Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

HCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8 weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection.     

Development of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis A virus

Most hepatitis A virus (HAV) replication in cell culture has been reported to be nonlytic and relatively slow. A rapidly replicating isolate of strain HM-175 from persistently infected, serially passed cell cultures (pHM-175) was found to induce a cytopathic effect. This observation allowed the development of a classic plaque assay for pHM-175 in FRhK-4 cells. The plaques were neutralized by polyclonal and monoclonal antisera to HAV.     

Variants of measles virus

When tested on Vero cells the Edmonston strain of measles virus contained a mixture of plaque variants and variants which produced different kinds of cytopathic effect under liquid culture. A persistently infected culture of HEp2 cells which was derived from the Edmonston virus yielded low titers of infectious virus and was temperature sensitive (Gould, 1974). This persistent virus was less heterogenous than the parent Edmonston strain as judged by plaque morphology and type of cytopathic effect produced under liquid culture. An attempt has been made to isolate pure populations of each variant and to study their temperature sensitivity and cytopathogenicity. The possible origin of the persistent variant is discussed.     

Fisetin and rutin as 3C protease inhibitors of enterovirus A71

Enterovirus A71 (EV-A71) causes severe complications: encephalitis, pulmonary edema, and death. No effective drug has been approved for clinical use. This study investigated the antiviral effects of flavonoids against EV-A71. An in vitro inhibitor screening assay using recombinant EV-A71 3C protease (3Cpro) demonstrated fisetin and rutin inhibiting 3Cpro enzymatic activity in a dose-dependent manner. Cell-based fluorescence resonance energy transfer (FRET) assay with an EV-A71 3Cpro cleavage motif probe also confirmed that fisetin and rutin inhibited the replication of EV-A71 in cells. A virus replication assay indicated that fisetin and rutin reduced significantly the EV-A71-induced cytopathic effect and viral plaque titers in RD cells culture. The IC(50) values of plaque reduction against EV-A71 were 85 μM for fisetin and 110 μM for rutin. Therapeutic indices (CC50/IC50 of plaque reduction assays) of fisetin and rutin exceeded 10. The study suggests that fisetin and rutin inhibit the replication of EV-A71.     

A colorimetric assay for viral agents that produce cytopathic effects

Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.     

Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses

A novel method was developed for the precise quantitation of viruses using infrared fluorescent detection of foci of infection in conventional cell culture plates. In this assay, termed the infrared fluorescent immunofocus assay (IR-FIFA), appropriate cell cultures were infected with serial dilutions of hepatitis A virus (HAV) or measles virus (MV) and maintained with a semi-solid overlay for 1-5 days. Cell monolayers were fixed with formaldehyde, and then stained in succession with a primary monoclonal antibody and an Alexa Fluor 680 conjugate. Foci of infection (analogous to plaques) were detected by scanning culture plates using the Odyssey infrared imaging system and counted to determine the virus titre, expressed as focus forming units (FFU) per mL, as is done for conventional plaque assays. HAV and MV were used as models of minimally cytopathic viruses, and showed a linear dose-response between focus formation and virus dilution. Viral titres calculated using this method were comparable to conventionally used methods. The IR-FIFA was also successfully adapted to quantify duck hepatitis B virus (DHBV) as a model for a non-cytopathic virus. This simple and sensitive assay will have wide use for the quantitation of non-cytopathic and minimally cytopathic viruses.     

反义硫代修饰寡核苷酸体外抗甲型流感病毒感染的效应研究

目的 在感染甲型流感病毒(influenza A virus,IFAV)的人气道上皮细胞(9HTEO)中观察与IFAV PB1、M2、NS、PB2、HA基因瓦补的反义硫代修饰寡核苷酸(ASODN)的抗病毒活性.方法 细胞外体系观察没计的ASODN效率,筛选对IFAV有效的3个ASODN进行9HTEO的抗病毒效应.倒置显微镜下观察IFAV的敛细胞病变作用和ASODN的细胞保护作用.MTT方法测细胞存活率,空斑试验、RT-PCR、Westem blot、免疫荧光检测ASODN特异性抗病毒效应,MTT法检测ASODN对细胞的保护作用.结果 IFAV感染复数(MOI)在0.1以上时,培养5 d后IFAV感染的9HTEO细胞全部死亡.设计的ASODN能够提高感染细胞的存活率,且呈量效依赖关系.空斑试验、RT-PCR、Western blot、免疫荧光试验证实在细胞水平、基因转录水平、蛋白水平针对IFAV PB1、M2、NS基因mRNA转录起始点保守序列没计的ASODN具有特异性抗IFAV作用.结论 ASODN具有较明显的抗病毒活性,为进一步研究和开发新型抗IFAV药物提供了实验依据.为高致病性禽流感(H5N1)的基因治疗提供了新的思路和理论依据.     

