Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus.

Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells.

Growth Kinetics of Kyasanur Forest Disease Virus in Mammalian Cell Lines and Development of Plaque Reduction Neutralization Test.

Kyasanur forest disease virus (KFDV) is a tick-borne flavivirus identified in 1957 in the Karnataka state of India causing fatalities in monkeys and humans. Even after the introduction of a vaccine in the endemic areas, hundreds of cases are reported every year. Being a high-risk category pathogen, the studies on this virus in India were limited till the past decade. The growth characteristics of this virus in various mammalian cell lines have not yet been studied. In this study, we have demonstrated the growth pattern of virus in BHK-21, Vero E6, Vero CCL81, rhabdomyosarcoma, porcine stable kidney, and Pipistrellus ceylonicus bat embryo cell lines, and found BHK-21 to be the best. We have developed KFDV plaque reduction neutralization test for the first time.

Antiviral activity of pinocembrin against Zika virus replication.

Zika virus (ZIKV) is a mosquito-borne virus that has garnered a lot of attention in recent years, due to the explosive epidemic from 2014 to 2016. Since its introduction in the Americas in late 2014, ZIKV has spread at an unprecedented rate and scale throughout the world and infected millions of people. Its infection has also been associated with severe neurological disorders like Guillain-Barré syndrome and microcephaly in fetuses. Despite these, there is currently no approved antiviral against ZIKV. In this study, an immunofluorescence-based high throughput screen was conducted on a library of 483 flavonoid derivatives to identify potential anti-ZIKV compounds. Flavonoids, which are natural polyphenolic compounds found in plants, represent an attractive source of antivirals due to their abundance in food and expected low toxicity. From the primary screen, three hits were selected for validation by cell viability and viral plaque reduction assays. Pinocembrin, a flavanone found in honey, tea and red wine, was chosen for downstream studies as it exhibited the strongest inhibition of ZIKV infection in human placental JEG-3 cells (IC50 = 17.4 µM). Time-course studies revealed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. Furthermore, pinocembrin inhibits viral RNA production and envelope protein synthesis based on quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses. This study has demonstrated for the first time the in vitro anti-ZIKV activity of pinocembrin.

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.

Development and application of a higher throughput RSV plaque assay by immunofluorescent imaging.

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent "objects". The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.

In vitro infectivity of oncolytic Newcastle Disease Virus: Correlation between plaque and fluorescent focus assays.

Newcastle Disease Virus (NDV) is an avian paramyxovirus that has no significant pathogenicity in humans. Cancer cells with impaired immune defense mechanisms are susceptible to infection and lysis by NDV. A recombinant construct of a lentogenic form of NDV (rNDV) containing an insertion of granulocyte macrophage colony stimulating factor (GMCSF) transgene was earlier reported and shown to have acceptably low avian pathogenicity as well as oncolytic potential. Reliable measurement of infectious titer is key to determining the effectiveness of virus preparations to infect and lyse cells. We report here a comparative evaluation of two infectious titer assays as applied to rNDV: plaque assay and fluorescent focus assay (FFA). Optimization of assay conditions for both titer methods has produced concordant results spanning several orders of magnitude. While plaque formation is the gold standard measure of virus titer, FFA provides higher throughput and faster turn-around. FFA has been further evaluated on two different instrument platforms, for automated versus manual foci recognition and counting, with equivalent results. These results point to amenability of FFA to transfer between different laboratories and analysts, without introducing significant subjectivity in data analysis.

Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research.

Chikungunya virus (CHIKV) is becoming an increasing global health issue which has spread across the globe and as far north as southern Europe. There is currently no vaccine or anti-viral treatment available. Although there has been a recent increase in CHIKV research, many of these in vitro studies have used a wide range of cell lines which are not physiologically relevant to CHIKV infection in vivo. In this study, we aimed to evaluate a panel of cell lines to identify a subset that would be both representative of the infectious cycle of CHIKV in vivo, and amenable to in vitro applications such as transfection, luciferase assays, immunofluorescence, western blotting and virus infection. Based on these parameters we selected four mammalian and two mosquito cell lines, and further characterised these as potential tools in CHIKV research.

