口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK-15细胞凋亡的研究结果。采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的缺口末端标记（TUNEL）技术均检测到了典型的细胞凋亡。结果显示使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK-15细胞,在培养32?h后荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,凋亡率约为20%;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

Structure of Foot-and-Mouth Disease Virus Mutant Polymerases with Reduced Sensitivity to Ribavirin

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R) selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop β9-α11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 Å, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop β9-α11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.Ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) (R) is a clinically important nucleoside analogue that exhibits antiviral activity against a broad spectrum of RNA viruses (17). R displays several antiviral mechanisms of action, including lethal mutagenesis (loss of infectivity associated with an increase in the mutation rate) (7, 9, 21, 23). The 5′-triphosphorylated form of R (RTP) can be incorporated by the viral polymerases into the nascent RNA, acting as either an adenylate or a guanylate analogue, inducing base transitions. Ambiguous utilization of RTP by RNA-dependent RNA polymerases during genome replication may lead to virus extinction (1, 6, 7, 33).As extensively documented for nonmutagenic antiviral inhibitors, selection of mutagen-resistant viruses may be a problem for the efficacy of antiviral treatments based on lethal mutagenesis. Serial passages of foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of R resulted in the selection of a mutant virus containing the amino acid substitution M296I in polymerase 3D. Measurements of viral fitness and progeny production suggested that M296I was selected because it decreased the mutagenic activity of R on FMDV (28). The mutant polymerase restricted the incorporation of RTP during RNA synthesis, relative to the wild-type enzyme, without an increase in average copying fidelity. Rather, the mutant enzyme displayed an about 2-fold lower RTP incorporation frequency and an about 2.5-fold increase in the A-to-G transition frequency (3). The substitution M296I in 3D conferred upon FMDV resistance to extinction by high R concentrations, but extinction of the mutant was achieved by an alternative mutagenic treatment (22).In contrast to passage of FMDV, passage of poliovirus (PV) in the presence of R selected a mutant virus that included the replacement G64S in 3D (25). This substitution conferred upon 3D a higher average copying fidelity, allowing the enzyme to restrict the incorporation of RTP in the place of ATP or GTP (4, 6). The increased copying fidelity gave rise to PV populations that were less adaptable than wild-type populations to a complex environment, represented by PV-susceptible mice (24, 32). In FMDV 3D, the substitution equivalent to G64S in PV is G62S. This replacement was never selected in FMDV passaged in the presence of R and was never detected as a minority component in mutant spectra of FMDV that replicated in the absence or presence of R or other mutagenic agents (1, 28, 29).To interpret the selection of disparate R resistance mutations in FMDV and PV and to gain insight into the molecular basis of R resistance, we have engineered FMDVs encoding 3D with G62S, alone and together with M296I and compared the behavior of the mutants with that of wild-type FMDV. We have purified the corresponding 3Ds with the G62S, the M296I, or both substitutions and determined their polymerase activities and three-dimensional structures alone and in several catalytic complexes. The results show that FMDV expressing 3D with G62S is genetically unstable and that the reason for its instability probably lies in impaired polymerase activity associated with the conformation acquired by a loop located close to motif B (loop β9-α11, residues 294 to 304) which is involved in interactions with the template RNA and with the incoming nucleotide. Comparison of the structures revealed that the mutated residues, G62S and M296I, are involved in an extensive network of interactions that affect residues directly required for the catalytic function of the enzyme.     

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG₁ antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.     

Inhibition of Human Immunodeficiency Virus Type 1 Virion Entry by Dominant-Negative Hck

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa–CD4–LTR–β-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Immunosuppression during Acute Infection with Foot-and-Mouth Disease Virus in Swine Is Mediated by IL-10

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses, causing a devastating disease in cloven-hoofed animals with enormous economic consequences. Identification of the different parameters involved in the immune response elicited against FMDV remains unclear, and it is fundamental the understanding of such parameters before effective control measures can be put in place. In the present study, we show that interleukin-10 (IL-10) production by dendritic cells (DCs) is drastically increased during acute infection with FMDV in swine. In vitro blockade of IL-10 with a neutralizing antibody against porcine IL-10 restores T cell activation by DCs. Additionally, we describe that FMDV infects DC precursors and interferes with DC maturation and antigen presentation capacity. Thus, we propose a new mechanism of virus immunity in which a non-persistent virus, FMDV, induces immunosuppression by an increment in the production of IL-10, which in turn, reduces T cell function. This reduction of T cell activity may result in a more potent induction of neutralizing antibody responses, clearing the viral infection.     

Chemically Characterized Media for Study of Foot-and-Mouth Disease Virus in Baby Hamster Kidney Cells

Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.     

Effect of Foot-and-Mouth Disease Virus Infection on the Frequency,Phenotype and Function of Circulating Dendritic Cells in Cattle

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes one of the most devastating diseases in cloven-hoofed animals. Disease symptoms develop within 2 to 3 days of exposure and include fever and vesicular lesions on the tongue and hooves. Dendritic cells (DC) play an essential role in protective immune responses against pathogens. Therefore, investigating their role during FMDV infection would lead to a better understanding of host-pathogen interactions. In this study, following infection of cattle with FMDV, we investigated the frequency and function of conventional (cDC) and plasmacytoid DC (pDC) in blood by using multi-color flow cytometry. We show that the frequency of cDC and pDC increased following FMDV infection and peaked 3 to 4 days post-infection. During peak viremia, the cattle became lymphopenic, the expression of MHC class II molecules on cDC and pDC was dramatically down-regulated, the processing of exogenous antigen by cDC and pDC was impaired, and there was an increase in IL-10 production by DC and monocytes. Notably, after clearance of FMDV from the blood, MHC class II expression returned to pre-infection levels. Altogether, our study demonstrates that in cattle, FMDV inhibits the function of DC, thereby retarding the initiation of adaptive immune responses, potentially enhancing virus shedding during the acute phase of infection.     

