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2013年28卷3期

West Nile virus (WNV) is widely distributed throughout the world. So far, there is no effective antiviral therapy. In this issue, Shan C and colleagues used the secreted Gaussia luciferase as the reporter, and developed a fast, sensitive and efficient replicon assay system (Gluc-WNV-Rep). This system will be useful in antiviral drug screening programs, as well as in viral replication studies and pathogenesis of WNV.

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Meeting Report

The Mérieux Chinese Research Network in Wuhan: the 4th Chapter of a Success Story

Arnaud Favry, Jiali Si

2013, 28(3): 127 doi: 10.1007/s12250-013-3346-1

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On April 17-18th 2013, the 4th meeting of the Mérieux Chinese Research Network was held in Wuhan, an event organized by Institut Mérieux, together with the Wuhan Institute of Virology, Chinese Academy of Sciences, Huangzhong University of Science and Technology, and the Chinese Society for Microbiology.

Research Article

The Identification of Three Sizes of Core Proteins during the Establishment of Persistent Hepatitis C Virus Infection in vitro

Qingjiao Liao, Jiansheng Tian, Yang Wu, Xulin Chen

2013, 28(3): 129 doi: 10.1007/s12250-013-3296-7

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Similar to Hepatitis C virus (HCV) infection in humans, HCVcc infection can also result in persistent and chronic infection. The core protein is a variable protein and exists in several sizes. Some sizes of core proteins have been reported to be related to chronic HCV infection. To study the possible role of the core protein in persistent HCV infection, a persistent HCVcc infection was established, and the expression of the core protein was analysed over the course of the infection. The results show that there are three sizes of core proteins (p24, p21 and p19) expressed during the establishment of persistent HCVcc infection. Of these, the p21 core protein is the mature form of the HCV core protein. The p24 core protein is the phosphorylated form of p21. The p19 core protein appears to be a functional by-product generated during the course of infection. These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion. The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.

Enhanced Protective Efficacy of H5 Subtype Influenza Vaccine with Modification of the Multibasic Cleavage Site of Hemagglutinin in Retroviral Pseudotypes

Ling Tao, JianJun Chen, Jin Meng, Yao Chen, Hongxia Li, Yan Liu, Zhenhua Zheng, Hanzhong Wang

2013, 28(3): 136 doi: 10.1007/s12250-013-3326-5

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Traditionally, the multibasic cleavage site (MBCS) of surface protein H5-hemagglutinin (HA) is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines. Whether such modification benefits new candidate vaccines has not been adequately investigated. We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA (wtH5) from A/swine/Anhui/ca/2004 (H5N1) virus. Here, we generated mtH5N1 pseudotypes, which contained a mutant-type HA (mtH5) with a modified monobasic cleavage site. Groups of mice were subcutaneously injected with the two types of influenza pseudotypes. Compared to the group immunized with wtH5N1 pseudotypes, the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-γ in immunized mice, and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henan/12/2004 (H5N1). This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N1 influenza viruses.

Epidemic and Maintenance of Rabies in Chinese Ferret Badgers (Melogale moschata) indicated by Epidemiology and the Molecular Signatures of Rabies Viruses

Shoufeng Zhang, Ye Liu, Yanli Hou, Jinghui Zhao, Fei Zhang, Ying Wang, Rongliang Hu

2013, 28(3): 146 doi: 10.1007/s12250-013-3316-7

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An epidemic of Chinese ferret badger-associated human rabies was investigated in Wuyuan county, Jiangxi province and rabies viruses isolates from ferret badgers in different districts in Jiangxi and Zhejiang provinces were sequenced with their nucleotides and amino acids and aligned for epidemiological analysis. The results showed that the human rabies in Wuyuan are only associated with ferret badger bites; the rabies virus can be isolated in a high percentage of ferret badgers in the epidemic areas in Jiangxi and Zhejiang provinces; the isolates share the same molecular features in nucleotides and have characteristic amino acid signatures, i.e., 2 sites in the nucleoprotein and 3 sites in the glycoprotein, that are distinct from virus isolates from dogs in the same region. We conclude that rabies in Chinese ferret badgers has formed an independent transmission cycle and ferret badgers may serve as another important rabies reservoir independent of dog rabies in China.

