs) are oral pills that can be easily taken. In addition, DAAs are more effective and have less adverse reactions compared to the previously used drugs. Chronic hepatitis C is hard to treat because the virus is error-prone virus. Host immunity is helpless against the hepatitis C virus since it evades the host immunity through various complex mechanisms. There are 6 genotypes. Quasispecies can co-exist even in the same patients. The treatment strategy is based on the combination of the individual drug corresponding to each step of viral replication process. NS5B nucleosides are the most powerful and effective drug available until now. Other drugs with different mechanisms of action can be used to provide synergy. NS5A and NS5B inhibition drugs currently belong to the leading group amongst many DAAs. These drugs will soon be available in Korea. We have to know the merits and adverse drug reactions of the new drug.
Antiviral Agents/*therapeutic use ; Drug Therapy, Combination ; Enzyme Inhibitors/therapeutic use ; Genotype ; Guidelines as Topic ; Hepacivirus/genetics ; Hepatitis C, Chronic/*drug therapy/immunology/virology ; Humans ; Viral Nonstructural Proteins/antagonists & inhibitors/metabolism

Dengue virus (DENV) is a re-emerging disease transmitted by the Aedes mosquitoes and has become a major public health problem in southern China. Currently, no antiviral drug or effective vaccine exist to control this disease. The chimeric DENV structural protein vaccine cannot elicit balanced levels of protective immunity to each of the four viral serotypes; therefore, non-structural protein components may be required to construct an effective DENV vaccine. The Dengue virus non-structural 1 (DENV NS1) protein plays a critical role in viral pathogenesis and protective immunity. Therefore, immunity to Dengue 1-4 NS1 subtypes may be crucial for the prevention of severe disease. This review attempts to provide an overview about the structure and function of DENV NS1.
Animals ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; chemistry ; genetics ; immunology ; Dengue Virus ; chemistry ; genetics ; immunology ; Humans ; Viral Nonstructural Proteins ; chemistry ; genetics ; immunology

NS2 and NS3 are two post-transcriptional gene silencing suppressors that are encoded by Rice stripe virus. Gene silencing suppressors are always related to the pathogenicity of viruses. In this study, the cDNA of NS2 and NS3 were recombined by overlapping PCR assays, ligated to the RNAi vector, and inserted into the PXQ expression vector using Pst I; the expressed vector was transferred into calluses induced from seeds of the japonica rice cultivar, 'Nipponbare', using an Agrobacterium-mediated method. Thirty-one T0 transgenic plants were selected by G418 screening. PCR and southern blot analyses confirmed that the target gene was transformed into transgenic rice successfully, and different transgenic plants contained various copies of the gene. The disease resistance assay revealed that T0 transgenic rice had a delayed onset of RSV for approximately 10-20 d, and the accumulation of virus in the transgenic plants was reduced by 30%-50%. This was related to the delayed onset of disease.
Disease Resistance ; Oryza ; genetics ; immunology ; virology ; Plant Diseases ; genetics ; immunology ; virology ; Plants, Genetically Modified ; genetics ; immunology ; virology ; RNA Interference ; Tenuivirus ; genetics ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology

To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revealed 94.2% and 92.1% agreement respectively. The assay demonstrates good specificity and sensitivity, and can be applied in the detection of porcine parvovirus.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Parvoviridae Infections ; diagnosis ; immunology ; veterinary ; virology ; Parvovirus, Porcine ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; genetics ; immunology ; Swine ; Swine Diseases ; diagnosis ; immunology ; virology ; Viral Nonstructural Proteins ; genetics ; immunology

Heat shock protein 27 (HSP27) is a member of the small heat shock proteins (sHSP) and has multiple functions, it also plays an important role in the life cycle of some viruses. To investigate the regulatory effect of HSP27 during influenza virus infection, we cloned and expressed human HSP27 in both prokaryotic and eukaryotic cells, and demonstrated that HSP27 interacted with influenza A virus NS1 protein both in vivo and in vitro. Luciferase assay showed that HSP27 inhibited the expression of interferon-beta (IFN-beta) in infected cells, and independent of its phosphorylation. Moreover, HSP27 enhanced the inhibitory effect of NS1 on the expression of IFN-beta. Further analysis indicated that HSP27 exerted the inhibitory effect probably through influencing MDA5 of the RIG-I like helicase (RLH) pathway. The results suggested that HSP27 play a role in the innate immunity of infected cells, contributed to our understanding of the regulatory effect of host factors during influenza virus infection.
Cell Line ; Gene Expression Regulation, Viral ; HEK293 Cells ; HSP27 Heat-Shock Proteins ; genetics ; pharmacology ; Humans ; Influenza, Human ; genetics ; immunology ; Interferon-beta ; antagonists & inhibitors ; metabolism ; Viral Nonstructural Proteins ; genetics ; pharmacology

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; metabolism ; Encephalitis, Japanese ; immunology ; virology ; Female ; Glycosylation ; Humans ; Mice ; Mice, Inbred BALB C ; Viral Nonstructural Proteins ; genetics ; immunology ; metabolism

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

To develop a new Hepatitis C virus (HCV) DNA vaccine ,We explored strategies for optimizing the immunogenicity of HCV DNA vaccine in fusion with dendritic cell-targeting molecules (scDEC, a single-chain antibody against the murine DC cell surface molecule DEC205). We constructed the DNA vaccine plasmids expressing the HCV non-structural protein NS3 alone or in combination with DEC205 as a fusion protein and identified the expression of the molecules of interest by transient transfection of 293 cells with the resultant DNA vaccine plasmids. Then BALB/C mice were immunized with these plasmids by the injection in combination with electroporation. The NS3-specific IgG antibody(ELISA) and cellular immunity (IFN-gamma ELISPOT) were analyzed post twice immunization. Our results showed that: the single-chain antibody against DEC205 fused with vaccine antigen NS3 significantly enhanced the immunogenicity of new HCV DNA vaccine, the intradermal injection in combination with electroporation using caliper electrodes resulted in most robust NS3-specific antibody and T cell immune response. In conclusion,immune response for the HCV NS3 protein-encoding DNA vaccine was enhanced significantly when targeting antigen NS3 to DCs by scDEC. The present strategy could merit further study in the context of other prophylactic and therapeutic DNA based vaccines.
Animals ; Cell Line ; Dendritic Cells ; immunology ; virology ; Enzyme-Linked Immunospot Assay ; Female ; HIV Antibodies ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology