Publications (42)178.7 Total impact
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Article: Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.
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ABSTRACT: The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor -
Article: A novel cis-acting element within the capsid-coding region enhances flavivirus vRNA replication by regulating genome cyclization.
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ABSTRACT: Cis-acting elements in the viral genome RNA (vRNA) are essential for the translation, replication and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved Stem 1 and Loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted Loop 1-Stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.Journal of Virology 04/2013; · 5.40 Impact Factor -
Article: Identification and characterization of a linearized B-cell epitope on the pr protein of dengue virus.
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ABSTRACT: The four serotypes of dengue viruses (DENV) represent one of the major mosquito-borne pathogen globally, so far no vaccine or specific antiviral is available. During virion maturation, the pr protein is cleaved from its precursor form prM protein on the surface of immature DENV by host protease. Recent findings have demonstrated that the pr protein not only played critical roles in virion assembly and maturation, but also involved in antibodies dependent enhancement of DENV infection. However, the B cell epitopes on the pr protein of DENV have not been well characterized at present. In this study, a set of 11 partially overlapping peptides spanning the entire pr protein of DENV-2 were fused with GST and expressed in E.coli, and ELISA screening with murine hyper immune antiserum against immature DENV identified the P8 peptide (57KQNEPEDIDCWCNST71) in the pr protein as the major immunodominant epitope. Fine mapping by truncated protein assays confirmed the 8-e peptide 57KQNEPEDI64 represented as the minimal requirement unit responsible for antibody binding. Importantly, the 8-e epitope could react with sera from dengue fever patients. Site-directed mutagenesis revealed the Asparagine residue at position 59 was important for epitope recognition. The 8-e epitope well coincided with the predicted B-cell epitopes by IEDB analysis, and 3D structural modeling mapped the 8-e peptide on the surface of prM-E heterodimers. Overall, our findings characterized a linearized B-cell epitope on the pr protein of DENV, which will help understand the life cycle of DENV and pathogenesis of dengue infections in human.Journal of General Virology 04/2013; · 3.36 Impact Factor -
Article: Japanese Encephalitis Virus RNA not Detected in Urine.
Clinical Infectious Diseases 03/2013; · 9.15 Impact Factor -
Article: Rational design of a flavivirus vaccine through abolishing viral RNA 2' -O methylation.
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ABSTRACT: Viruses that replicate in the cytoplasm cannot access to the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2' -O cap of their RNA; alternatively, they 'snatch' host mRNA cap to form the 5' -end of viral RNA. The function of 2' -O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2' -O methylation is replicative; but its viral RNA lacks 2' -O methylation, and is recognized and eliminated by host immune response. Such mutant virus could be rationally designed as a live attenuated vaccine. Here we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N7 and 2' -O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2' -O methylation was stable in cell culture after passaging for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune response, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2' -O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and non-flaviviruses that encode their own viral 2' -O methyltransferases.Journal of Virology 03/2013; · 5.40 Impact Factor -
Article: Severe dengue due to secondary DENV-1 infection in Mainland China.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2013; · 3.12 Impact Factor -
Article: Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice.
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ABSTRACT: Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R 2 = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.Applied Microbiology and Biotechnology 03/2013; · 3.42 Impact Factor -
Article: Development and characterization of the replicon system of Japanese encephalitis live vaccine virus SA14-14-2.
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ABSTRACT: BACKGROUND: Viral self-replicating sub-genomic replicons represent a powerful tool for studying viral genome replication, antiviral screening and chimeric vaccine development. Many kinds of flavivirus replicons have been developed with broad applications. FINDINGS: The replicon system of JEV live vaccine strain SA14-14-2 was successfully developed in this study. Two kinds of replicons that express enhanced green fluorescent protein (EGFP) and Renilla luciferase (R.luc) were constructed under the control of SP6 promoter, respectively. Robust EGFP and R.luc signals could be detected in the replicon-transfected BHK-21 cells. Furthermore, the potential effects of selected amino acids in the C-terminal of envelope protein on replication were characterized using the replicon system. CONCLUSIONS: Our results provide a useful platform not only for the study of JEV replication, but also for antiviral screening and chimeric vaccine development.Virology Journal 02/2013; 10(1):64. · 2.34 Impact Factor -
Article: Human IgG Subclasses against Enterovirus Type 71: Neutralization versus Antibody Dependent Enhancement of Infection.
