assay Antiviral Res. 45 spike glycoprotein are of extreme importance. Each serum dilution was measured in duplicate and the lower asymptote of the nonlinear regression curve was constrained to 0 (A) or left unconstrained (B). virus neutralization assay relies on visual grading of virus infected or uninfected cells to determine the level of neutralization 4. This volume covers multidisciplinary approaches on various aspects of Chikungunya Virus (CHIKV) research that was brought together from leading laboratories across the globe. Second report. Forty hours later, the A549 cells were harvested for the ICW assay to assess NP expression. Acad. Methods 81, 193197. ORIGINAL: ENGLISH IVB Bethesda, MD 20894, Help Notably, the NP spotted and exhibited in the ICW results forms obvious stains that mimic plaque-forming units. SARS-CoV-2 Spike Pseudoviruses: A Useful tool to study A panel, Figure 4. Data are presented as the geometric mean 95% CI. Details of the studies are described in the S1 File. HUVECs and A549 cells grown in 24-well plates were infected with HTNV. The 95% quantile for negative human serum was found to be 2.02 log10EC50, for negative rhesus macaque serum 2.12 log10EC50, and for CD-1 mouse serum 2.18 log10EC50 (Fig 7 and Table 3). The reproducibility of the PBNA was determined by testing the two internal control samples in three independent runs, with each run the two samples being tested 3 times on 10 individual plates. Mouse ascitic fluid generated by i.p. Neat or diluted Z-RVP (7.5 L) was transferred to a 384-well plate and Vero cells in assay media added to a final volume of 22.5 L/well. These authors have contributed equally to this work. The stock virus 50% endpoint must be between 100 and 500 TCID 50 /0.1 mL for a test to be valid. The matrix effect of CD-1 mouse serum was significantly higher than both human and rhesus macaque sera (p < 0.0001 for both) and the matrix effect of rhesus macaque serum was higher than that of human serum (P = 0.0002). Dengue and Dengue Hemorrhagic Fever, 2nd Edition All in-life practices, including husbandry and environmental enrichment, were approved and conducted per IACUC of The University of Wisconsin-Madison, College of Letters and Science and Vice Chancellor for Research and Graduate Education Centers Institutional Animal Care and Use Committee under protocol number G005401-R01. Manenti A, Maggetti M, Casa E, Martinuzzi D, Torelli A, Trombetta CM, et al. The Signal Window (SW) was calculated for each plate , where n is the number of replicates of the test substance that will be used in the assay (n = 2) [35]. The viral titer was calculated as the TCID50 using the Reed and Muench formula. Antibody Engineering Ye, W., Lei, Y., Yu, M., Xu, Y., Cao, M., Yu, L., et al. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Biol. As there is no specific preventative or treatment for ZIKV infection, there is an urgent medical need for ZIKV vaccines capable of preventing infections, particularly during pregnancy. Febrile (n = 8), hypotensive (n = 8), oliguric (n = 16), diuretic (n = 16), and convalescent (n = 16) patients were included. Imaging results (G) and the intensity ratio analyzed by software (H) are presented. Among them, S protein is related to virus attachment and entry into target cells and is considered to be a key protein to start the process of virus infection. SARS-CoV-2 Serology Assays. (3) Z-RVP particles are added (7.5 L volume and Z-RVP concentration optimized by lot) and (4) the plate is incubated at 37C for 60 min in a 5% CO2 humidified incubator to allow neutralization. HTNV NP production was dose-dependent from an MOI of 0.01 to 5 in both the HUVECs (Figure 2D) and A549 cells (Figure 2E). A total of 150,000200,000 hantavirus infection cases are reported annually worldwide, with mortality rates of 15% for HFRS and 50% for HPS during the natural infection process (Hussein et al., 2012). Immunogenicity, efficacy and safety in nonclinical models is an important aspect of development of ZIKV vaccine candidates [48, 49] as the current low prevalence of ZIKV infections make Phase III efficacy studies difficult to conduct. Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector. We also confirmed the poor effects of the current vaccine on NAb stimulation. Vaccine 28, 71407142, doi: 10.1016/j.vaccine.2010.08.082 (2010). Quantile plots of log10EC50 values are shown for 124 human, 84 rhesus macaque, and 69 CD-1 mouse sera. Geometric mean titers (GMT) were utilized to compare the neutralizing activity among all groups. Briefly, assay plates were left to come to ambient temperature (room temperature, 2022C) for 15 min. The scanned imaging results (C) and the intensity ratio (GP/-actin) (D) are presented. These findings indicated a clear positive relationship between NP production and the infection time or dose within limits. These findings implied that the ICW assay was highly accurate for the evaluation of HTNV replication compared with the qRT-PCR and Western blotting assays. Flat lines indicates no predicted interaction. Front. The inactivated bivalent Hantaan virus (HTNV, type I) and Seoul virus (SEOV, type II) vaccines are universally applied in China to prevent HFRS. GENLISA SARS-CoV-2 (Covid-19) Surrogate Virus Neutralization Test (sVNT) ELISA : KBVH400. When inoculated into or out used for influenza virus neutralization field, 7:535. doi: 10.3389/fimmu.2016.00535, Yasuda-Inoue, M., Kuroki, M., and Ariumi, Y. 3D8 and 3G1 prepared by our lab had high neutralizing activity (Xu et al., 2002), with titers measured by ELISA of 1/1585 (0.0063 g/l) and 1/2240 (0.0045 g/l), respectively (Figures 8B,D). We developed several acceptance criteria based on Z-RVP-384 assay performance characterization: required assay signal (RLU) and positive and negative control ranges. Gene ontology analysis, which is organized into three independent hierarchies for cellular components, molecular functions, and biological processes, was applied to analyze the main functions of the differentially expressed genes. Virus neutralization test (VNT): Of 793 Malian sera, 432 (45 doubtful and 387 positive, according to anti-CHIK IgG ELISA results) were selected to perform cytopathic effect (CPE) based VNT. No, Is the Subject Area "Chemiluminescence" applicable to this article? J. Mol. These data demonstrate specificity of the Z-RVP-384 assay for neutralizing antibodies elicited by the candidate ZIKV vaccine when compared to YFV, JEV, WNV, and TBE vaccines. Equal amounts of protein (20 g protein/lane) were electrophoresed in a 10% SDS polyacrylamide gel and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Patients with prompt production of NAbs tended to have favorable disease outcomes. Cell Biol. (2016). A panel of fifty negative human and thirty mouse serum samples were used to determine the limit of detection (LOD). Neutralizing antibody (NAb) assays for human immunodeficiency virus (HIV) are used to study the immune response in infected individuals, to examine monoclonal antibodies and viral diversity, and to judge the potential value of candidate vaccine immunogens in preclinical and clinical trials. Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection. doi: 10.3390/v5092272, McCaughey, C., Shi, X., Elliot, R. M., Wyatt, D. E., O'neill, H. J., and Coyle, P. V. (1999). DENV neutralizing monoclonal antibody (mAb) 4G2 neutralized all four serotypes of DENV-RVPs while non-neutralizing mAb 15F3 did not neutralize any [24]. doi: 10.1016/j.jviromet.2013.06.022, Boveia, V., and Schutz-Geschwender, A. Biol. BSL-3 sparing neutralization assays We are working to develop neutralization assays via flow cytometry as well as advanced live cell imaging to assist SARS-CoV-2 vaccine and therapeutic development. To narrow this gap, in-cell Western (ICW) assays have been applied to monitor hantavirus replication kinetics and assess viral titers. Luciferase assays were performed 24 h after infection, and the results were expressed as the comparative ratio of firefly luciferase to Renilla luciferase activity compared to the untreated group. An important element of ZIKV neutralizing antibody assay development was capability for high throughput to enable rapid testing of large numbers of clinical samples. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. The effect of prior flavivirus exposure on ZIKV infection or ZIKV vaccination is not well understood and therefore further studies are required. ICH recommends in its guidelines that the linearity of the assay is evaluated across its range with a minimum of five concentrations tested [36]. This book provides researchers with a better understanding of what is currently known about these diseases, including whether there is a vaccine available or under development. In the present study, the ICW assay was used to detect HTNV NP expression and monitor viral replication kinetics, based on which viral and NAb titers were evaluated. Serum samples were assayed for West Nile virus (WNV)specific antibodies by using the plaque-reduction neutralization test as previously described (1). Versus MOI of 0, the ICW assay could detect NP production at MOIs of 0.01, 0.1, 0.5, and 1(Figure 2F). Pseudoviruses are useful virological tools because of their safety and versatility; however the low titer of these viruses substantially limits their wider applications. The experiments were performed independently at least three times with similar results. (2010). The book concludes with an assessment of the serum neutralization, indirect fluorescent antibody, and rapid fluorescent focus inhibition tests. This book is a valuable resource for virologists, pathologists, epidemiologists, and students. Front. Start a Free Trial Log In. HTNV Gc was also detected with either 3D8 (Figures 1C,D) or 3G1 (Figures 1E,F) at concentrations more than 1.00 103 g/l. Comprehensive and timely, Avian Influenza Virus equips diagnosticians and researchers with the current tools and information they need to learn more about this high impact disease. Serum samples were heatinactivated for 30 minutes at 56C; twofold serial dilutions, starting from 1:10, were then mixed with an equal volume of viral solution containing 100 TCID50 of SARSCoV2. Data are presented as the mean SD. 90, 36613675. (2016). The experiments were performed independently at least three times with similar results. Neutralization Titers in log10 are shown in (C) as calculated by nonlinear regression in GraphPad Prism. Interferon-Induced Transmembrane Protein 3 Inhibits Hantaan Virus Infection, and Its Single Nucleotide Polymorphism rs12252 Influences the Severity of Hemorrhagic Fever with Renal Syndrome. KM mice with body weight ranging from 8 to 26 grams were inoculated with 110, Panel 5A: selection of the pseudovirus dose used in the. