Research progress in the etiology of hepatitis type E virus / 中华肝脏病杂志

Hepatitis type E virus (HEV) is a significant infectious zoonotic disease that causes hepatitis E. The disease is primarily transmitted via the fecal-oral route through contaminated water or food and is transmissible between species and genera. The causative agent for the disease is the hepatitis type E virus, which is a member of the Hepadnaviridae family and a single-stranded RNA virus. Its 7.2 kb genome mainly contains three open reading frames (ORFs): ORF1 encodes a non-structural polyprotein that mediates viral replication and transcription; ORF2 encodes a capsid protein and free antigen that induce neutralizing antibodies; ORF3 partially overlaps with ORF2 and encodes a small multifunctional protein involved in virion formation and release. HEV has a unique dual life cycle: it is excreted into feces in the form of naked virions but circulates in the blood in the form of "quasi-enveloped" particles. The two kinds of virus particles adsorb and penetrate the host cell in distinct ways, then internalize and decapsulate to replicate the genome, thereby producing more virion and releasing it outside the cell to mediate the virus's spread. This paper reviews the morphological characteristics, genome structure, encoded proteins, and function of HEV virus-like particles in order to provide a theoretical basis for basic research and comprehensive disease prevention and control.

Safety and immunogenicity of a split-virion quadrivalent influenza vaccine / 预防医学

Objective @# To evaluate the safety and immunogenicity of a split-virion quadrivalent influenza vaccine.@*Methods @#The healthy people aged three years or over in Wuyang County and Xiping County of Henan Province were divided into the experimental group, control group 1 and control group 2, and were vaccinated with split-virion quadrivalent influenza vaccines, split-virion trivalent influenza vaccines (without B/Victoria) and a split-virion trivalent influenza vaccines (without B/Yamagata) , respectively. The hemagglutination inhibition (HI) antibodies were detected before and after immunization. The incidence rate of adverse events following immunization (AEFI) , HI antibody positive conversion rate, the protection rate of HI antibodies and the growth of geometric mean titer (GMT) were calculated and compared with the standard of Food and Drug Administration (FDA).@*Results@#Totally 2 924 people were recruited, with 975 in the experimental group, 974 in the control group 1 and 975 in control group 2. The incidence rate of AEFI in the experimental group was 11.7%, higher than 7.9% in control group 1 and 8.8% in control group 2 (P < 0.05) during 30 minutes and 8 days after inoculation. The positive conversion rates of HI antibodies of H1N1, H3N2, By and Bv in the experimental group were 78.5%, 53.3%, 78.3% and 62.9%, respectively. The rate differences of the positive conversion rates of HI antibodies of By between the experimental group and control group 2, and of Bv between the experimental group and control group 1 were 42.1% (95%CI: 38.0%-46.2%) and 33.2% (95%CI: 28.9%-37.5%) , with both lower limits of 95%CI more than -0.10. The GMT increase of HI antibodies was more than 2.5 times in the three groups. The protective rates of HI antibodies of H1N1, H3N2, By and Bv in the experimental group were 87.7%, 98.7%, 93.6% and 77.2%, respectively. The protective rates of HI antibodies of By in control group 2 and Bv in control group 1 were 71.1% and 51.0%, both lower than those in the experimental group (P < 0.05).@*Conclusions @#After the inoculation of the quadrivalent influenza vaccine, the positive conversion rates (>40%) , protection rates (>70%) and GMT increase (>2.5 times) of HI antibodies of H1N1, H3N2, By and Bv all meet the quality standards of FDA. The safety and immunogenicity of the quadrivalent influenza vaccine are not inferior to those of the trivalent influenza vaccine.

Determinación in silico de las propiedades inmunogénicas de la glicoproteína E de Zika virus / In silico determination of immunogenic properties of glycoprotein E from Zika Virus

Introducción: La infección por Zika virus (ZIKV) ha sido asociada a múltiples complicaciones y nuevas formas de transmisión. La descripción del genoma y la estructura cristalizada permiten desarrollar análisis moleculares, incluyendo las propiedades inmunológicas. Objetivos: En este trabajo, se analiza a la glicoproteína E de ZIKV, con el fin de determinar su utilidad en la creación de una vacuna proteica recombinante. Métodos: Se analizó la glicoproteína E, por medio del software DNASTAR, en base a su antigenicidad de epítopos de células B y MHC-II, estructura secundaria, hidrofilizada, flexibilidad y accesibilidad a solvente en el virión maduro e hidratado. Resultados: Se identificaron 14 sitios antigénicos para células B, de los cuales, 7 comparten su antigenicidad para MHC-II. Al tomar en cuenta los demás parámetros analizados, los sitios se reducen a 3, con longitudes de 13, 9 y 5 aminoácidos. Conclusiones: La glicoproteína E de ZIKV podría desencadenar una respuesta inmune T-dependiente, por tanto, ser útil para la creación de una vacuna proteica recombinante.