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ABSTRACT: The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.PLoS ONE 01/2013; 8(5):e64024. · 4.09 Impact Factor -
Article: Complete genome sequence analysis of human echovirus type 30 isolated in china.
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ABSTRACT: We report here the complete genome sequence of a human echovirus type 30 strain ECV30/GX10/05 isolated in Guangxi, China, in 2010. Phylogenetic analysis showed that ECV30/GX10/05 was closely related to a Korean strain isolated in 2008. The sequence information will help in an understanding of the molecular epidemiology and evolution of echovirus.Journal of Virology 12/2012; 86(24):13856-7. · 5.40 Impact Factor -
Article: Complete genome sequence of dengue virus serotype 2 cosmopolitan genotype strain in guangdong, china.
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ABSTRACT: Here we report the complete genome sequence of a dengue virus serotype 2 (DENV-2) strain, GZ40, isolated in Guangdong, China, in 2010. A phylogenetic analysis classified GZ40 into the Cosmopolitan genotype, while previous Chinese DENV-2 isolates belong to the Asian I genotype. The reemergence of the Cosmopolitan genotype of DENV-2 in China deserves further investigation.Journal of Virology 12/2012; 86(24):13808-9. · 5.40 Impact Factor -
Article: Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus.
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ABSTRACT: A new duck Tembusu virus (TMUV), also known as BYD virus, has been identified as the causative agent for the emerging duck egg-drop syndrome in mainland China. The rapid spread and wide distribution of the new TMUV in mainland China result in heavy loss to the poultry industry and pose great threats to public health. Rapid and sensitive detection methods are critical for prevention and control of TMUV infections. In this study, a reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) and an SYBR Green-I-based real-time RT-PCR assay specific for the duck TMUV were developed and validated with laboratory and field samples, respectively. The detection limits were 1 × 10(-4) and 1 × 10(-3) PFU per reaction for the RT-LAMP assay and real-time RT-PCR assay, respectively. The specificities were analyzed with other related members of the genus Flavivirus, and no cross-reaction was observed. Furthermore, both assays were evaluated with field samples, and they exhibited high sensitivity and specificity. In addition, the real-time RT-PCR assay worked well in viral load analysis, which revealed that the spleen may be the primary target for the replication of new duck TMUV in ducks. The TMUV-specific RT-LAMP and real-time RT-PCR assays will provide useful tools for the diagnosis and epidemiological surveillance of TMUV infection.Archives of Virology 08/2012; · 2.11 Impact Factor -
Article: Complete genome sequence of a chikungunya virus isolated in Guangdong, China.
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ABSTRACT: Chikungunya virus belongs to the genus Alphavirus in the family Togaviridae. Here we report the complete genome sequence of a chikungunya virus strain, GD05/2010, isolated in 2010 from a patient with chikungunya fever in Guangdong, China. The sequence information is important for surveillance of this emerging arboviral infection in China.Journal of Virology 08/2012; 86(16):8904-5. · 5.40 Impact Factor -
Article: A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.
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ABSTRACT: Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.Journal of General Virology 06/2012; 93(Pt 9):1959-64. · 3.36 Impact Factor -
Article: Co-circulation of two genotypes of dengue virus serotype 3 in Guangzhou, China, 2009.
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ABSTRACT: Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future.Virology Journal 06/2012; 9:125. · 2.34 Impact Factor -
Article: Complete genome sequence of a dengue virus serotype 4 strain isolated in Guangdong, China.
