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An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus

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Virologica Sinica 2015年第5期30卷 354-362页

作者： Ruikun Du Manli Wang Zhihong Hu Hualin Wang Fei Deng State Key Laboratory of virology Wuhan Institute of VirologyChinese Academy of Sciences

Japanese encephalitis virus(JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus–host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope(E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

关键词： Japanese encephalitis virus(JEV) reverse genetic s

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The roles of ebolavirus glycoproteins in viral pathogenesis

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Virologica Sinica 2017年第1期32卷 3-15页

作者： Yun-Jia Ning Fei Deng Zhihong Hu Hualin Wang State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

Ebolaviruses are highly dangerous pathogens exhibiting extreme virulence in humans and nonhuman primates. The majority of ebolavirus species, most notably Zaire ebolavirus, can cause Ebola virus disease(EVD), formerly known as Ebola hemorrhagic fever, in humans. EVD is associated with case-fatality rates as high as 90%, and there is currently no specific treatment or licensed vaccine available against EVD. Understanding the molecular biology and pathogenesis of ebolaviruses is important for the development of antiviral therapeutics. Ebolavirus encodes several forms of glycoproteins(GPs), which have some interesting characteristics, including the transcriptional editing coding strategy and extensive O-glycosylation modification, clustered in the mucin-like domain of GP1, full-length GP(GP_(1,2)), and shed GP. In addition to the canonical role of the spike protein, GP_(1,2), in viral entry, ebolavirus GPs appear to have multiple additional functions,likely contributing to the complex pathogenesis of the virus. Here, we review the roles of ebolavirus GPs in viral pathogenesis.

关键词： ebolavirus glycoprotein (GP) mucin-like domain (MLD) cytotoxicity immune evasion inflammation pathogenesis

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Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

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Virologica Sinica 2016年第2期31卷 160-167页

作者： Zheng Hou Zheng Zhou Zonglin Wang Gengfu Xiao State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

关键词： yeast plasmid shuttle DNA recombination Genome assembly inserted amplification homologous

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Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection

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Virologica Sinica 2016年第3期31卷 219-228页

作者： Guan-Hua Qiao Fei Zhao Shuang Cheng Min-Hua Luo State Key Laboratory of virology Wuhan Institute of VirologyChinese Academy of Sciences

Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth *** studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells(MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry,replication initiation, viral protein expression, and infectious virion release using western blotting,immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

关键词： human cytomegalovirus(HCMV) multipotent mesenchymal stromal cells(MSCs) susceptibility umbilical cord Wharton's jelly

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Structure-function relationship of the mammarenavirus envelope glycoprotein

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Virologica Sinica 2016年第5期31卷 380-394页

作者： Wei Wang Zheng Zhou Leike Zhang Shaobo Wang Gengfu Xiao State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

Mammarenaviruses, including lethal pathogens such as Lassa virus and Junin virus, can cause severe hemorrhagic fever in humans. Entry is a key step for virus infection, which starts with binding of the envelope glycoprotein （GP） to receptors on target cells and subsequent fusion of the virus with target cell membranes. The GP precursor is synthesized as a polypeptide, and maturation occurs by two cleavage events, yielding a tripartite GP complex （GPC） formed by a stable signal peptide （SSP）, GP1 and GP2. The unique retained SSP interacts with GP2 and plays essential roles in virion maturation and infectivity. GP1 is responsible for binding to the cell receptor, and GP2 is a class I fusion protein. The native structure of the tripartite GPC is unknown. GPC is critical for the receptor binding, membrane fusion and neutralization antibody recognition. Elucidating the molecular mechanisms underlining the structure-function relationship of the three subunits is the key for understanding their function and can facilitate novel avenues for combating virus infections. This review summarizes the basic aspects and recent research of the structure-function relationship of the three subunits. We discuss the structural basis of the receptor-binding domain in GP1, the interaction between SSP and GP2 and its role in virion maturation and membrane fusion, as well as the mechanism by which glycosylation stabilizes the GPC structure and facilitates immune evasion. Understanding the molecular mechanisms involved in these aspects will contribute to the development of novel vaccines and treatment strategies against mammarenaviruses infection.

关键词： Mammarenaviruses glycoprotein complex (GPC) stable signal peptide (SSP) membrane fusion glycosylation

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Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

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Virologica Sinica 2018年第3期33卷 213-226页

作者： Shiyu Dai Tao Zhang Yanfang Zhang Hualin Wang Fei Deng State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

关键词： ZIKV Baculovirus expression system. Virus-like particles (VLPs) Neutralizing antibodies

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Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus

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Virologica Sinica 2016年第2期31卷 118-130页

作者： Shu Shen Junming Shi Jun Wang Shuang Tang Hualin Wang Zhihong Hu Fei Deng State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