Introduction: Zika virus (ZIKV) infection have been associated with multiple complications and new ways of transmission. The description of the genome and the crystalized structure allow the performance of molecular analysis, including immunological properties. Objectives: In this paper, we analyze glycoprotein E from ZIKV to determine its utility in the development of a recombinant protein vaccine. Methods: The protein was analyzed with the software DNASTAR, through the following properties: B cells and MHC-II antigenicity, secondary structure, hydrophilicity, flexibility and solvent-accessibility in the mature and hydrated virion. Results: We identified 14 antigenic sites with B-cells antigenicity, 7 of which shared the antigenicity for MHC-II. Considering other parameters analyzed, sites were reduced to 3, with length of 13, 9 and 5 amino acids. Conclusions: Glycoprotein E, from ZIKV, could trigger a T-dependent immune response, and therefore, may be useful in the creation of a recombinant protein vaccine.

Determination of the contents of two types of hemagglutinin B in quadrivalent influenza vaccines (split virion) by single radial immunodiffusion assay / 中华实验和临床病毒学杂志

Objective@#Quadrivalent influenza vaccines contain two lineages of type B virus, this study aimed to assess whether the result of single radial immunodiffusion (SRID) are accurate. The cross-interference of two type B hemagglutinins remains unknown.@*Methods@#We detected the vaccine samples developed by Jiangsu GDK Biological Technology Co., ltd by SRID.@*Results@#There was no significant difference between the HA content of antigen reagent, bulk sample and mixed sample of two B bulk within 10 to 40 μg/ml (P>0.05). Then each hemagglutinin B was diluted respectively by other three HA, 30 μg/ml, the other hemagglutinin B or phosphate buffer solution were measured within 10-160 μg/ml. Within 10-40 μg/ml, the HA content was proportional to the diameters of immunodiffusion (R2=0.998), while within the higher content range, a ternary linear regression equation fitted best (R2=0.999).@*Conclusions@#No cross-interference between B/Brisbane and B/Phuket was found in SRID within both detection ranges.

Overexpression of syntenin-1 enhances production of low-density hepatitis C virus particles / 第二军医大学学报

Objective To investigate the effect of syntenin-1 on the assembly of hepatitis C virus (HCV) particles. Methods The content of syntenin-1 in human primary hepatocytes was detected by Western blotting analysis. Human hepatocellular carcinoma cell line Huh7.5.1 was transfected with lentiviral plasmids to construct syntenin-1 overexpressed cell line and green fluorescent protein (GFP) overexpressed cell line. After transferring HCV RNA into Huh7.5.1 cells, syntenin-1 overexpressed cells and GFP overexpressed cells by electroporation, the effects of syntenin-1 overexpression on HCV in the cells were investigated in terms of RNA replication, viral protein contents and secretion of infectious virus particles by luciferase analysis, Western blotting and virus titer, respectively. The density of viral particles was assessed by isopycnic ultracentrifugation to analyze the distributions of HCV RNA and infective titer. Results The contents of syntenin-1 in three human primary hepatocytes were higher than that in the Huh7.5.1 cells (P0.01). Syntenin-1 overexpression had no effect on HCV RNA replication in host cells; the infectivity of HCV derived from syntenin-1 overexpressed cells was not significantly different compared with the Huh7.5.1 cells or GFP overexpressed cells. In syntenin-1 overexpressed cell culture supernatants, some infectious HCV particles mainly concentrated in the region of concentration of 1.08-1.16 g/mL gradually transferred to the low-density region of 1.01-1.02 g/mL. Conclusion Syntenin-1 overexpression alters the distribution of density components of infectious HCV particles.