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ABSTRACT: Here we report the first complete genome sequence of a dengue virus serotype 4 genotype II strain, GZ30, isolated in Guangzhou, Guangdong Province, China, in 2010. The sequence information provided herein will help us to understand the molecular epidemiology of dengue virus and predict the risk of severe diseases in mainland China.Journal of Virology 06/2012; 86(12):7021-2. · 5.40 Impact Factor -
Article: Recombination of human coxsackievirus B5 in hand, foot, and mouth disease patients, China.
Emerging Infectious Diseases 02/2012; 18(2):351-3. · 6.79 Impact Factor -
Article: A DNA-based West Nile virus replicon elicits humoral and cellular immune responses in mice.
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ABSTRACT: While self-replicating, non-infectious subgenomic flavivirus replicons have been described, most of them are RNA transcripts under the control of an Sp6 or T7 promoter. In this study, using West Nile virus (WNV) as a model, a series of DNA-based reporter replicons under the control of a minimal cytomegalovirus (CMV) immediate-early promoter were constructed, and functional analysis showed that these reporter replicons replicate efficiently in mammalian cells. When the DNA-based WNV replicon was used to immunize mice, NS1-specific IgG antibodies and anti-WNV neutralizing antibodies were both induced. Additionally, immunization with this DNA-based WNV replicon induced high levels of lymphocyte proliferation and enhanced the secretion of IFN-γ. These results suggest that this type of DNA-based replicon can induce humoral and cellular immune responses in mice, indicating that this type of DNA-based replicon may serve as a useful platform for vaccine development and protein expression.Journal of virological methods 08/2011; 178(1-2):87-93. · 2.13 Impact Factor -
Article: In vitro and in vivo characterization of a new enterovirus type 71-specific human intravenous immunoglobulin manufactured from selected plasma donors.
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ABSTRACT: Enterovirus type 71 (EV71) causes large outbreaks with significant mortality among young children, and no specific antiviral treatment is currently available. Antibody-based therapy represents a promising alternative strategy for lethal EV71 infection. Our previous data has shown that anti-EV71 neutralization antibodies were present in a significant proportion of blood donors in China. To produce a new human intravenous immunoglobulin (IVIG) product containing high titer anti-EV71 neutralizing antibodies and investigate its therapeutic efficacy against lethal EV71 infection in a murine model. Plasma units that contained high titer neutralizing antibodies from selected Chinese donors were pooled and processed into pharmaceutical grade IVIG preparations according to the standard procedure. The efficacy of these EV71-specific IVIG product was characterized in vitro by neutralization assay and in vivo by suckling mouse protection testing. The therapeutic effects against lethal EV71 challenge were further assayed in a suckling mouse model. About 12% of the normal plasma units were selected and pooled to manufacture the EV71-IVIG preparations, and in vitro and in vivo efficacy data showed that these EV71-specific IVIG preparations were enriched with neutralizing antibodies against EV71. Furthermore, treatment with EV71-specific IVIG was evidenced to confer protection against lethal EV71 challenge in a dose- and time-dependent manner in the suckling mouse model. This preclinical study indicates that these "tailor-made" EV71-IVIG preparations manufactured from selected plasma donors in EV71-endemic areas may represent a promising therapeutic option for the lethal EV71 infections, and further clinical trials should be warranted in the future.Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 06/2011; 51(4):246-9. · 3.12 Impact Factor -
Article: Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China.
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ABSTRACT: Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3' untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3'UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.Archives of Virology 05/2011; 156(8):1485-8. · 2.11 Impact Factor
Top co-authors
Top Journals
Institutions
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2013
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Zhejiang University
Hangzhou, Zhejiang Sheng, China -
Guangzhou Medical University
Guangzhou, Guangdong Sheng, China
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2008–2013
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Beijing Institute of Microbiology and Epidemiology
Beijing, Beijing Shi, China
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2004–2008
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Academy of Military Medical Sciences
Tianjin, Tianjin Shi, China
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