Recent outbreaks of Zika virus （ZIKV） infections in Oceania＇s islands and the Americas were characterized by high numbers of cases and the spread of the virus to new areas. To better understand the origin of ZIKV, its epidemic history was reviewed. Although the available records and information are limited, two major genetic lineages of ZIKV were identified in previous studies. However, in this study, three lineages were identified based on a phylogenetic analysis of all virus sequences from GenBank, including those of the envelope protein （E） and non-structural protein 5 （NS5） coding regions. The spatial and temporal distributions of the three identified ZIKV lineages and the recombination events and mechanisms underlying their divergence and evolution were further elaborated. The potential migration pathway of ZIKV was also characterized. Our findings revealed the central roles of two African countries, Senegal and Cote d＇lvoire, in ZIKV evolution and genotypic divergence. Furthermore, our results suggested that the outbreaks in Asia and the Pacific islands originated from Africa. The results provide insights into the geographic origins of ZlKV outbreaks and the spread of the virus, and also contribute to a better understanding of ZlKV evolution, which is important for the prevention and control of ZlKV infections.

关键词： Flaviviridae Zika virus (ZIKV) phylogenetic analysis lineages recombination migration

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Tick-Borne Viruses

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Virologica Sinica 2018年第1期33卷 21-43页

作者： Junming Shi Zhihong Hu Fei Deng Shu Shen State Key Laboratory of virology Wuhan Institute of Virology Chinese Academy of Sciences

Ticks are important vectors for the transmission of pathogens including *** viruses carried by ticks also known as tick-borne viruses(TBVs),contain a large group of viruses with diverse genetic properties and are concluded in two orders,nine families,and at least 12 *** members of the TBVs are notorious agents causing severe diseases with high mortality rates in humans and livestock,while some others may pose risks to public health that are still unclear to ***,we review the current knowledge of TBVs with emphases on the history of virus isolation and identification,tick vectors,and potential pathogenicity to humans and animals,including assigned species as well as the recently discovered and unassigned *** these will promote our understanding of the diversity of TBVs,and will facilitate the further investigation of TBVs in association with both ticks and vertebrate hosts.

关键词： Ticks - Tick-borne viruses (TBVs) - Isolation - Identification

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Virus like particle-based vaccines against emerging infectious disease viruses

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Virologica Sinica 2016年第4期31卷 279-287页

作者： Jinliang Liu Shiyu Dai Manli Wang Zhihong Hu Hualin Wang Fei Deng State Key Laboratory of virology Wuhan Institute of VirologyChinese Academy of Sciences

Emerging infectious diseases are major threats to human *** severe viral disease outbreaks occur in developing regions where health conditions are *** increased international travel and business,the possibility of eventually transmitting infectious viruses between different countries is *** most effective approach in preventing viral diseases is ***,vaccines are not currently available for numerous viral *** particles(VLPs) are engineered vaccine candidates that have been studied for *** are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus *** have antigenicity similar to that of the native virus,but are non-infectious as they lack key viral genetic *** vaccines have attracted considerable research interest because they offer several advantages over traditional *** have shown that VLP vaccines can stimulate both humoral and cellular immune responses,which may offer effective antiviral *** we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious *** infectious agents discussed include RNA viruses from different virus families,such as the Arenaviridae,Bunyaviridae,Caliciviridae,Coronaviridae,Filoviridae,Flaviviridae,Orthomyxoviridae,Paramyxoviridae,and Togaviridae families.

关键词： emerging infectious disease self-assembly vaccine virus virus-like particle(VLP)

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The V1 region of gp120 is preferentially selected during SIV/HIV transmission and is indispensable for envelope function and virus infection

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Virologica Sinica 2016年第3期31卷 207-218页

作者： Yanpeng Li Ulf Dittmer Yan Wang Jiping Song Binlian Sun Rongge Yang Research Group of HIV Molecular Epidemiology and virology Center for Emerging Infectious DiseasesState Key Laboratory of VirologyWuhan Institute of VirologyChinese Academy of Sciences institute for virology University Hospital EssenUniversity of Duisburg-Essen

A transmission bottleneck occurs during each human immunodeficiency virus(HIV) transmission event, which allows only a few viruses to establish new infection. However, the genetic characteristics of the transmitted viruses that are preferentially selected have not been fully elucidated. Here, we analyzed amino acids changes in the envelope protein during simian immunodeficiency virus(SIV)/HIV deep transmission history and current HIV evolution within the last 15–20 years. Our results confirmed that the V1V2 region of gp120 protein, particularly V1, was preferentially selected. A shorter V1 region was preferred during transmission history, while during epidemic, HIV may evolve to an expanded V1 region gradually and thus escape immune recognition. We then constructed different HIV-1 V1 mutants using different HIV-1 subtypes to elucidate the role of the V1 region in envelope function. We found that the V1 region, although highly variable, was indispensable for virus entry and infection, probably because V1 deletion mutants exhibited impaired processing of gp160 into mature gp120 and gp41. Additionally, the V1 region affected Env incorporation. These results indicated that the V1 region played a critical role in HIV transmission and infection.

关键词： simian immunodeficiency virus(SIV) human immunodeficiency virus(HIV) transmission selection V1 loop Env function

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