树鼩大便中腺病毒的实验研究

本文报道用聚丙烯酰胺凝胶电泳(PAGE)和电镜(EM)技术,从不含轮状病毒的野生成年云南树鼩(Tupain belangeri chinensis)大便中检出腺病毒的实验研究结果。从90份样品中检出12份含腺病毒,阳性率为13％。这些腺病毒DNA的电泳图型类似于肠道腺病毒。电镜检查7份PAGE阳性的样品,证实全都含有典型腺病毒颗粒。这些腺病毒具有低滴度的凝聚人“O”型、“B”型红血细胞以及猴和树鼩红血细胞的作用。作者认为这些腺病毒可能是树鼩自身的肠道腺病毒。     

树鼩实验感染后血清中抗基孔肯雅病毒IgM和IgG抗体检查

成年树经人工感染基孔肯雅病毒后，能产生2～6天的病毒血症。感染后第6天能产生特异性IgM抗体，第14～21天为高峰，以后逐渐下降。感染后第12天，IgG抗体开始出现，第30～60天为高峰，并持续不降，表明树对基孔肯雅病毒敏感。     

Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes

Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus-cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase-3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations.     

HBV动物模型研究进展

HBV感染动物模型是研究HBV致病机制、筛选新型有效抗HBV药物和治疗方法的重要工具,然而HBV感染具有高度组织特异性以及种属特异性,这给HBV感染动物模型的建立带来了困难。近年来,随着分子生物学、实验动物学、病毒学及免疫学等相关学科技术的进步,HBV感染或复制动物模型取得了明显进展。目前应用于HBV(包括与HBV具有相似特性的动物肝炎病毒)研究的动物模型主要包括黑猩猩、树鼩、土拨鼠及鸭HBV感染模型,HBV转基因小鼠、高压水动力注射介导的小鼠HBV复制模型和重组腺相关病毒载体介导的小鼠HBV复制模型,以及人源化人-鼠嵌合肝脏HBV感染模型,此外,钠离子-牛磺胆酸共转运蛋白转基因小鼠感染模型是近年的研究热点。本文就上述HBV动物模型的研究进展进行综述。     

丙型肝炎病毒体外可感染树鼩肝细胞

目的 探讨丙型肝炎病毒（HCV）体外感染树目句原代肝细胞。方法 用HCV RNA阳性血清感染原代树鼩肝细胞，并用感染后细胞培养上清液进行传代感染，通过检测受染肝细胞正、负链HCV RNA、培养上清液中包装后HCV RNA，并对比分析感染前后病毒准种变化等指标，评价感染是否成功。结果 受染树鼩肝细胞自第5～10天可检出负链HCV RNA，而正链RNA至感染后第14天仍可检出；感染后3～14d培养上清液中可检出HCV RNA，且呈RNA酶抗性；培养上清液中病毒可传代感染新的树鼩肝细胞；感染前后HCV准种分析显示树鼩肝细胞可被特定的准种选择性感染，而传代感染后肝细胞可检出新的准种。结论 原代树鼩肝细胞体外可被HCV感染且支持病毒复制。     

Human bocavirus: Current knowledge and future challenges

Human bocavirus(HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate(HBoV 1), namely HBoV 2, HBoV 3 and HBoV 4, have been detected principally in human faeces. HBo Vs are small nonenveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1(NS1) and nuclear phosphoprotein(NP1) and the third the viral capsid proteins 1 and 2(VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBo V infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzymelinked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoV s as true pathogen or innocent bystander is also emphasized.     

Dendritic Cell Apoptosis and the Pathogenesis of Dengue

Dengue viruses and other members of the Flaviviridae family are emerging human pathogens. Dengue is transmitted to humans by Aedes aegypti female mosquitoes. Following infection through the bite, cells of the hematopoietic lineage, like dendritic cells, are the first targets of dengue virus infection. Dendritic cells (DCs) are key antigen presenting cells, sensing pathogens, processing and presenting the antigens to T lymphocytes, and triggering an adaptive immune response. Infection of DCs by dengue virus may induce apoptosis, impairing their ability to present antigens to T cells, and thereby contributing to dengue pathogenesis. This review focuses on general mechanisms by which dengue virus triggers apoptosis, and possible influence of DC-apoptosis on dengue disease severity.     

