Evaluation of three alternative methods to the plaque reduction neutralizing assay for measuring neutralizing antibodies to dengue virus serotype 2.

BACKGROUND: Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT). METHODS: Twenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples. RESULTS: Positive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% (r = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation (r = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay (r = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses. CONCLUSION: The xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

Amino Acids at Positions 156 and 332 in the E Protein of the West Nile Virus Subtype Kunjin Virus Classical Strain OR393 Are Involved in Plaque Size, Growth, and Pathogenicity in Mice.

The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.

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.

Characterization of a Small Plaque Variant Derived from Genotype V Japanese Encephalitis Virus Clinical Isolate K15P38.

Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.

Cytokinins Reduce Viral Replication and Alter Plaque Morphology of Frog Virus 3 In Vitro.

Cytokinins (CKs) are a group of N6-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N6-isopentenyladenine (iP), N6-isopentenyladenosine (iPR), N6-isopentenyladenosine-5'monophosphate (iPMP), and 2-methylthiol-N6-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

A simplified method of bacteriophage preparation for transmission electron microscope.

Bacteriophages are viruses that infect bacteria. Researchers use different methods to study the characteristics of bacteriophages. Transmission electron microscope (TEM) is considered the best method to analyze these characteristics. However, the quality of TEM micrographs is significantly influenced by the preparation methods used to prepare the bacteriophages sample. In this study, researchers compared two different methods for preparing the bacteriophage samples. In one method was used SM buffer, while in the other used deionized water. The results were analyzed by TEM and compared with each other. Additionally, the viability of bacteriophage in deionized water and SM buffer at 4°C was determined through plaque assay within 72 hours. TEM micrographs showed that the quality of bacteriophage sample prepared with deionized water is superior to those prepared with SM buffer. Furthermore, the titer of the bacteriophages did not show a significant reduction during 72 hours in both SM and deionized water. In conclusion, the results suggested that preparation method can significantly impact the quality of TEM micrographs. Using sterile deionized water for the preparation of bacteriophages is a simple way to improve the quality of TEM micrographs and it is advisable to send the samples to the laboratory within 72 hours.

Measles Foci Reduction Neutralization Test (FRNT).

The plaque reduction neutralization test (PRNT) and the enzyme-linked immunosorbent assay (ELISA) are both widely used to assess immunity to infectious diseases such as measles, but they use two different measurement principles: ELISA measures the ability of antibodies to bind to virus components, while the PRNT detects the aptitude of antibodies to prevent the infection of a susceptible cell. As a result, detection of measles virus (MV) neutralizing antibodies is the gold standard for assessing immunity to measles. However, the assay is laborious and requires experience and excellent technical skills. In addition, the result is only available after several days. Therefore, the classical PRNT is not suitable for high-throughput testing. By using an immunocolorimetric assay (ICA) to detect MV-infected cells, the standard PRNT has been developed into a focus reduction neutralization test (FRNT). This assay is faster and has improved specificity. The FRNT described here is extremely useful when immunity to measles virus needs to be assessed in patients with a specific medical condition, such as immunocompromised individuals in whom presumed residual immunity needs to be assessed. The FRNT is not generally recommended for use with large numbers of specimens, such as in a seroprevalence study.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Antiviral and synergistic effects of photo-energy with acyclovir on herpes simplex virus type 1 infection.

This experimental study aimed to evaluate the antiviral and synergistic effects of photoenergy irradiation on human herpes simplex virus type I (HSV-1) infection. We assessed viral replication, plaque formation, and relevant viral gene expression to examine the antiviral and synergistic effects of blue light (BL) with acyclovir treatment. Our results showed that daily BL (10 J/cm2) irradiation inhibited plaque-forming ability and decreased viral copy numbers in HSV-1-infected monkey kidney epithelial Vero cells and primary human oral keratinocyte (HOK) cells. Combined treatment with the antiviral agent acyclovir and BL irradiation increased anti-viral activity, reducing viral titers and copy numbers. In particular, accumulated BL irradiation suppressed characteristic viral genes including UL19 and US6, and viral DNA replication-essential genes including UL9, UL30, UL42, and UL52 in HOK cells. Our results suggest that BL irradiation has anti-viral and synergistic properties, making it a promising therapeutic candidate for suppressing viral infections in clinical trials.

Screening and verification of antiviral compounds against HSV-1 using a method based on a plaque inhibition assay.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) infection is a common viral disease that mainly causes oral lesions, but can also cause genital lesions in some instances. Current treatments with nucleoside analogs are limited by the emergence of drug resistance. Therefore, novel anti-HSV-1 drugs are urgently needed. METHODS: In this study, we screened a library of 2080 compounds for anti-HSV-1 activity using a plaque formation assay. We selected 11 potential inhibitors of HSV-1 and further evaluated their antiviral effects by plaque reduction assay and real-time polymerase chain reaction (qPCR). RESULTS: Five compounds, namely ginsenoside Rd, brassinolide, rosamultin, 3'-hydroxy puerarin, and clinafloxacin HCl, showed potent anti-HSV-1 activity and completely suppressed plaque formation at a concentration of 10 µM. Among them, clinafloxacin HCl, a fluoroquinolone antibiotic, exhibited a high selectivity index for HSV-1. CONCLUSIONS: Our findings suggest that these five compounds have potential antiviral properties against HSV-1 and may have different mechanisms of action. Further studies are warranted to elucidate the antiviral mechanisms of these compounds and to explore their therapeutic potential for HSV-1 infection.

CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro.