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. No, Is the Subject Area "Vaccines" applicable to this article? Clipboard, Search History, and several other advanced features are temporarily unavailable. The experiments were performed independently at least three times with similar results. A., et al. We would like to thank the Integral Molecular team for their scientific support on Z-RVP production and QC. The midpoint of 2.5 L was chosen as optimal for this Z-RVP lot and assay conditions. NLRP3 inflammasome is responsible for Hantavirus inducing interleukin-1beta in THP-1 cells. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses in swine to measure the titer of neutralizing antibodies post-infection or after vaccination. Rabbits were given Ketamine and Xylazine (anesthesia and analgesia) before euthanization by CO2 inhalation and exsanguination. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Am. Nevertheless, DDX3 was considered a host factor that was hijacked by viruses and favored the replication of flaviviruses (e.g., HCV, dengue virus and West Nile virus) and retroviruses (e.g., HIV-1; Brai et al., 2016). PLoS neglected tropical diseases 9, e0003709, doi: 10.1371/journal.pntd.0003709 (2015). Sustained high level of serum VEGF at convalescent stage contributes to the renal recovery after HTNV infection in patients with hemorrhagic fever with renal syndrome. Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (Hantavax) in healthy adults. Yes DExD/H-box RNA helicases as mediators of anti-viral innate immunity and essential host factors for viral replication. Assay Principle of Creative Diagnostics SARS-CoV-2 Neutralizing Antibody ELISA Kit (CD Cat# DEIA-WZ021) Introduction. The intention of this volume is to comprehensively review neutralization and where possible to construct a unifying theory which can be tested by experimentation. Application of an In-Cell Western assay for measurement of influenza A virus replication. The DExD/H box helicases play vital roles in RNA metabolism, and some of these helicases can regulate host IFN responses and inhibit viral infection (Li et al., 2015; Diot et al., 2016). 2016YFC1202903) and the National Natural Science Foundation of China (nos. Over the past 60 years, almost 1.7 million cases and 47,000 deaths have been reported in China (Jiang et al., 2016). For SARS-CoV-2, viral neutralization assays are performed using either live virus (7) or viral vectors pseudotyped with the spike protein (8). The same lot of rabies pseudoviruses were titrated six times in each cell lines. Micro-neutralization tests are conventionally used to assess HTNV NAb titers through ELISA, which requires at least 10 days for HTNV propagation in Vero cells. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. DDX60L is an interferon-stimulated gene product restricting hepatitis C virus replication in cell culture. The book covers advancements in the classical methods described as well as recent methods and approaches pertaining to rabies diagnosis and research. The upper limit of quantitation (ULOQ) of the Z-RVP-384 assay was dependent on determination of the upper asymptote of the non-linear regression curve (no neutralization, maximum RLU). Two ZIKV antibody positive cynomolgus macaque sera and two ZIKV antibody positive human sera were serially diluted in two-fold steps in negative human serum or cynomolgus macaque serum respectively. Hoechst 33258 (100 ng/ml; Beyotime) was utilized to stain the cell nuclei, and the cells were observed using a BX60 fluorescence microscope (Olympus, Tokyo, Japan). serum-virus neutralization assay. The linearity of an assay is the capability to generate results directly proportional to the expected quantity of analyte in the sample. Ye, W., Xu, Y., Wang, Y., Dong, Y., Xi, Q., Cao, M., et al. Figure 1. Some viruses, such as the new coronavirus SARS-CoV-2 (2019-nCoV), SARS-CoV, Ebola virus, H5N1, etc., are highly infectious and highly pathogenic, which Vero cells were obtained from ATCC (CCL-81.4) and expanded into a research cell bank (non-CGMP). The positive serum used throughout assay development was generated by Takeda by immunizing rabbits with live ZIKV preparation using strain Dakar41524 and strain PRVABC59 (anti-Dakar L777P16, anti-Dakar L821P119, anti-PRVABC59 L773P08). All of the following experiments were performed with 1A8 (0.25 103 g/l) in the ICW assay to assess the amount of HTNV NP. The serumvirus mixture was incubated for 1 hour at 37C in a humidified atmosphere with 5% CO 2. and H.J.D and reviewed of the study design, data and manuscript. P < 0.05 were considered significant. Both the MNT and PRNT performed in 96 well plates are largely manual, labor-intensive assays, which make them challenging and expensive to use in settings such as large clinical