Bing du xue bao = Chinese journal of virology最新文献

To study the replication and antiviral innate immunity of EV-A71 in mouse macrophages, we selected the mouse macrophage RAW264.7cell line as a model. An absolute quantitative PCR detection method was constructed to detect the viral load of EV-A71 in RAW264.7cells.RT-qPCR detected the fold changes of the proinflammatory cytokine, chemokine, and pattern recognition receptors at different time points post-infection in RAW264.7cells infected with EV-A71 and UV-inactivated EV-A71.The results revealed that the viral load of EV-A71 in RAW264.7cells decreased as the time post-infection increased. Proinflammatory cytokines, including IL-1β,IL-6,TNF-α,and chemokines, including IP-10,MCP-1,and MIP-1αwere induced, and the mRNA expression levels of TLR2,TLR1,TLR6,MDA5,and RIG-I were up-regulated. These results indicate that EV-A71 could replicate in mouse macrophages at a lower level, and proinflammatory cytokine and chemokine responses were induced.

To study the genotype and genomic and molecular organization of a GI norovirus isolate from Huzhou, China, the genomic sequence of 2008/Huzhou/N11 was amplified by RT-PCR, then the PCR product was purified,sequenced, and input into phylogenetic and Simplot analyses. The complete genomic sequence of the 2008/Huzhou/N11 strain was 7691nucleotides (nt) long. It contained three open reading frames(ORFs):ORF1,ORF2,and ORF3,with lengths of 5367,1623,and 630 nt, respectively. Phylogenetic analysis revealed that the RdRp region of 2008/Huzhou/N11 belonged to genotype GI.2,and the VP1 and VP2regions belonged to genotype GI.6.The SimPlot analysis indicated that potential recombination points in 2008/Huzhou/N11 were located upstream of the ORF1/ORF2 overlap. The complete genomic sequence of this recombinant GI.2/GI.6 strain can serve as a reference sequence for the phylogenetic analysis of norovirus evolution.

Nipah virus (NiV) is the pathogen of an emerging zoonotic disease that is highly lethal and infectious. NiV seriously impacts human lives and the property in the epidemic areas and poses a great threat to the global public health security. This paper provides a brief introduction to the morphological structure, and physiological function of the attachment protein G and the fusion protein F, which are both crucial glycoproteins located on the surface of the virus envelope. Moreover, recent advances in domestic and international research are reviewed. We will consider tissue tropism to elaborate the interactive mechanism between the attachment protein and its protein receptors ephrin-B2/B3,as well as the interactions between the two glycoproteins in detail. Avenues of future research are also discussed.

Human immunodeficiency virus type 1(HIV-1)-specific CD8 cytotoxic T-lymphocytes(CTL)are essential components of the protective immunity against HIV-1infection.However,due to heterogeneous responses of CTL to HIV-1,our general understanding of CTL efficacy in the context of HIV-1infection remains limited. To better understand the factors that determine the potency of HIV-1specific CTL responses, this study directly investigated the relationship between different functional attributes associated with CTL response at the single cell level by using HIV-1specific CTL clones isolated in vitro. Twelve selected HIV-1CTL clones with various HLA restriction and specific antigen epitopes were comprehensively evaluated by several functional assays(e.g., killing capacity, degranulation, production of multiple cytokines and polyfunctionality, as well as the expression of lytic granule components and exhaustion molecules).Our principal findings were that the killing capacity of the CTL response was most closely associated with their degranulation capacity. Additionally, the killing and the degranulation capacity of CTL was associated with the levels and polyfunctionality of the cytokines secreted later. These findings implicate that multiple functional CTL responses are coordinately regulated and determined.

Hepatitis E, an acute self-limited disease is caused by hepatitis E virus(HEV)and is a public-health concern for people worldwide. HEV is transmitted primarily via the fecal-oral route while direct evidence for blood-borne transmission has been reported. So the risk of blood transfusion safety caused by HEV has been widely paid attention. Here, we aim to provide some references to HEV screening for blood donors through analyzing the existing diagnostic methods for HEV, which are of great significance for the prevention and control of HEV infection. Currently, the primary detection indexes for HEV primarily include HEV RNA,HEV antigen, anti-HEV IgM, and anti-HEV IgG. HEV RNA testing is considered to be the "gold standard" for the detection of HEV infection. This test takes advantage of patients with chronic Hepatitis E, immunosuppressed people, and patients with nonhepatic manifestations of hepatitis E.HEV antigen testing is regarded as a current infection index for HEV, which could be used to detect HEV in blood donors and diagnose acute HEV infection. Anti-HEV IgM is a mark of HEV recent infection, but not a single index to diagnose a current infection with HEV. Anti-HEV IgG indicates that HEV was the previous infection, and it is not suitable to diagnose acute HEV infection. At present, blood donors screening for HEV were mainly based on nucleic acid detection, and an antigen test possibly could cover its’ shortage. To confirm the value of the antigen and antibody tests for blood donor screening, further studies are required in the future.