A new look at viruses in type 1 diabetes

Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells. Genetic factors are believed to be a major component for the development of T1D, but the concordance rate for the development of diabetes in identical twins is only about 40%, suggesting that nongenetic factors play an important role in the expression of the disease. Viruses are one environmental factor that is implicated in the pathogenesis of T1D. To date, 14 different viruses have been reported to be associated with the development of T1D in humans and animal models. Viruses may be involved in the pathogenesis of T1D in at least two distinct ways: by inducing beta cell-specific autoimmunity, with or without infection of the beta cells, [e.g. Kilham rat virus (KRV)] and by cytolytic infection and destruction of the beta cells (e.g. encephalomyocarditis virus in mice). With respect to virus-mediated autoimmunity, retrovirus, reovirus, KRV, bovine viral diarrhoea-mucosal disease virus, mumps virus, rubella virus, cytomegalovirus and Epstein-Barr virus (EBV) are discussed. With respect to the destruction of beta cells by cytolytic infection, encephalomyocarditis virus, mengovirus and Coxsackie B viruses are discussed. In addition, a review of transgenic animal models for virus-induced autoimmune diabetes is included, particularly with regard to lymphocytic choriomeningitis virus, influenza viral proteins and the Epstein-Barr viral receptor. Finally, the prevention of autoimmune diabetes by infection of viruses such as lymphocytic choriomeningitis virus is discussed.     

Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes

BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.     

Antiviral Compounds for Blocking Arboviral Transmission in Mosquitoes

Mosquito-borne arthropod-borne viruses (arboviruses) such as the dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are important human pathogens that are responsible for significant global morbidity and mortality. The recent emergence and re-emergence of mosquito-borne viral diseases (MBVDs) highlight the urgent need for safe and effective vaccines, therapeutics, and vector-control approaches to prevent MBVD outbreaks. In nature, arboviruses circulate between vertebrate hosts and arthropod vectors; therefore, disrupting the virus lifecycle in mosquitoes is a major approach for combating MBVDs. Several strategies were proposed to render mosquitoes that are refractory to arboviral infection, for example, those involving the generation of genetically modified mosquitoes or infection with the symbiotic bacterium Wolbachia. Due to the recent development of high-throughput screening methods, an increasing number of drugs with inhibitory effects on mosquito-borne arboviruses in mammalian cells were identified. These antivirals are useful resources that can impede the circulation of arboviruses between arthropods and humans by either rendering viruses more vulnerable in humans or suppressing viral infection by reducing the expression of host factors in mosquitoes. In this review, we summarize recent advances in small-molecule antiarboviral drugs in mammalian and mosquito cells, and discuss how to use these antivirals to block the transmission of MBVDs.     

Targeting Host Factors to Treat West Nile and Dengue Viral Infections

West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.     

Animal model for study of human hepatitis viruses

Human hepatitis B virus (HBV) and hepatitis C virus (HCV) infect only chimpanzees and humans. Analysis of both viruses has long been hampered by the absence of a small animal model. The recent development of human hepatocyte chimeric mice has enabled us to carry out studies on viral replication and cellular changes induced by replication of human hepatitis viruses. Various therapeutic agents have also been tested using this model. In the present review, we summarize published studies using chimeric mice and discuss the merits and shortcomings of this model.     

Immune Responses and Lassa Virus Infection

Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and nonhuman primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.     

