Production of Venezuelan equine encephalitis virus in cells grown on artificial capillaries

Primary cell cultures, a continuous cell line, and a diploid cell line were grown on an artificial capillary system. The cells were subsequently infected with Venezuelan equine encephalitis virus, and viral replication was studied. Extracellular fluids harvested from this system contained high titers of virus and were relatively free of cell debris.     

Adaptation of Swinepox Virus to an Established Cell Line

Swinepox virus was successfully adapted to the PK-15 cell line, but not the MDBK cell line. Virus titers obtained in the PK-15 host cell system were comparable to diploid swine kidney cell cultures.     

Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures

The aim of our study was to establish an efficient system for thein vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.Abbreviations Ac-NPV Autographa californica Nuclear Polyhedrosis Virus - BV Baculovirus - MOI Multiplicity Of Infection - ECV Extracellular Virus     

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.     

Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells.

To investigate the antiviral CD4+ T cell response in coronavirus MHV-JHM-induced encephalomyelitis, spleen and thymic lymphocytes from diseased rats were stimulated in culture with virus Ag, expanded and tested for their specificity to viral proteins and nucleocapsid (N) and spike (S) proteins that had been expressed in bacteria. A strong T cell response specific for N was measurable during acute disease, whereas S-specific T cells were only detectable in rats with a later onset of disease. CD4+ T cell lines with specificity for virus and either N or S protein were established and their influence on the course of a mouse hepatitis virus-JHM infection was investigated. All lines were of the CD4+ phenotype. Both N and S protein-specific CD4+ T cells conferred protection to infected Lewis rats and reduced the amount of infectious virus in the central nervous system. After transfer of CD4+ T cells and challenge with virus, an increase in the antiviral IgM response occurred, but neutralizing antibodies were not detectable during the period of virus clearance. Previous CD8+ cell depletion did not abrogate protection mediated by CD4+ T cell line transfer.     

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.     

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.     

肝癌细胞系（HepG2）感染HCV RNA的研究

为建立丙型肝炎病毒（HCV）体外感染和细胞培养系统,用定量的HCV RNA阳性血清感染人肝癌细胞系（HepG2细胞系）,应用地高辛标记HCV RNA探针原位杂交技术和RT－PCR方法对感染后的细胞和上清液听 HCV RNA进行了检测。在感染后的第一代至第七代的细胞中出现特异性杂交阳性信号,第一代、第二代和第六代检测出HCV RNA正链,并在感染后第一、二代检测出HCV RNA负链。显示HCV不仅能在体外感染HepG2细胞系,而且在基因的复制,证明HepG2细胞能作为HCV的体外细胞培育系。     

Packaging cell lines for simian foamy virus type 1 vectors

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.     

Establishment of a Vero cell line persistently infected with African swine fever virus.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.     

The specificity of Helicoverpa armigera stunt virus infectivity.

Helicoverpa armigera stunt virus (HaSV) is a member of the Tetraviridae family of RNA viruses whose replication and expression strategies are not well understood due to the absence of an in vitro cell culture system. We set out to find such a system for HaSV by screening an array of 13 insect and 1 mammalian cell culture lines with both virus particle infection and genomic RNA transfection. No cell line was found to be permissive for replication, although entry of genomic RNA was verified. The apparent specificity of this virus for its in vivo midgut target site was strongly corroborated by studies involving Northern blots of RNA extracted from infected insects. Only larval midgut RNA showed the presence of virus after hosts were infected per os or by injection which exposed other host cell types to the virus. The absence of replication in cell culture was due to a lack, or presence, of host factors important to replicase activity and also the likely absence of virus particle binding and entry. We thus provide both in vitro- and in vivo-based evidence demonstrating that this virus is extremely specific in the type of cells in which it will initiate an infection.     

Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl.

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Establishment and characterization of a testicular cell line from the half-smooth tongue sole, Cynoglossus semilaevis

Spermatogenesis within the adult testis is an excellent system for studying stem cell renewal and differentiation, which is under the control of testicular somatic cells. In order to understanding spermatogenesis in the half-smooth tongue sole (Cynoglossus semilaevis) as a marine fish model of aquaculture importance, we established a cell line called CSGC from a juvenile gonad of this organism. CSGC is composed of fibroblast-like cells, retains a diploid karyotype of 42 chromosomes, lacks the heterogametic W chromosome, lacks a female specific marker and expresses the dmrt, a marker for testicular somatic cells. Therefore, CSGC appears to consist of testicular somatic cell cells. We show that this cell line is effective for infection by the turbot reddish body iridovirus and flounder lymphocystis disease virus as evidenced by the appearance of cytopathic effect and virus propagation in the virus-infected cells, and most convincingly, the observation of viral particles by electon microscopy, demonstrateing that CSGC is suitable to study interactions between virus and host cells. As a first fish testicular somatic cell line of the ZZ-ZW genetic sex determination system, CSGC will be a useful tool to study sex-related events and interactions between somatic cells and germ cells during spermatogenesis.     

In vivo and in vitro models of demyelinating diseases: tropism of the JHM strain of murine hepatitis virus for cells of glial origin.

Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV₃ strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV₃ is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells.This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.     

干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.     

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.