Simplexvirus humanalpha1 (HSV-1) affects approximately 67% of the world's population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into the plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyses revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas 9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

Rapid and stain-free quantification of viral plaque via lens-free holography and deep learning.

A plaque assay-the gold-standard method for measuring the concentration of replication-competent lytic virions-requires staining and usually more than 48 h of runtime. Here we show that lens-free holographic imaging and deep learning can be combined to expedite and automate the assay. The compact imaging device captures phase information label-free at a rate of approximately 0.32 gigapixels per hour per well, covers an area of about 30 × 30 mm2 and a 10-fold larger dynamic range of virus concentration than standard assays, and quantifies the infected area and the number of plaque-forming units. For the vesicular stomatitis virus, the automated plaque assay detected the first cell-lysing events caused by viral replication as early as 5 h after incubation, and in less than 20 h it detected plaque-forming units at rates higher than 90% at 100% specificity. Furthermore, it reduced the incubation time of the herpes simplex virus type 1 by about 48 h and that of the encephalomyocarditis virus by about 20 h. The stain-free assay should be amenable for use in virology research, vaccine development and clinical diagnosis.

The host phylogeny determines viral infectivity and replication across Staphylococcus host species.

Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts.

Rapid, simple, and cost-effective plaque assay for murine norovirus using microcrystalline cellulose.

Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.

Quantification of West Nile Virus by Plaque-Forming Assay.

The plaque-forming assay is a gold standard technique to determine the concentration of infectious viral particles. In this assay, lytic viruses infect and lyse the cells but are immobilized due to the presence of an agarose-containing overlay medium. The progeny viruses can only spread locally to and kill the adjacent cells and finally form a clear zone or plaque after staining the live cells. The number of plaques formed can be theoretically considered as the initial number of the infectious viral particles present in the sample and hence can be expressed as plaque-forming units (PFU) in a volume of the sample. Here, we provide a step-by-step method to carry out a plaque-forming assay to determine the titer of West Nile virus in a cell culture medium, which also can be adapted to other lytic viruses of eukaryotic cells.

Viral Titer Quantification of West Nile Virus by Immunostaining Plaque Assay.

Immunostained plaque assay based on the specific antibody binding to viral antigen enables the detection and titration of virus infectivity, especially for viruses that could not form plaques using the classical crystal violet or neutral red staining methods. Here we describe the application of this method to quantify viral titers of wild-type West Nile virus (WNV-WT) and replication-defective WNV-ΔNS1 virus.

Comparison of plaque reduction and focus reduction neutralization tests for the measurement of neutralizing antibody titers against japanese encephalitis virus.

Japanese encephalitis is prevalent throughout the temperate and tropical regions of Asia and is caused by the Japanese encephalitis virus (JEV), a mosquito-borne viral pathogen. The plaque reduction neutralization test (PRNT) is currently recommended as the gold standard test for detecting human antibodies against JEV. The plaque assay is the most widely used method for detecting infectious virions and involves counting discrete plaques in cells. However, it is time-consuming, and results can be subjective (owing to analyst variability during manual plaque counting). The focus reduction neutralization test (FRNT), which is based on an immuno-colorimetric assay, can be used to automatically count foci formed by the JEV. Here, we compared the efficacy of PRNT and FRNT in measuring the neutralizing antibody titers using 102 serum samples from vaccinated and unvaccinated individuals. We observed positive correlations between these neutralization assays against the Nakayama and Beijing strains (R2 = 0.98 and 0.77, respectively). Thus, FRNT may be preferable to PRNT for evaluating the efficacy of JEV vaccines in large-scale serological studies.

Neutralizing Antibodies Limit Cell-Associated Spread of Human Cytomegalovirus in Epithelial Cells and Fibroblasts.

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.

Virus-like Particles: Measures and Biological Functions.

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.

Dengue virus seroprevalence study in Bangphae district, Ratchaburi, Thailand: A cohort study in 2012-2015.

BACKGROUND: To determine the seroprevalence and transmission dynamics of dengue virus (DENV), age-stratified longitudinal serological surveys were conducted in Bangphae district, Ratchaburi province, Thailand, for 3 years between April 2012 and April 2015. METHODOLOGY: The surveys enrolled 2012 healthy children and adults between 1 and 55 years-of-age, and a longitudinal serosurvey of six repeated bleeds of the same cohort of individuals was conducted every 8 months for the first 2 years (M0, M8, M16) and every half a year (M24, M30, M36) for the rest of the study period. All samples were tested using in-house indirect sandwich dengue IgG ELISA to determine DENV antibody titer, and 640 paired samples which showed rising of DENV IgG titers in paired serum were further tested using in-house neutralization assay, Plaque Reduction Neutralization Test (PRNT50). PRINCIPAL FINDINGS: When compared against the gold standard based on the results of PRNT50, sensitivity and specificity of indirect ELISA were found to be both about 85%. The overall DENV IgG positivity determined by ELISA was 74.3% in 2012 and increased to 79.4% by the final sample collection in 2015. In our study sample, more than 98% of subjects older than 25 years were found to be seropositive. Among 518 IgG negative subjects at enrollment, the seroconversion rates were measured in paired bleeds; the rates (between successive visits, approximately 6 months) ranged between 4.8% (between M16 and M24) and 14.7% (between M0 and M8). The dominant serotype of primary DENV infection cases based on seroconversion was identified from the PRNT results and it was DENV-2. CONCLUSIONS: Our study documented high levels of seroprevalence and rate of transmission. Given the importance of the serostatus and disease burden in consideration for dengue vaccine introduction, our data could be used in decision-making on implementation of various dengue control and preventive measures.