Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture.

Virus and cells evolve during serial passage of cloned BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV). These carrier cells, termed C1-BHK-Rc1 (J.C. de la Torre, M. Dávila, F. Sobrino, J. Ortín, and E. Domingo, Virology 145:24-35, 1985), become constitutively resistant to the parental FMDV C-S8c1. Curing of late-passage C1-BHK-Rc1 cells of FMDV by ribavirin treatment (J.C. de la Torre, B. Alarcón, E. Martínez-Salas, L. Carrasco, and E. Domingo, J. Virol. 61:233-235, 1987) did not restore sensitivity to FMDV C-S8c1. The resistance of C1-BHK-Rc1 cells to FMDV C-S8c1 was not due to an impairment of attachment, penetration, or uncoating of the particles but to some intracellular block that resulted in a 100-fold decrease in the amount of FMDV RNA in the infected cells. FMDV R59, the virus isolated from late-passage carrier cells, partly overcame the cellular block and was more cytolytic than FMDV C-S8c1 for BHK-21 cells. Sequencing of the VP1 gene from nine viral clones from C1-BHK-Rc1 cells showed genetic heterogeneity of 5 X 10(-4) substitutions per nucleotide. Mutations were sequentially fixed during persistence. In addition to resistance to FMDV C-S8c1, C1-BHK-Rc1 cells showed a characteristic round cell morphology, and compared with BHK-21 cells, they grew faster in liquid culture, were less subject to contact inhibition of growth, and had an increased ability to form colonies in semisolid agar. Reconstitution of a persistent infection was readily attained with late-passage C1-BHK-Rc1 cells and FMDV C-S8c1 or FMDV R59. The results suggest that coevolution of BHK-21 cells and FMDV contributes to the maintenance of persistence in cell culture.     

miRNA干扰口蹄疫病毒基因组早期复制的初步研究

根据miRNA的设计要求,以miR-31为模型通过软件设计出潜在的能够使3D基因沉默的特异性miRNA序列3D-1203,并以pcDNA3.1(+)质粒为载体构建能够表达成熟miRNA的重组质粒,将其转染到BHK-21细胞后感染口蹄疫病毒,研究3D-1203的miRNA的干扰作用。蚀斑实验结果显示特异性miRNA可以在早期抑制口蹄疫病毒的复制,与阳性对照相比,病毒复制效率降低53.0%。实时TaqMan荧光定量RT-PCR检测数据表明特异性miRNA干扰病毒基因组增殖的效率达66.7%。本研究证明,利用miR-31设计的特异性miRNA在病毒复制早期能够特异的干扰口蹄疫的复制。     

口蹄疫病毒感染BHK—21细胞的代谢热谱研究

本文应用高灵敏度的LKB2277生物活性检测仪测定FMDV感染BHK-21细胞的代谢热谱,并与传统方法测定的一步生长曲线进行比较,二者具有显著的相似性。结果表明,微量热法通过对细胞及其受病毒感染的细胞体系代谢热的测定,能有效地监测病毒在宿主细胞内增殖的过程。该方法还提供了一种动态连续分析病毒感染增殖的新手段。     

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.     

Antiapoptotic but Not Antiviral Function of Human bcl-2 Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human bcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog, bcl-X_L, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced bcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 which plays a role in the establishment of JEV persistence.     

口蹄疫病毒持续感染细胞系的快速选择及其特性研究

采用NH4Cl弱碱法处理感染口蹄疫病毒的叙利亚仓鼠肾细胞系(BHK—21),存活细胞经过单克隆和选择,获得32株阳性克隆细胞株。随机选取一株(BHK—ROp),常规传代并采用RT-PCR,透射电子显微镜,流式细胞仪进行持续感染特性分析,结果表明,BHK—ROp第4,16,36代细胞中病毒持续存在,但不影响细胞的生长特性,表现出病毒持续感染的基本特征。可见,NH4Cl弱碱法用于建立病毒持续感染细胞系是十分有效的。     

口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达

利用定点突变的原理,获得包含有口蹄疫病毒P1,2A,3C及部分2B编码区的目的基因片段,KpnⅠ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1（+）,经筛选、鉴定及DNA序列分析后,将重组质粒pcDNA3.1/P12X3C转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法,检测细胞中表达的口蹄疫病毒抗原。结果表明,口蹄疫病毒基因片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3.1/P12X3C可在BHK-21细胞中表达FMDV目的蛋白。     

A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.     

The antiapoptotic protein Mcl-1 controls the type of cell death in Theiler's virus-infected BHK-21 cells

Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Resistance to foot-and-mouth disease virus mediated by trans-acting cellular products.

Upon serial passage of BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) C-S8c1, cells with increased resistance to the virus were selected (J. C. de la Torre, E. Martinez-Salas, J. Diez, and E. Domingo, J. Virol. 63:59-63, 1989). Two highly resistant cell clones, 74A11 and 74D12, were transformed to puromycin resistance (Purr) and were fused to BHK-21 cells transformed to neomycin resistance (Neor). The hybrid Neor Purr cells showed the specific resistance to FMDV C-S8c1 characteristic of clones 74A11 and 74D12. The results suggest that resistance to FMDV C-S8c1 is mediated by trans-acting cellular products. The possibility of engineering constitutive resistance to FMDV is discussed.     

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies.     

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8(+) T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8(+) T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8(+) T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.     

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells

Background

Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding

Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID₅₀ of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.

Conclusion

RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.