A simple screening assay for receptor switching of avian influenza viruses.

BACKGROUND: Adaptation of the receptor-binding preference from alpha2,3- to alpha2,6-linked sialic acid is an essential step for an avian influenza virus to transmit efficiently in human population and become a pandemic virus. The currently available assays for receptor-binding preference are complex and not widely available. OBJECTIVES: A simple high-throughput screening assay will facilitate early detection of a potential pandemic virus, which is crucial for the prevention and control of the possible pandemic. We wanted to develop a simple assay to differentiate influenza viruses with alpha2,3- or alpha2,6-linked receptor-binding preference. STUDY DESIGN: The assay employs a specific sialidase (from Salmonella thyphimurium) that can eliminate alpha2,3-linked sialic acid from red blood cells. A reduction of hemagglutination titer indicates alpha2,3-linked receptor preference in this assay. RESULTS: Using a panel of H5N1 avian influenza isolates and H1/H3 human influenza isolates, as well as mutated H5 reverse genetics virus, the assay could accurately differentiate the viruses according to their receptor-binding preference. Furthermore, the assay was sufficiently sensitive to detect a minor variant with alpha2,6-linkage-specificity in a background of alpha2,3-linkage-specific virus. CONCLUSIONS: We have developed a simple screening assay capable of detecting avian influenza viruses that have switched their receptor-binding preference.     

A simplified plaque assay for influenza viruses in Madin-Darby kidney (MDCK) cells

A variety of influenza A and B viruses plaque in MDCK cell in trypsin is added only at the time of viral adsorption to the monolayer. Therefore, a conventional soft-agar overlay can be employed without addition of proteolytic enzymes. Plaquing efficiency was comparable to that when embryonated eggs were used to determine infectivity. Finally the method is simple and economical.     

A sensitive plaque inhibition technique for assay of antibodies to influenza virus: Use to detect previous antigenic priming with influenza viruses

Plaque reduction test performed in MDCK cells was found to be a more sensitive assay than hemagglutination inhibition, for revealing previous immunological experience with influenza A virus in the population. Based on the preimmunization antibody levels, determined by plaque reduction tests, it was possible to distinguish between previously primed and unprimed subjects among vaccinees.     

A modified plaque assay method for accurate analysis of infectivity of influenza viruses with uncleaved hemagglutinin

Summary A modified plaque assay method for evaluation of infectivity of influenza viruses is described. The modification consists of a double agar overlay, trypsin being included in the second layer added 24 hours after infection. The modified method allows one to determine accuratelly the infectivity titer of influenza viruses possessing uncleaved hemagglutinin, and to compare the infectivity of viruses possessing cleaved or uncleaved forms of hemagglutinin. The standard method in which trypsin is included into agar overlay just after infection artifically increases the infectivity titer of viruses with uncleaved hemagglutinin and therefore is not suitable for these purposes.With 2 Figures     

Hemagglutinin-specific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies present in human serum or nasal washes directed against influenza A or B hemagglutinin glycoproteins. The assay was modified to measure the immunoglobulin isotype specificity of the anti-hemagglutinin response in serum and nasal secretions. In the postinfection sera anti-hemagglutinin of the immunoglobulin G isotype was predominant, whereas in nasal secretions the antibody was predominantly immunoglobulin A. The antibody response detected by the ELISA manifested hemagglutinin subgroup specificity. In addition, there was a good correlation between the ELISA antibody titer and the hemagglutination-inhibition or neutralizing antibody titer. The ELISA was more sensitive than the hemagglutination-inhibition assay, and the range of antibody titers measurable by ELISA in human serum was from less than 1:20 for children who had never experienced influenza infection to 1:400,000 for adults convalescing from a secondary infection. With more sensitive tests to detect antibody to the influenza hemagglutinin it should be possible to determine the relative contribution of local and systemic immunity to resistance to influenza virus infection.     

Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses

A wide range of influenza B virus strains with various passage histories uniformly formed well-defined clear plaques with high efficiency in cultures of an established line of canine kidney cells (MDCK). PFU titers of the viruses assayed in MDCK exceeded the titers assayedin ovo. With recently isolated strains such as B/Hong Kong/5/72 and Gifu/2/73, the PFU/EID₅₀ ratios were as high as 100 to 400. MDCK cells have been successfully employed for primary isolation of influenza B viruses from throat washings of patients by direct plaquing.     