Inhibition of Japanese Encephalitis Virus Infection by Flavivirus Recombinant E Protein Domain III

Jingjing Fan, Yi Liu, Xuping Xie, Bo Zhang, Zhiming Yuan

2013, 28(3): 152 doi: 10.1007/s12250-013-3331-8

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Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.

Development and Characterization of West Nile Virus Replicon Expressing Secreted Gaussia Luciferase

Chao Shan, Xiaodan Li, Chenglin Deng, Baodi Shang, Linlin Xu, Hanqing Ye, Zhiming Yuan, Bo Zhang

2013, 28(3): 161 doi: 10.1007/s12250-013-3332-7

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We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

Interleukin-12 as a Genetic Adjuvant Enhances Hepatitis C Virus NS3 DNA Vaccine Immunogenicity

Malihe Naderi, Atefeh Saeedi, Abdolvahab Moradi, Mishar Kleshadi, Mohammad Reza Zolfaghari, Ali Gorji, Amir Ghaemi

2013, 28(3): 167 doi: 10.1007/s12250-013-3291-z

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Hepatitis C virus (HCV) chronic infection is a worldwide health problem, and numerous efforts have been invested to develop novel vaccines. An efficient vaccine requires broad immune response induction against viral proteins. To achieve this goal, we constructed a DNA vaccine expressing nonstructural 3 (NS3) gene (pcDNA3.1-HCV-NS3) and assessed the immune response in C57BL/6 mice. In this study, the NS3 gene was amplified with a nested-reverse transcriptase-polymerase chain reaction (RT-PCR) method using sera of HCV-infected patients with genotype 1a. The resulting NS3 gene was subcloned into a pcDNA3.1 eukaryotic expression vector, and gene expression was detected by western blot. The resultant DNA vaccine was co-administered with interleukin-12 (IL-12) as an adjuvant to female C57BL/6 mice. After the final immunizations, lymphocyte proliferation, cytotoxicity, and cytokine levels were assessed to measure immune responses. Our data suggest that co-administration of HCV NS3 DNA vaccine with IL-12 induces production of significant levels of both IL-4 and interferon (IFN)-γ (p<0.05). Cytotoxicity and lymphocyte proliferation responses of vaccinated mice were significantly increased compared to control (p<0.05). Collectively, our results demonstrated that co-administration of HCV NS3 and IL-12 displayed strong immunogenicity in a murine model.

Immunization with Cytomegalovirus Envelope Glycoprotein M and Glycoprotein N DNA Vaccines can Provide Mice with Complete Protection against a Lethal Murine Cytomegalovirus Challenge

Huadong Wang, Yanfeng Yao, Chaoyang Huang, Quanjiao Chen, Jianjun Chen, Ze Chen

2013, 28(3): 174 doi: 10.1007/s12250-013-3330-9

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Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.

Letter

Investigation and Analysis of Rabies Viral Infection and Distribution in China in 2005-2012

Wentao Jiao, Hao Li, Xiaoyan Tao, Miao Song, Xinxin Shen, Zhenyang Guo, Yunjiao Zhao, Qing Tang, Guodong Liang

2013, 28(3): 183 doi: 10.1007/s12250-013-3324-7

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We report the results of a preliminary investigation of data collected between 2005 and 2012. A National Human Rabies Surveillance program was initiated in high rabies incidence regions in 2005, and subsequently carried out nationally to monitor rabies situation. Our work presents a summary of epidemiological and etiologic rabies surveillance data collected since the implementation of the program.

Investigation of the Evolutionary History of the Lyssaviruses

Xiaoyan Tao, Zhenyang Guo, Hao Li, Na Han, Qing Tang, Guodong Liang

2013, 28(3): 186 doi: 10.1007/s12250-013-3334-5

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We report the results of evolutionary history estimation of the lyssaviruses based on an analysis of the Glycoprotein (G) sequences gene using the BEAST software package. The most recent common ancestor (TMRCA) of all the lyssavirus strains was estimated to be approximately 5030 years (95% HPD 3988-6069 years), and there was a significant spread of the rabies virus throughout the world range in the last 200 years, consistent with significant time points in development and migration of human civilizations. We speculate that increased and expansion of human migration during this time period may have promote the increase in lyssavirus diversity. In addition, evidence of host switching in lyssavirus history from the Chiroptera to the Carnivora orders was also identified.