Our objective was to establish a robust method for the expression and purification of hepatitis E virus(HEV)p495protein using a baculovirus-based insect cell expression system; to determine the properties and cryo-EM structure of the resulting virus-like particles(VLPs);and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine (Hecolin). The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein. ELISA, analytical ultracentrifugation, size-exclusion chromatography and negative-staining transmission electron microscopy(TEM)were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of>95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions. The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature(PDB ID:2ZZQ).In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin. Thus, a robust and scalable approach to obtain homogeneous, immunogenic HEV p495 VLPs has been established. This study may assist investigations of HEV receptors, epitope mapping, vaccine improvement and so on.

This study was designed to prepare a monoclonal antibody against Orf virus 118 protein and explore the biological properties of ORFV118 using this antibody. We constructed a recombinant plasmid pET33b-ORFV118 that contained a full-length ORFV118 gene. The plasmid was transformed into E.coli BL21,and the expression of ORFV118 was induced by isopropyl-β-d-thiogalactoside (IPTG).Prokaryotic ORFV118 was purified via Ni-NTA affinity chromatography and was subsequently used as an antigen to immunize mice. An anti-ORFV118 antibody was prepared using hybridoma technology. The titer and specificity of this antibody were tested by an indirect ELISA and Western blot/immunohistochemistry, respectively. We successfully obtained three antibody-secreting hybridomas,1A2,3B5,and 5D10.The titers of the three hybridomas were 1:10000,1:6400,and 1:8000.The monoclonal antibody (mAb),1A2 and the highest titer was selected for further research. The mAb 1A2,an IgG1 type antibody was bonded to its immunizing antigen, both the eukaryotic and natural ORFV118 with high specificity. The immunohistochemical analysis showed that the focal specific staining was restricted to the epidermal layer and subcutaneous tissue, which conformed to the characteristics of an ORFV infection. The mAb 1A2 recognized ORFV118with high specificity. Further study of mAb 1A2 will facilitate our understanding of ORFV118 and provide potentially novel methods for the diagnosis, prevention, and treatment of Orf.

This study aimed at investigating the molecular epidemiology and genetic variation of PRRSV based on the detection of 250 clinical samples collected from 118 farms from 2014 to 2015in different regions of Henan Province by RT-PCR. The NSP2 and ORF5genes of the PRRSV-positive samples were sequenced and analyzed. The results showed that 58 samples were positive for PRRSV, with a positive rate of 23.2%.A total of 29NSP2 and 31ORF5genes were obtained. The phylogenetic analysis revealed that most of the prevalent strains belonged to the North American genotype. Among the 58 positive samples,14 strains were highly homologous with HP-PRRSV, another 15 samples were highly homologous with the North American prevalent strain, and NADC30 contained a discontinuous deletion of 131 amino acids in Nsp2,which had been recently reported in China and Korea. This study showed that the HP-PRRSV and NADC30-Like strains are presently the dominant strains in Henan,particularly in comparison with the results from 2012 to 2013.Moreover,the NADC30-Like strains accounted for a higher percentage. In addition, both the NSP2 and ORF5genes had significant variations, suggesting that more attention should be continuously paid to monitor the pathogenic epidemiology and genetic variation of PRRSV. Furthermore, additional research should be conducted regarding the mechanism of pathogenicity and immunological suppression of PRRSV to provide a reference for the research and development of vaccines and antiviral drugs.

To prepare an epitope-based recombinant Rubella virus (RV) recombinant diagnostic antigen(designated ‘H29’) and preliminarily evaluate its antigenicity. With Glutathione S-Transferase (GST) located at the N-terminal, and the His tag at the C-terminal, the epitope-based RV recombinant diagnostic antigen (designated‘H29’) was expressed in Escherichia coli (E.coli) and purified by affinity and anion exchange chromatography. Based on the antigenicity of H29 identified by Western blot (WB), we constructed and evaluated a novel early diagnostic ELISA for RV infection. The soluble H29 protein with a high homogeneity was obtained; the WB analysis demonstrated that the H29 protein could bind to a monoclonal antibody for RV-E1 and GST antigens, as well as detect RV acute-phase serum. Using the novel ELISA, the serum from 48 cases with positive RV infection,48 cases with negative RV infection, and 48 healthy people was detected, displaying the excellent consistency. Using prokaryotic expression and chromatography purification, the epitope-based recombinant RV-IgM diagnostic antigen was obtained with excellent antigenicity, which could be applied for the serological detection of the early infection with RV.

P[4], P[6] and P[8] rotaviruses (RVs) are the most prevalent RV genotypes in the population. In order to further investigate the receptor binding and structural character of P[4], P[6] and P[8] RVs, VP8 * core proteins of the P[4], P[6] and P[8] RV strains isolated directly in the stool samples in China were expressed and purified with the GST and His-tag respectively. The GST-fusion protein was approximately 46 kDa while the His-tag proteins approximately 20 kDa. In conclusion, the recombinant plasmids of PGEX4T-1-VP8 * core and pET30a-VP8 * core were constructed and the VP8 * core proteins were successfully expressed in the soluble form by using E.coli expression system. These findings provide the basis for the futhure functional and structural studies of VP8 * proteins.