Human hepatitis B virus and hepatocellular carcinoma I. Experimental infection of tree shrews with hepatitis B virus

Tree shrews (Tupaia belangeri chinenesis) can be experimentally infected with human hepatitis B virus (HBV) by inoculation with human serum positive for HBV, the experimental infection rate being 55.21%. Successive infections have been passed through five generations among the tree shrews inoculated with HBV-positive sera from the infected animals, the average infection rate being 94.0%. The experimental infection of tree shrews with HBV may be prevented by immunization with hepatitis B vaccine, the protection rate being 88.89%. Standard serum containing HBV at 10⁸ CID (chimpanzee infection dose)/ml, was diluted 10^–6, 10^–7, 10^–8, 10^–9, and 10^–10 and produced infection rates of 80.0%, 88.8%, 66.7%, 55.6% and 42.9% respectively. Thus the CID₅₀ in tree shrews may reach a dilution of 10^–9, which shows that tree shrews are sensitive to HBV infection. These results successfully establish tree shrews as a reliable and useful animal model for research on HBV infection and its relation to hepatocarcinogenesis.Abbreviations HCC Hepatocellular carcinoma - HBV human hepatitis B virus     

Viral gastrointestinal infections.

Numerous disease-causing viruses of the gastrointestinal tract have been described. The diseases that they induce range from subclinical to fatal gastrointestinal or systemic organ infection. Recent advances in molecular biology and virus culture techniques, as well as improvements in animal models of these infections, have increased our understanding of pathologic processes associated with these viruses. Although our overall understanding of these viruses has improved to the extent that many more viruses can be identified as pathogens, we are still a long way from achieving a thorough understanding of the pathogenesis of viral infection and the mechanisms that mediate immunity.     

Inhibitory effect of adefovir and lamivudine on the initiation of hepatitis B virus infection in primary tupaia hepatocytes

Adefovir dipivoxil and lamivudine are two safe and efficacious drugs licensed for the treatment of chronic hepatitis B virus (HBV) infection. Both drugs inhibit the viral polymerase, resulting in a profound suppression of virus production. Blocking the viral polymerase may also affect the initiation of HBV infection, because HBV virions harbor a partially double-stranded genome and productive infection requires completion of viral plus-strand DNA synthesis with subsequent formation of covalently closed circular DNA (cccDNA). To address this issue, we used primary hepatocytes from the tree shrew Tupaia belangeri that were recently shown to be susceptible to HBV infection. Treatment of cells with either drug partially inhibited initial HBV cccDNA formation. Adefovir was more effective than lamivudine, resulting in a 3-fold reduction of RNA synthesis and viral surface antigen production. However, prevention of initial cccDNA formation was incomplete even after combined treatment, whereas de novo synthesis of viral replicative intermediates was completely suppressed. A possible explanation for this observation is the genomic plus-strand gap of less than 200 bases in some virions, limiting the window for antiviral action. In conclusion, nucleos(t)ide analogues can target initial plus-strand DNA repair and reduce but not completely block HBV infection.     

Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses

Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.     

儿科重症监护室先天性心脏病合并呼吸道感染患儿的病毒性病原学研究

目的：总结儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病毒性病原谱。方法收集2010年6月至2012年6月因呼吸道感染入住本院儿科重症监护室的患儿咽拭子标本622份,其中先天性心脏病合并呼吸道感染患儿咽拭子34份。应用多重聚合酶链反应(PCR)技术对呼吸道病毒进行检测,并对照分析合并先天性心脏病患儿的病毒性病原学特点。结果①34份先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性20份(58.8%),588份非先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性368份(62.6%)。②先天性心脏病组中,最常见的病毒分别为人鼻病毒(human rhinovirus,HRV)8份,呼吸道合胞病毒(respiratory syncytial virus, RSV)6份,人博卡病毒(human bocavirus,HBoV)4份,腺病毒(adenovirus,ADV)2份;非先天性心脏病组中,最常见的病毒分别为 HRV 160份,RSV 104份,ADV 72份,HBoV50份;其他病毒阳性率较低。③先天性心脏病组中,混合病毒感染有2份(2/20,10.0%),非先天性心脏病组中,混合病毒感染有110份(110/368,29.9%)。结论本地区儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病原体中病毒性病原体检出率高,以鼻病毒、呼吸道合胞病毒、人博卡病毒和腺病毒最常见,病毒谱和非先天性心脏病组相似。