Replication and plaque formation of swine hemagglutinating encephalomyelitis virus (67N) in swine cell line, SK-K culture

Swine hemagglutinating encephalomyelitis virus (HEV), 67N strain, adapted to suckling mouse brain, grew readily in a porcine cell line, SK-K cell culture with cytopathic effect (CPE) consisting of syncytium formation and detachment of fused cells and round cells from glass surface. After further passages in SK-K cell monolayers with undiluted culture fluid, CPE developed earlier and became complete within 48 h postinoculation (p.i.). Viral specific antigen was detected in the cytoplasm of the infected SK-K cells by indirect immunofluorescence using rabbit antiserum against the mouse-passaged virus. The SK-K-passaged virus as well as the original mouse-passaged virus formed clear plaques on SK-K cell monolayers under simple overlay medium. The plaque assay system for HEV 67N was established by studying various factors influencing the plaque formation in the SK-K cell cultures. By this system more than 10(6) PFU/0.2 ml of the virus yield was detected in the fluid phase of the infected cultures at 48 h p.i. The SK-K-passaged virus caused fatal infection in 4-week-old mice by intracerebral inoculation, but was inhibited by rabbit antiserum against the mouse-passaged virus. Plaque formation and hemagglutinating activity of the virus were specifically inhibited by antisera against the mouse-passaged and SK-K-passaged 67N virus.     

Enhanced detection of infectious airborne influenza virus

Current screening methodologies for detecting infectious airborne influenza virus are limited and lack sensitivity. To increase the sensitivity for detecting infectious influenza virus in an aerosol sample, the viral replication assay was developed. With this assay, influenza virus is first amplified by replication in Madin-Darby canine kidney (MDCK) cells followed by detection with quantitative PCR (qPCR). Spanning a 20-h replication period, matrix gene expression levels from infectious virus were measured at several time points using qPCR and found to exponentially increase. Compared with the traditional culture-based viral plaque assay, the viral replication assay resulted in a 4.6 × 10(5) fold increase in influenza virus detection. Furthermore, viral replication assay results were obtained in half the time of the viral plaque assay. To demonstrate that the viral replication assay is capable of detecting airborne influenza virus, dilute preparations of strain A/WS/33 were loaded into a nebulizer, aerosolized within a calm-air settling chamber and subsequently collected using NIOSH Two-Stage Bioaerosol Samplers. At the most diluted concentration corresponding to a chicken embryo infectious dose 50% endpoint (CEID(50)) of 2.8E+02/ml, the viral replication assay was able to detect infectious influenza virus that was otherwise undetectable by viral plaque assay. The results obtained demonstrate that the viral replication assay is highly sensitive at detecting infectious influenza virus from aerosol samples.     

Replication of sialodacryoadenitis virus of rat in LBC cell culture

Summary Sialodacryoadenitis virus of rat readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provides a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus.With 5 Figures     

脱氧核酶在培养人气道上皮细胞体系内的抗甲型流感病毒NS基因效应

目的 探讨针对甲型病毒(influenza A virus,IFV)NS基因特异性的脱氧核酶(DNAzyme,DZ-NS)在培养的人气道上皮细胞(9HTEo)体系内对甲型流感病毒复制的抑制效应,方法 光镜观察DZ-NS对IFV致9HTEo细胞病变效应变化的影响;病毒空斑形成阻断试验及四甲基偶氮唑盐比色试验(MTT)检测脱氧核酶DZ-NS对IFV复制的抑制和对感染IFV的9HTEo细胞的保护作用;RT-PCR检测DZ-NS对IFV mRNA表达的抑制作用.结果 DZ-NS可明显改善IFV所致的细胞病变并能明显提高细胞感染IFV后的存活率(P＜0.01);可显著抑制IFV-NS基因mRNA的表达;空斑实验病毒抑制率可达80.11%;DZ-NS在细胞内对IFA病毒表达的抑制效率,明显高于作为对照的反义寡核苷酸(antisense oligonucleotides,AS-NS)(P＜0.05).结论 在9HTEo 感染IFV细胞模型系统,DZ-NS能高效阻断IFV的NS基因的表达,是一种特异性的、高效的抗IFV基因治疗剂.     

Cell surface changes associated with mutation of visna virus in antibody-treated cell cultures.

Sheep cells inoculated with a portion of a plaque suspension of visna virus developed acute cytopathic changes and produced progeny virus which was antigenically identical to the parental strain. In contrast, cultures inoculated with other portions of the same suspension and maintained in medium containing specific viral antiserum developed cytopathic changes more slowly and yielded genetically stable mutant strains of virus which were poorly neutralized by the same antiserum. Scanning and transmission electron microscopy showed that, in the absence of antibody, cell surfaces of infected cells were covered diffusely with viral buds, whereas in the presence of antibody, viral buds were often grouped in caps and progressive aggregation of released virus occurred. Viral aggregates were strongly anchored to viral buds or long villi and sometimes became engulfed by infected cells. In addition, single maturing virions were continuously detected close to large aggregates. These morphological events suggest that selective replication of mutant virus in antibody-treated cells is favored by (1) elimination of parental virus through aggregation and endocytosis and (2) patching and capping of viral buds, which leave free cell surface areas for maturation of mutant virus.     