Tetrazolium-based plaque assay for HIV-1 and HIV-2, and its use in the evaluation of antiviral compounds

A modification of the MT-4 cell plaque assay for human immunodeficiency virus (HIV) is described, which gave reproducible results with all 4 HIV-1 strains and the two HIV-2 strains that were used. The main feature of this new method is the use of a tetrazolium (MTT) staining procedure. The number of plaques read after 4-6 days was essentially the same as the number of infectious units derived from the 50% cell culture infective dose (CCID50) in MT-4 suspension cultures. For a selected group of antiviral compounds the 50% plaque-inhibitory doses were comparable with the 50% inhibitory doses (ID50) in suspension cultures. In the plaque assay HIV-1 (HTLV-IIIB) and HIV-2 (LAV-2ROD) were equally susceptible to azidothymidine (AZT), and the same was true for didehydrodideoxythymidine (D4T). For these compounds it was irrelevant whether they were already present during the initial HIV adsorption phase or added immediately thereafter. Pentosan polysulfate proved about 20-fold more inhibitory to HIV-2 than HIV-1. There was a 5-fold increase in activity if present during the virus adsorption stage.     

An antiviral drug screening system for enterovirus 71 based on an improved plaque assay: A potential high-throughput method

Plaque assay plays an irreplaceable role in a variety of virological studies, including determining titers of viruses. Our previous study showed that a simple and highly repeatable plaque assay could be used for enterovirus 71 (EV-A71). Now, we show that using a subclone of a clinical EV-A71 isolate and a rhabdomyosarcoma cell line (RD), a plaque assay based on an EV-A71/RD model could exhibit the most rapid formation of plaques (<2 days), with much higher repeatability and consistency. Inspired by a plaque inhibitory test for testing ribavirin and interferon, as well as a plaque reduction neutralization test, this modified method has been used to establish a convenient system by using 96-well plates for screening anti-EV-A71 drugs from a 130-compound library containing multiple types of inhibitors. Nine candidate effective compounds for EV-A71 have been screened out, and among them, nobiletin (flavonoid) was found to be a novel effective compound at the concentration of 10 μM. Our findings imply that this improved method based on an EV-A71/RD model proved to be a potential high-throughput method in screening novel antiviral drugs for EV-A71. Undoubtedly, this method can also be applied to other viruses that can produce an obvious cytopathic effect.     

Multiplication and plaque assay of influenza viruses in a continuous cell line (G2) of human origin

Summary Serial propagation of strains of all types of influenza viruses proved to be possible in a continuous cell line derived from a human bone carcinoma. This is the second continuous human cell line in which influenza viruses can be serially propagated. The concentration of influenza viruses in this cell line, G2, was essentially equivalent to the infectivity obtained in embryonated eggs. Clear-cut cytopathogenicity of the infected G2 cells did not occur. Viral antigenicity as determined by serum-virus hemagglutination-inhibition tests was not altered by the cell culture passage. Plaque assay of influenza viruses which had been serially propagated in G2 cell cultures was a simple procedure and the results were reproducible enough for practical application.     

胶体金免疫层析试纸条快速检测乙型流感病毒

目的建立有效、简便的胶体金免疫层析试纸条快速检测乙型流感病毒感染的方法。方法通过对乙型流感病毒核蛋白单克隆抗体进行胶体金标记,成功研制了乙型流感病毒免疫层析检测试纸条。结果该试纸条操作简单,肉眼于10～15 min内判定结果,对乙型流感病毒具有高度特异性,与甲型H1N1、H3N2亚型流感病毒等其他重要呼吸道病毒无交叉反应。试纸条在室温保存12个月、2～8℃保存18个月,其特异性和灵敏度无明显变化。对从内蒙自治区医院收集的流感样症状病人的702份鼻咽部分泌物进行检测,与美国Quidel公司同类产品的符合率为95%。结论建立的乙型流感病毒免疫层析检测方法具有简便、快速、特异、敏感和稳定等特点,对乙型流感病毒感染疾病的临床检测与早期诊断具有重要意义。     

Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform

The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory-adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY-HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N-terminus of the viral nuclear-export protein (NEP). Here, we show that this 11-amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY-HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT-tagged virus can be measured by a simple Nano-Glo assay, providing a robust high-throughput screening (THS) platform. We used this platform to evaluate a collection of the pre-purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti-IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.     