A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry

Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin–moesin–radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.     

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.     

A Similar Pattern of Interaction for Different Antibodies with a Major Antigenic Site of Foot-and-Mouth Disease Virus: Implications for Intratypic Antigenic Variation

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C₁, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.     

口蹄疫病毒RT-LAMP检测方法的建立

利用逆转录环介导等温核酸扩增技术(RT-LAMP),建立了口蹄疫病毒快速检测方法,同时评价了该方法的灵敏性和特异性。结果表明,根据口蹄疫病毒多聚蛋白基因保守区段设计的LAMP引物能够在65℃下,1 h内实现目标核酸区段的大量扩增,检测结果可直接用肉眼判断。该检测体系具有极高的特异性,只能检测到目标病毒,与其他类似病毒如猪水泡病病毒、猪瘟病毒、猪细小病毒等无交叉反应,可检测到10-5稀释度的目标病毒核酸量,比普通RT-PCR的灵敏性高100倍,比荧光PCR高10倍。     

Pathogenesis of Primary Foot-and-Mouth Disease Virus Infection in the Nasopharynx of Vaccinated and Non-Vaccinated Cattle

A time-course pathogenesis study was performed to compare and contrast primary foot-and-mouth disease virus (FMDV) infection following simulated-natural (intra-nasopharyngeal) virus exposure of cattle that were non-vaccinated or vaccinated using a recombinant adenovirus-vectored FMDV vaccine. FMDV genome and infectious virus were detected during the initial phase of infection in both categories of animals with consistent predilection for the nasopharyngeal mucosa. A rapid progression of infection with viremia and widespread dissemination of virus occurred in non-vaccinated animals whilst vaccinated cattle were protected from viremia and clinical FMD. Analysis of micro-anatomic distribution of virus during early infection by lasercapture microdissection localized FMDV RNA to follicle-associated epithelium of the nasopharyngeal mucosa in both groups of animals, with concurrent detection of viral genome in nasopharyngeal MALT follicles in vaccinated cattle only. FMDV structural and non-structural proteins were detected in epithelial cells of the nasopharyngeal mucosa by immunomicroscopy 24 hours after inoculation in both non-vaccinated and vaccinated steers. Co-localization of CD11c⁺/MHC II⁺ cells with viral protein occurred early at primary infection sites in vaccinated steers while similar host-virus interactions were observed at later time points in non-vaccinated steers. Additionally, numerous CD8⁺/CD3^- host cells, representing presumptive natural killer cells, were observed in association with foci of primary FMDV infection in the nasopharyngeal mucosa of vaccinated steers but were absent in non-vaccinated steers. Immunomicroscopic evidence of an activated antiviral response at primary infection sites of vaccinated cattle was corroborated by a relative induction of interferon -α, -β, -γ and -λ mRNA in micro-dissected samples of nasopharyngeal mucosa. Although vaccination protected cattle from viremia and clinical FMD, there was subclinical infection of epithelial cells of the nasopharyngeal mucosa that could enable shedding and long-term persistence of infectious virus. Additionally, these data indicate different mechanisms within the immediate host response to infection between non-vaccinated and vaccinated cattle.     

Complement-Fixation Analysis of Four Subtypes of Foot-and-Mouth Disease Virus Type A

Complement-fixation patterns were established for four subtypes of foot-and-mouth disease virus by block assays against homologous and heterologous antiserum. Inhibition of fixation by excess antigen was observed in most homologous systems but rarely in the heterologous systems. The heterologous antibody titers were, in all instances, considerably lower than those for the homologous systems. Although relatively high dilutions of antiserum may be desirable for subtyping, higher concentrations of antibody should be used for determining serological types.     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.     

Inactivation of Foot-and-Mouth Disease Virus by Interaction of Dye and Visible Light

The inactivation of foot-and-mouth disease virus was studied by means of the interaction of neutral red, Toluidine Blue, and methylene blue with visible light. The virus, Type A, strain 1, CANEFA of Argentine origin, was grown in tissue culture and tested in the crude and clarified state. Virus and dye were mixed and incubated together at 4 C for 45 min in the dark, or were mixed and immediately exposed to the visible light source without prior incubation together. Mixtures of crude virus and dye, under any of the experimental conditions used, did not inactivate more than 1 to 2 logs of viral infectivity when held in the dark or when exposed to light during a period of 45 min. Complete inactivation of virus was achieved when clarified virus and dye were mixed and immediately exposed to the visible light source for 15 min. Prior incubation of clarified virus and dye permitted inactivation by methylene blue only, whereas no incubation prior to exposure resulted in three of the dyes contributing to inactivation. A concentration of 6 μg of neutral red, Toluidine Blue, methylene blue, and crystal violet was used per milliliter of virus suspension. Crystal violet was not a good viral inactivator under the conditions of the experimentation. Inactive virus induced the formation of neutralizing antibodies in adult chickens and mice. The antibody titer stimulated by the antigen treated with methylene blue and visible light was probably significant.