Infectivity assays for the Autographa californica nuclear polyhedrosis virus using Spodoptera littoralis cells

Tissue culture ID50 and plaque assays for the Autographa californica nuclear polyhedrosis virus, using the Spodoptera littoralis cell line HPB-SL, were developed. Direct comparison using these assay methods showed that these cells were as susceptible to infection as the more commonly used Spodoptera frugiperda cell line IPLB-SF-21. Both infectious tissue culture supernatants or virus isolated directly from polyhedra could be titrated. It was important to use low cell seeding densities in the assays so that clear centres of infection formed. Dose-response curves indicated that one infectious particle was capable of initiating an infection. Virus could be cloned using either method even though, for the plaque assay, plates had been stained. The tissue culture ID50 assay was performed using 96-well plates and required an incubation period of about 10 days. The plaque assay used a simple nutrient agarose overlay and an incubation period of 5-6 days. Easily countable plaques of 0.3-1.2 mm diameter were detected after staining with iodonitrote-trazolium chloride. The plaques comprised areas of inhibited cell division and round or dead cells. Most plaques contained only some cells with polyhedra and yields averaged about 1/cell. Occasionally plaques or infected wells were found in which no polyhedra could be seen. These infectivity assays are therefore not dependent on polyhedra formation.     

A simplified method for the extraction of baculoviral DNA for PCR analysis: a practical application

There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.     

Assay of human interferon in Vero cells by several methods.

Four methods for the assay of human interferon in Vero cells were compared based on the inhibition of viral cytopathic effect (CPE) in tubes, the inhibition of CPE in microplates, the reduction of plaques, and the inhibition of quantitative hemadsorption. For inhibition of CPE, Sindbis virus, vesicular stomatitis virus, poliovirus type 2, and vaccinia virus were used for challenge. In the plaque reduction method, Sindbis virus, vesicular stomatitis virus, and poliovirus were employed, and Newcastle disease virus was used in the quantitative hemadsorption assay. Sindbis virus was most susceptible to interferon in those tests measuring inhibition of CPE, but vesicular stomatitis virus was as sensitive in the plaque reduction method. Highest titers of interferon were recorded in microplates, especially with Sindbis virus as the challenge agent, followed by the quantitative inhibition assay. The CPE inhibition method was the simplest, and the quantitative hemadsorption assay was the most rapid to perform. Reproducibilities, as shown by the coefficient of variation, were 15, 39, and 59% for plaque reduction, CPE inhibition in tubes, and CPE inhibition in microplates, respectively.     

Comparison of in situ hybridization and immunologic staining with cytopathology for detection and identification of herpes simplex virus infection in cultured cells.

Two recently developed sensitive techniques, in situ hybridization with a biotinylated cloned DNA probe and an avidin-biotin complex immunoperoxidase assay, were compared with the appearance of cytopathic changes for the early detection of herpes simplex virus infection in cell culture. By using commercially made reagents, these detection methods were evaluated in two different cell culture systems inoculated with both high- and low-input multiplicity of virus. The results revealed that both viral antigen and viral DNA detection methods could shorten the time to diagnosis of herpes simplex virus infection in cell culture; however, these methods were most useful in specimens containing low titers of virus when a less sensitive cell system was used. In this study, the avidin-biotin immunoperoxidase method was more sensitive and much cheaper than hybridization with a biotinylated probe. Significantly, when a highly sensitive cell system was used, cytopathic changes alone were comparable in rapidity and sensitivity to viral antigen or DNA detection methods applied in a less sensitive cell system.     

Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain HM175

Hepatitis A virus (HAV) strain HM175 derived after repeated subculture of persistently infected B-SC-1 cells caused a specific cytopathic effect upon acute infection of B-SC-1 cells. The virus formed visible plaques on B-SC-1 cell monolayers after 9 to 14 days of incubation at 34 degrees C, and virus can therefore be titrated by plaque assay. Virus could be re-isolated from plaques of infected cells, which allows the clonal isolation of HAV variants. The stability of a plaque-purified variant of HAV at elevated temperatures exceeded that of poliovirus type 1, but this variant is less stable than previously reported strains of HAV.     

HIV replication elicits little cytopathic effects in vivo: analysis of surrogate markers for virus production, cytotoxic T cell response and infected cell death

Several potential mechanisms for viral destruction of HIV-infected cells have been described. The hypothesis was examined that if HIV were cytopathic, a positive relation between the in vivo virus production or CTL activity and infected cell death should be observed. In a regression analysis no significant relation was found between surrogate markers for in vivo virus production or the virus-specific CTL response and death rates of productively infected cells. In a subgroup of patients the hypothesis is rejected that HIV replication elicits a large (R(2) > 0.25) cytopathic effect (P < 0.05, N = 36). It is concluded that HIV replication elicits little cytopathic effect in productively infected cells and that CD4(+) T lymphocytes are eroded by other mechanisms.