A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE

For antiviral screenings purposes, infection of cell cultures with the virus under study, should ideally result in the induction, within just a few days, of (nearly) complete CPE and allow the calculation of acceptable Z' factors (>0.5). The human Corona virus NL63 (HCoV-NL63) causes only limited CPE on different cell lines (Schildgen et al., 2006). Following infection of Vero118 cells, virus-induced CPE was too low to allow readout based on classical colorimetric methods (such as the MTS assay), even following prolonged incubation times (>7 days). To develop an antiviral screenings-assay against HCoV-NL63, we explored whether a dead-cell protease substrate could be used instead. The substrate used is a quenched peptide (bis-AAF-R110) that releases a fluorophore upon proteolytic-cleavage by proteases; the latter released from dead cells. Following different rounds of optimization a screening protocol was developed: Vero118 cells in 96-well plate format were infected with HCoV-NL63 (MOI=0.01; 200μL cell culture; 2.10(4)cells/mL, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35°C after which 20μL of the peptide solution was added. Fluorescence was quantitated 2 hr after incubation at 37°C. A roughly 3-fold increase in fluorescence intensity in the infected cultures was observed as compared to the uninfected cultures with a low well-to-well variability. Z' factors calculated from different experiments were in the range of 0.6-0.8, indicating excellent assay quality. An anti-ACE2 polyclonal antiserum (that prevents coronavirus infection in cell cultures) was used as a positive control and allowed to validate the assay for antiviral screening purposes. In conclusion, in conditions where a viability staining is inadequate to quantitate virus-induced CPE, a novel simple and convenient method that detects cell-death and that is suitable for high-throughput screening purposes can be employed.     

Probable association of plaque size with neuraminidase subtype among H3N2 influenza A viruses

Summary The present study demonstrates that the plaque size of certain influenza A H3N2 virus recombinants is dependent on their containing a specific neuraminidase glycoprotein, but is independent of the genes coding for the HA, P3, and NP proteins.     

A simple microtiter plate screening assay for bacterial invasion or adherence

Many bacteriologic studies, including cellular invasion and adherence assays, require enumeration of viable organisms or colony forming units. When attempting to screen large numbers of clinical or environmental isolates or laboratory-derived mutants for differences in invasion or adherence phenotype, standard plating methods can be cumbersome and severely limit the number of organisms or conditions which can be tested. As a potential alternative, we describe a simple, rapid and inexpensive soft agar-based technique for semi-quantitative determination of bacterial colony counts directly within the wells of a 96-well microtiter plate.     

Cross-protective immunity to influenza A viruses

Antigenic changes in influenza virus occur gradually, owing to mutations (antigenic drift), and abruptly, owing to reassortment among subtypes (antigenic shift). Availability of strain-matched vaccines often lags behind these changes, resulting in a shortfall in public health. In animal models, cross-protection by vaccines based on conserved antigens does not completely prevent infection, but greatly reduces morbidity, mortality, virus replication and, thus, viral shedding and spread. Such immunity is especially effective and long-lasting with mucosal administration. Cross-protective immunity in humans is controversial, but is suggested by some epidemiological findings. 'Universal' vaccines protective against all influenza A viruses might substantially reduce severity of infection and limit spread of disease during outbreaks. These vaccines could be used 'off the shelf' early in an outbreak or pandemic, before strain-matched vaccines are available.     

A sensitive plaque assay for influenza virus A2 Japan in chick embryo fibroblast cultures

Summary Growth of influenza virus type A₂, strain Japan/305/57 (formerly grown in the allantoic cavity of 11-day old chick embryos), was obtained in cultures of chick embryo fibroblasts. Virus, passed four times in chick embryo fibroblasts, followed by one passage in the allantoic sac of 11-day old embryonated eggs, gave clear plaques in a chick embryo monolayer under a serum-free agar overlay with an added DEAE-dextran concentration of 0.5 mg./ml. After three days incubation at 37° C in stoppered flasks the plaques reached a diameter of 2–3 mm. An approximately straight-line dose-response relationship was observed between the square root of the number of plaques produced and the relative concentration of virus. Specificity was demonstrated by quantitative neutralization with immune serum.