Visualization and Detection of Infectious Coxsackievirus Replication Using a Combined Cell Culture-Molecular Beacon Assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5′ noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.     

Detection of hepatitis a virus by using a combined cell culture-molecular beacon assay

Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5' noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.     

Human Astrocytic Cells Support Persistent Coxsackievirus B3 Infection

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.     

An Attenuated Coxsackievirus B3 Vector: A Potential Tool for Viral Tracking Study and Gene Delivery

Cardiomyocytes are quite resistant to gene transfer using standard techniques. We developed an expression vector carrying an attenuated but infectious and replicative coxsackievirus B3 (CVB3) genome, and unique ClaI-StuI cloning sites for an exogenous gene, whose product can be released from the nascent viral polyprotein by 2A^pro cleavage. This vector was tested as an expression vehicle for green fluorescent protein (GFP). The vector transiently expressed GFP in cell cultures for at least ten passages and delivered functional GFP to the infected cardiomyocytes for at least 6 days. Moreover, the recombinant viruses showed virulence attenuation in vitro and in vivo. The findings suggest that the recombinant CVB3 vector could be a useful tool for viral tracking study and delivering exogenous proteins to cardiomyocytes.     

Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro

Enteroviral persistence has been implicated in the pathogenesis of several chronic human diseases, including dilated cardiomyopathy, insulin-dependent diabetes mellitus, and chronic inflammatory myopathy. However, these viruses are considered highly cytolytic, and it is unclear what mechanisms might permit their long-term survival. Here, we describe the generation of a recombinant coxsackievirus B3 (CVB3) expressing the enhanced green fluorescent protein (eGFP), which we used to mark and track infected cells in vitro. Following exposure of quiescent tissue culture cells to either wild-type CVB3 or eGFP-CVB3, virus production was very limited but increased dramatically after cells were permitted to divide. Studies with cell cycle inhibitors revealed that cells arrested at the G(1) or G(1)/S phase could express high levels of viral polyprotein and produced abundant infectious virus. In contrast, both protein expression and virus yield were markedly reduced in quiescent cells (i.e., cells in G(0)) and in cells blocked at the G(2)/M phase. Following infection with eGFP-CVB3, quiescent cells retained viral RNA for several days in the absence of infectious virus production. Furthermore, RNA extracted from nonproductive quiescent cells was infectious when transfected into dividing cells, indicating that CVB3 appears to be capable of establishing a latent infection in G(0) cells, at least in tissue culture. Finally, wounding of infected quiescent cells resulted in viral protein expression limited to cells in and adjacent to the lesion. We suggest that (i) cell cycle status determines the distribution of CVB3 during acute infection and (ii) the persistence of CVB3 in vivo may rely on infection of quiescent (G(0)) cells incapable of supporting viral replication; a subsequent change in the cell cycle status may lead to virus reactivation, triggering chronic viral and/or immune-mediated pathology in the host.     

Mapping of a neutralizing antigenic site of Coxsackievirus B4 by construction of an antigen chimera.

A neutralizing antigenic site of coxsackievirus B4 (CVB4) was identified by construction of an antigen chimera between coxsackievirus B3 (CVB3) and CVB4. This chimera, designated CVB3/4, was constructed by inserting five amino acids of the putative BC loop of the structural protein VP1 of CVB4 into the corresponding loop of CVB3 by site-directed mutagenesis of infectious recombinant CVB3 cDNA. The chimeric cDNA was capable of inducing an infectious cycle upon transfection of permissive host cells. The resulting chimeric virus CVB3/4 was neutralized and precipitated by CVB4 and CVB3 serotype-specific polyclonal antisera, demonstrating that it unifies antigenic properties of both coxsackievirus serotypes. In addition, the chimera elicited antibodies in rabbits which were capable of neutralizing the two coxsackievirus serotypes CVB3 and CVB4. The insertion of the CVB4-specific antigenic site into the BC loop of CVB3 reduces the efficiency of viral replication, resulting in a small-plaque morphology of the virus chimera. In summary, these data give evidence for the presence of a serotype-specific neutralizing antigenic site in the BC loop of VP1 of CVB4 (amino acids 81 to 89). Our findings suggest that the construction of intertypic chimeras can be used as a tool for the identification of antigenic sites of coxsackieviruses. The retained immunogenicity of the mapped CVB4-specific antigenic epitope, when expressed in CVB3, indicates that CVB3 can be used as a RNA virus vector for heterologous antigenic sites.     

Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells

Enteroviruses, including coxsackieviruses, exhibit significant tropism for the central nervous system, and these viruses are commonly associated with viral meningitis and encephalitis. Previously, we described the ability of coxsackievirus B3 (CVB3) to infect proliferating neuronal progenitor cells located in the neonatal subventricular zone and persist in the adult murine central nervous system (CNS). Here, we demonstrate that cultured murine neurospheres, which comprise neural stem cells and their progeny at different stages of development, were highly susceptible to CVB3 infection. Neurospheres, or neural progenitor and stem cells (NPSCs), isolated from neonatal C57BL/6 mice, supported high levels of infectious virus production and high viral protein expression levels following infection with a recombinant CVB3 expressing enhanced green fluorescent protein (eGFP) protein. Similarly, NPSCs isolated from neonatal actin-promoter-GFP transgenic mice (actin-GFP NPSCs) were highly susceptible to infection with a recombinant CVB3 expressing DsRed (Discosoma sp. red fluorescent protein). Both nestin-positive and NG2(+) progenitor cells within neurospheres were shown to preferentially express high levels of viral protein as soon as 24 h postinfection (p.i.). By day 3 p.i., viral protein expression and viral titers increased dramatically in NPSCs with resultant cytopathic effects (CPE) and eventual cell death. In contrast, reduced viral replication, lower levels of CPE, and diminished viral protein expression levels were observed in NPSCs differentiated for 5 or 16 days in the presence of fetal bovine serum (FBS). Despite the presence of CPE and high levels of cell death following early CVB3 infection, surviving neurospheres were readily observed and continued to express detectable levels of viral protein as long as 37 days after initial infection. Also, CVB3 infection of actin-GFP NPSCs increased the percentage of cells expressing neuronal class III β-tubulin following their differentiation in the presence of FBS. These results suggest that neural stem cells may be preferentially targeted by CVB3 and that neurogenic regions of the CNS may support persistent viral replication in the surviving host. In addition, normal progenitor cell differentiation may be altered in the host following infection.     

Multiple viral determinants mediate myopathogenicity in coxsackievirus B1-induced chronic inflammatory myopathy

Mice infected with myopathic coxsackievirus B1 Tucson (CVB1(T)) develop chronic inflammatory myopathy (CIM) consisting of hind limb weakness and inflammation. Amyopathic virus variants are infectious but attenuated for CIM. In this report, viral clones, chimeras, and sequencing were used to identify viral determinants of CIM. Chimeras identified several regions involved in CIM and localized a weakness determinant to nucleotides 2493 to 3200 of VP1. Sequencing of multiple clones and viruses identified five candidate determinants that were strictly conserved in myopathic viruses with one located in the 5' untranslated region (UTR), three in the VP1 capsid, and one in the 3C protease. Taken together, these studies implicate Tyr-87 and/or Val-136 as candidate determinants of weakness. They also indicate that there are at least two determinants of inflammation and one additional determinant of weakness encoded by myopathic CVB1(T).     

5'-Terminal deletions occur in coxsackievirus B3 during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negative-strand viral RNA

Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5' termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5'-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5'-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5' CG or 5' AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.     

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.     

Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.     

Production of cross-reactive peptide antibodies against viral capsid proteins of human enterovirus B to apply diagnostic reagent

The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.     

Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.     

A proline-rich region in the coxsackievirus 3A protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.     

细胞自噬促进柯萨奇病毒B3复制的研究

本研究探索柯萨奇病毒B3(Coxsackievirus B3,CVB3)感染引起的自噬与病毒复制之间的关系。CVB3感染HeLa细胞,并在病毒感染后6 h、8 h和10 h时检测LC3-Ⅰ蛋白、LC3-Ⅱ蛋白和p62蛋白的表达水平。结果显示CVB3病毒感染促使LC3-Ⅱ/LC3-Ⅰ比值升高,同时降低p62蛋白的表达。分别将自噬诱导剂雷帕霉素(Rapamy-cin)、自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3MA)或溶酶体抑制剂阿洛司他丁(Aloxistatin,E46D)预处理HeLa细胞2 h,CVB3感染药物处理细胞并在病毒感染6 h后收集细胞、检测CVB3病毒VP1蛋白的表达。结果显示雷帕霉素和E64D促使CVB3病毒VP1蛋白表达增加,而3MA降低CVB3病毒VP1蛋白的表达。本研究得出结论 CVB3病毒感染诱导自噬进而促进病毒复制。