trials in which hundreds or thousands of samples need to be tested under good clinical laboratory practice conditions [18, 19]. Rep. 6:33763. doi: 10.1038/srep33763, Egorina, E. M., Sovershaev, M. A., and Osterud, B. doi: 10.1128/JVI.01297-15, Guardado-Calvo, P., Bignon, E. A., Stettner, E., Jeffers, S. A., Perez-Vargas, J., Pehau-Arnaudet, G., et al. (2011). Another challenge is that both authentic and pseudovirus virus assays depend on host cells for infection which adds variability to the assay. Next, HTNV was propagated in Vero E6 cells or mouse brains, and the titers were determined by both the ICW assay and ELISA. The emergence of severe acute respiratory syndrome (SARS) in late 2002 and 2003 challenged the global public health community to confront a novel epidemic that spread rapidly from its origins in southern China until it had reached more than https://doi.org/10.1371/journal.pone.0250516.g009. Efficacy evaluation of inactive HTNV vaccines by detecting NAb titers with ICW (A,B) The NAb titers of vaccinees without boost (two primary doses, n = 8) were determined by the ICW assay (A) and ELISA (B). The oligonucleotide primers used for the qRT-PCR were synthetized at Sangon (Shanghai, China); the sequences are listed in Table 1. Potential cell toxicity of the Z-RVP reagent lot 229A on Vero cells plated at 4625 cells/well was assessed using the CellTiter-Glo detection System in parallel to the Z-RVP infectivity. Thus, the well-established ICW assay offers a novel platform for screening antiviral molecules and developing therapeutic drugs. Get Unlimited Access. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Availability of these assays has helped The amount of NP production detected by the ICW assay indicated that IFITM3 but not IFITM1 and IFITM2 significantly suppressed HTNV replication (Figures 5C,D), which was consistent as we previously reported (Xu-Yang et al., 2017). Detection of bacillus Galmette-Guerin (Mycobacterium bovis BCG) DNA in urine and blood specimens after intravesical immunotherapy for bladder carcinoma. The singlecycle virus neutralization assays allow to directly read out the proportion of virus prevented from entering in a single round of infection and measure the neutralization activity of the inoculum. This book intends to review the latest updates on dengue fever, the tools available for its study and control, and promising technologies currently in the pipeline. Each lot number represents a different production run, each letter of the lot represents a different harvest date. MI. Moreover, the in vivo PBNA assay was also found to be superior to the live virus based assay. We performed a correlation analysis of the Z-RVP-384 assay with a ZIKV PRNT assay (Q2 Solutions, San Juan Capistrano, CA). doi: 10.1016/j.jviromet.2010.08.005, Wang, K., Singh, D., Zeng, Z., Coleman, S. J., Huang, Y., Savich, G. L., et al. Following intravenous administration, the pseudovirus effectively infected the mice, with dynamic viral distributions being sequentially observed in spleen, liver and brain. This volume provides various techniques and methodologies currently used in the study of MERS-CoV. 30 L of the diluted sample was transferred to a 96-well plate including negative, low and high positive controls. In summary, our study applied the ICW assay to detect viral protein expression, thereby establishing a novel approach to evaluate viral replication and detect viral titers, especially targeted at non-lytic viruses such as HTNV. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. Figure 5. https://doi.org/10.1371/journal.pone.0250516.g006. FCM and IFA can be used to rapidly monitor HTNV replication kinetics. No use, distribution or reproduction is permitted which does not comply with these terms. The traditional flavivirus neutralization assays, the PRNT and MNT assays, are low throughput, imprecise, manual labor intensive and represents a major bottleneck in testing large numbers of sera for clinical studies [18, 19, 21]. These findings implied that IFN responses might be necessary for the DDX3, DDX21 or DDX60-mediated antiviral influence. The procedures involve the use of a known dilution of specific neutralizing antiserum mixed with multiple dilutions of the homologous and suspect virus and subsequent observation (2) Serially diluted HI-serum is then transferred to a white tissue-culture treated 384-well flat bottom plate in a final volume of 7.5 L. Sixteen healthy individuals aged 2535 years form the Centers for Disease Control (CDC) of Shaanxi were inoculated with inactivated bivalent vaccines for Hantaan virus (HTNV, type I) and Seoul virus (SEOV, type II) using different immune strategies (three-dose schedule at 0, 1, and 13 months). These detected levels of matrix effect in the four dilution series (Fig 5, Table 2) were the result of the matrix effects of the individual sera (presumed negative) used for the dilution series. We established a minimum chemiluminescence signal of 100,000 RLU to allow for adequate range across the neutralization curves, with a maximum signal predetermined by the chemiluminescence reader capacity of 1x107 RLU.
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