A rapid, simple, and accurate plaque assay for human respiratory syncytial virus (HRSV).

Plaque assays of human respiratory syncytial virus (HRSV) are time-consuming, requiring 4 to 7 days for plaque formation and several hours for dye staining. Here, we describe a simple method by which RSV plaques can be visualized and counted with the naked eye only 2 days after infection of HEp-2 cells. In this assay, the infected cells are stained with monoclonal antibodies and the plaques are developed using diaminobenzidine (DAB). We tested the accuracy of this new plaque assay by comparing the results obtained on days 1, 2, 3, 4, 5, and 6 post-infection. The whole procedure is significantly simpler than the traditional method, with an immunostaining process of around 1.5h. Our method is rapid, accurate, and simple; thus, it has the potential to significantly contribute to studies related to RSV disease.

A neutralization assay with a severe fever with thrombocytopenia syndrome virus strain that makes plaques in inoculated cells.

Severe fever with thrombocytopenia syndrome (SFTS) is a recently-discovered, potentially fatal infectious disease caused by SFTS virus (SFTSV). Due to the inability of SFTSV to make clear cytopathic effects (CPE) in cell culture, titration and neutralization assays of the virus require immunostaining of inoculated cells; consequently, the assays are time-consuming and expensive. In this report, we demonstrate the use of a highly-passaged SFTSV strain, p50-2, in a neutralization assay, which made clear plaques in inoculated Vero cells under neutral red staining. Furthermore, we performed molecular analyses to determine the characteristics of the strain. The results suggested that a single amino acid mutation within the viral glycoprotein conferred the ability to make clear plaques to SFTSV.

Quantification of RSV Infectious Particles by Plaque Assay and Immunostaining Assay.

One of the most commonly used approaches for determining the quantity of infectious RSV particles in a given sample is the plaque assay. RSV infectious particles can be quantified by various direct and indirect methods. Here, we explain two simple methods for RSV titration: plaque assay and immunostaining assay.

Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods.

The consumption of organic fresh produce has increased in recent years due to consumer demand for healthy foods without chemical additives. However, the number of foodborne outbreaks associated with fresh produce has also increased. Contamination of food with enteric viruses is a major concern because the viruses have a low infectious dose and high persistence in the environment. Human adenovirus (HAdV) has been proposed as a good marker of faecal contamination. Therefore, the aim of this study was to evaluate the efficiency of the plaque assay (PA), real time PCR (qPCR) and integrated cell culture-RT-qPCR (ICC-RT-qPCR) for the recovery of HAdV from artificially and naturally contaminated fresh produce. Organic lettuce, strawberries and green onions were selected because these fresh products are frequently associated with foodborne outbreaks. The virus extraction efficiencies from artificially contaminated samples varied from 2.8% to 32.8% depending on the food matrix and the quantification method used. Although the HAdV recoveries determined by qPCR were higher than those determined by PA and ICC-RT-qPCR, PA was defined as the most reproducible method. The qPCR assays were more sensitive than the PA and ICC-RT-qPCR assays; however, this technique alone did not provide information about the viability of the pathogen. ICC-RT-qPCR was more sensitive than PA for detecting infectious particles in fresh produce samples. HAdV genome copies were detected in 93.3% of the analysed naturally contaminated samples, attesting to the common faecal contamination of the fresh produce tested. However, only 33.3% of the total samples were positive for infectious HAdV particles based on ICC-RT-qPCR. In conclusion, this study reported that HAdV can be an efficient viral marker for fresh produce contamination. Good detection of infectious HAdV was obtained with the ICC-RT-qPCR and PA assays. Thus, we suggest that the ICC-RT-qPCR and PA assays should be considered when quantitative microbial risk assessment (QMRA) studies are required and to establish reliable food safety guidelines.

Plaque2.0-A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion.

Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

Detection of viable murine norovirus using the plaque assay and propidium-monoazide-combined real-time reverse transcription-polymerase chain reaction.

Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21log10PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90°C in a water bath for 1min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71log10PFU/mL at temperatures of 65, 70, and 75°C, respectively. MNV was reduced >4.21log10PFU/mL at 80, 85, and 90°C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value=-logRN-logRP (RN: the relative quantity value of the not-treated sample, and RP: the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75°C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90°C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs.

The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

Fresh ginger (Zingiber officinale) has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginger, Zingiber officinale Roscoe, is a common spice and also a widely used medicinal plant in ancient China. Ginger is an ingredient of Ge-Gen-Tang (Kakkon-to; GGT). GGT has been proved to have antiviral activity against human respiratory syncytial virus (HRSV). However, it is unknown whether ginger is effective against HRSV. AIM OF THE STUDY: To find a readily available agent to manage HRSV infection, the authors tested the hypothesis that ginger can effectively decrease HRSV-induced plaque formation in respiratory mucosal cell lines. MATERIALS AND METHODS: Effect of hot water extracts of fresh and dried gingers on HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Ability of ginger to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Fresh ginger dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (p<0.0001). In contrast, dried ginger didn't show any dose-dependent inhibition. 300 µg/ml fresh ginger could decrease the plaque counts to 19.7% (A549) and 27.0% (HEp-2) of that of the control group. Fresh ginger was more effective when given before viral inoculation (p<0.0001), particularly on A549 cells. 300 µg/ml fresh ginger could decrease the plaque formation to 12.9% when given before viral inoculation. Fresh ginger dose-dependently inhibited viral attachment (p<0.0001) and internalization (p<0.0001). Fresh ginger of high concentration could stimulate mucosal cells to secrete IFN-ß that possibly contributed to counteracting viral infection. CONCLUSIONS: Fresh, but not dried, ginger is effective against HRSV-induced plaque formation on airway epithelium by blocking viral attachment and internalization.

Plaque assay for murine norovirus.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration. However, its limit of detection is higher compared to a plaque assay. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample.

Comparison of plaque- and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein.

The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC(50)) of these MAbs. Using PRNT as the reference and treating IC(50) values higher than 50 µg/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R(2) = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV.

Methods for growing and titrating African swine fever virus: field and laboratory samples.

Growing African swine fever virus (ASFV) isolates obtained mainly from the field, but also engineered in the laboratory, is a critical step for diagnosis, titration, or virus infection studies. This unit describes a set of methods and protocols to produce and titrate any ASFV strain in cell cultures. The procedures include (1) basic techniques to prepare virus-sensitive target cells; (2) strategies for growth, concentration, and purification of virus stocks; and (3) the semi-quantitative (end dilution) and quantitative (plaque) assays for the determination of viral titers, and the use of different ASFV-sensitive cells as targets for virus production and titration.

Development and evaluation of an automatable focus reduction neutralisation test for the detection of measles virus antibodies using imaging analysis.

A plaque reduction neutralisation test (PRNT) is still regarded as the gold standard for the investigation of anti-measles immunity. In this study, an alternative simplified automatable focus reduction neutralisation test (AFRNT) based on the classical PRNT was developed. The AFRNT uses the conventional Edmonston strain of measles, immunoperoxidase staining with monoclonal antibodies, and automated plaque counts performed with AID ViruSpot software. The assay is performed in 96-well plates, requires 2 days, and is fully automatable. The AFRNT was evaluated in comparison with PRNT and Enzygnost anti-measles enzyme immunoassay (EIA). A total of 130 samples, which included two available WHO international anti-measles standards, sera from 90 patients, and 38 different lots of immunoglobulin products, were tested. Overall, good agreement was observed between EIA and both neutralisation tests; however, the EIA values for the immunoglobulin products and international standards were slightly but significantly higher than those of the neutralisation tests. The Bland-Altman analysis showed excellent agreement between AFRNT and PRNT. AFRNT is a fully automatable high-throughput neutralisation assay, which can be performed with measles and other types of viruses, including wild-type strains. It is perfectly suited for epidemiological and vaccine studies.