Safety and immunogenicity of influenza vaccine (split virion) in humans / 中华微生物学和免疫学杂志

Objective To evaluate the immunogenicity and safety of a split-virion influenza vac-cine after its manufacturing process was improved. Methods The immunological non-inferiority of trial to control vaccines was evaluated in 240 subjects aged 3-<18 years. Another 360 subjects aged 18-<60 years were randomly divided into three groups that were respectively given three consecutive lots of trail vaccine to assess the consistency of immunogenicity. Results There were 4. 17% of the subjects aged 3-<18 years showed adverse reactions following immunization with trail vaccine and it was not significantly different from that of the control group (P>0. 05). No significant difference in seroconversion rate, geometric mean titer (GMT) of haemagglutination inhibition antibodies(HIAb) or protection rate was found between trial and control groups (P>0. 05). No significant difference in seroconversion rate or HIAb GMT was found among the three lots (P>0. 05). Conclusion The trial influenza vaccine has good safety, immunogenicity and lot-to-lot consistency after the manufacturing process was improved.

N-terminal Extension of Coat Protein of Turnip Yellow Mosaic Virus has Variable Effects on Replication, RNA Packaging, and Virion Assembly Depending on the Inserted Sequence

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus composed of 20 kDa single coat proteins. In this study, we modified the TYMV coat protein (CP) ORF by inserting an oligonucleotide linker corresponding to T7, HSV, Tat, (Arg)9, or (RxR)4 peptide at the 5'-end of the CP ORF and examined its effect on replication, RNA packaging, and virion assembly. The results showed that the constructs containing (Arg)9 and (RxR)4 sequences were barely capable of replication. The TYMV constructs containing T7 and Tat peptide produced virions that co-migrated with wild-type virions. However, the insertion of T7 and Tat sequences impaired genomic RNA (gRNA) accumulation and packaging, respectively. When only the CP gene was expressed, CPs with (Arg)9 or (RxR)4 successfully produced virus-like particles whose mobility was comparable to that of wild type. In the case of CP having a HSV tag, the virion band was not detected, although a sufficient amount of CP was produced. This indicates that CP with the HSV tag failed to assemble into virions. Overall, the results suggest that TYMV replication, RNA packaging and virion assembly are strongly influenced by the insertion sequence.

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1 (DHAV-1) Isolates in Shandong Province of China / 中国病毒学·英文版

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD50s) and the median lethal doses(LD50s),respectively.The results showed that the ELD5s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 106/mL to 1.44 × 107/mL,while the LD50s were 2.39 × 105/mL to 6.15 × 106/mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8％-99.7％ similarity at the nucleotide level and 92.4％-99.6％ at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(as 158-160,180-193 and 205-219) and other variable points in VPI protein,but which didn't cause virulence of DHAV-1 change.

Virion Proteomics of Large DNA Viruses / 中国病毒学·英文版

Large DNA viruses normally have complex structures with many of protein components derived from both viral and host origins. The development in proteomics, especially mass spectrometry identification techniques provide powerful tools for analyzing large viruses. In this review, we have summarized the recent achievements on proteomic studies of large DNA viruses, such as herpesvirus, poxvirus, nimavirus and baculoviruse. The proteomics of baculovirus occlusion-derived virions (ODV) were emphasized. Different mass spectrometry techniques used on ,carious baculoviruses were introduced, and the identified structurally associated proteins of baculoviruses are summarized.

The Dicistroviridae: An Emerging Family of Invertebrate Viruses / 中国病毒学·英文版

Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In this review I provide an overview of the Dicistroviridae and describe progress made toward the understanding and practical application of dicistroviruses, including (i) construction of the first infectious clone of a dicistrovirus, (ii) use of the baculovirus expression system for production of an infectious dicistrovirus, (iii) the use of Drosophila C virus for analysis of host response to virus infection, and (iv) correlation of the presence of Israeli acute paralysis virus with honey bee colony collapse disorder. The potential use of dicistroviruses for insect pest management is also discussed. The structure, mechanism and practical use of the internal ribosome entry site (IRES) elements has recently been reviewed elsewhere.

Construction and expression of ribonuclease-resistant virus-like particles containing long chimeric RNA sequence / 中华检验医学杂志

Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop.Methods 1.7 kb maturase and coat protein gene of ms2 were amplified.We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine.The 1700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac.The five target DNA sequences were spliced by overlapping extension(including 3 fragment of SARS-CoV,one fragment of HCV and one fragment of HSN1).PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM(R) T-easy vector and then excised with the Not Ⅰ restriction enzymes from the resulting recombinant plasmid.simoutaniously the pET-MC-pac plasmid was digested with Not Ⅰ restriction enzymes,fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V.Simoutaniously,we constructed three recombinant expression vector for control,including N-P3V-pET-P、N-P3V-pET-Cand P-3V-pET-P.N-P3V-pET-P contained one wild type pacsite located behind ms2 sequence:N-P3V-pET-C contained one C-variant pacsite located behind ms2 sequence:P-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence,another is located between SARS-CoV 3 and HCV sequence.Then these four expression vector were transformed into E.coli strain BL21(DE3),respectively.The 3V Armored RNA was expressed and purified.A260 absorbance value of expression product was determined.Results Four expression plasmids were constructed successfully.The Armored RNA with 1891 bases was snccessfully expressed by the two-paesite expression system of pET-ms2-3V and P-3V-pET-P;for N-P3V-pET-P、N-P3V-pET-C,only 1 200 bp target sequence was packaged into virus-like particles.Expression efficiency of N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P and N-P3V-pET-P was 0.23,0.35,0.35,0.51 mg/ml,respectively.Armored RNA was shown to have the charaeterization of Ribonuclease-resistant and stable at 4℃,37℃ and 25℃ respectively.Conclusion The expression plasmids containing two pacsites were constructed successfully and prokaryotic expression system can be used as an expressing plasmid platform for preparation of Ribonuclease-resistant virus-like particles containing long chimeric RNA sequence.

Nuclease-Resistance Assay for the Identification of Cell-free Herpesviral Virions: Comparison with Other Four Assays Including Electron Microscopy

To identify herpesviral virions secreted by viral replication, we have established the nuclease-resistance assay (NRA) as a comparative analysis to other conventional assays, including electron microscopy (EM). For this study, we used an efficient experimental in vitro infection model for Alcelaphine herpesvirus 1 (AlHV-1), a gamma herpesvirus, to propagate the virus. The NRA could identify extracellular, cell-free, enveloped virions in the supernatants after 24 hours post inoculation (h.p.i.) of AlHV-1. The results of EM observation were correlated with those of NRA. Mature virions were observed in the clarified, concentrated supernatants from 24 h.p.i. by EM. These results show that sensitivity of the NRA is comparable with that of EM for the identification of mature enveloped virions, which directly presents evidence of herpesviral lytic replication. NRA allows us to differentiate the virus from other member of Herpesviridae, and has extended the possibility of analysis for quantification of shedding viruses when used in conjunction with real-time polymerase chain reaction (PCR).

Construction and expression of RNase-resisting virus-like particles containing PSA mRNA / 中华老年医学杂志

Objective To construct an expression system to produce the virus-like particles containing a part of the sequence of PSA mRNA, which are ribonuclease-resistant due to the encapsulation of the mRNA by bacteriophage MS2 coat proteins. Methods The PCR products of PSA cDNA fragments were cloned to TA vector pBS-T, then the targeted segments could be obtained when the pBS-T-PSA were digested by restriction endonuclease Hind Ⅲ and cloned to prokaryocytic expression vector pNCCL1. The recombinant plasmids named PNCCL1-PSA were transfected into E. Coli BL21-DE3 and induced to express with IPTG. Results The recombinant plasmids were successfully constructed. The bacteriophage MS2 coat protein which expressed in BL21 can self- assemble to form ribonuclease resistant virus-like particles and the PSA mRNA was encapsulated into virus-like particles. Conclusions The virus-like particle containing PSA mRNA can be expressed in prokaryocyte and it can be used as standard and control in detecting PSA mRNA. It provides a new, stable and ribonuclease-resistant RNA standard in RNA detection.

CTL Response Induction in Mice Immunised with Chimeric BPVL1/HPV16 E7 Virus-Like Particles / 中华皮肤科杂志

Objective To study immunological property of chimeric BPVL1 / HPV16 E7 virus-like particles (VLPs) in mice. Methods HPV16 E7 gene was cut into three fragments, ligated to BPVL1 sequence, respectively, and then expressed as chimeric VLPs. The ability of the chimeric VLPs to stimulate in vivo cytotoxic response was analysed with lymphocytes taken from mice C57BL/6J lymph nodes. Results A strong CTL response against C-2 cells (HPV16 E7 aa47-59 transfected EL-4 cell) was induced in mice immunised with chimeric VLPs BPVL1 / HPV16 E7(b). Furthermore, no CTL response was detected against EL-4 cells in immunized mice. Conclusions Chimeric BPVL1/HPV16 E7 VLPs, serving as antigens, can activate mouse T lymphocytes and elicit a strong antigen-specific CTL response. Chimeric BPVL1/HPV16 E7 VLPs could be used as an efficient antigen delivery system, and might provide a novel strategy for HPV16 vaccine design.