Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Madison, WI USA. The purpose of this book is to provide a synthesis of the ever evolving field of pediatric viral infections. The most common and exotic pediatric viral infections are fully reviewed in this first one of a two volume set. The text features detailed experimental protocols with comprehensive sections on materials and preparations for all exercises, plus introductory material, discussion questions, and further reading. the use of few viruses and cell lines ... eCollection 2018 Dec. Ma X, Niezgoda M, Blanton JD, Recuenco S, Rupprecht CE. https://doi.org/10.1371/journal.pone.0250516.t002. This text then describes blood group antibodies that agglutinate red blood cells suspended in saline. Other chapters consider the classical pathway of complement utilization. All in-life practices, including husbandry and environmental enrichment, were approved and conducted per IACUC of Inotiv under protocol #2384–14376. Two-fold dilution series of the serum was mixed with ZIKV (200 TCID50) in DMEM 2%FBS. May 23, 2020 2:22 AM GMT. (B) The altered expression levels of DDX3, DDX5, DDX6, DDX21, DDX50, and DDX60 at the different time intervals post-HTNV infection in HUVECs as determined by qRT-PCR (n = 3). Sp2/0-derived mouse ascitic fluid was used as the negative control (NC). Digital gene expression (DGE) tags were annotated to the human transcriptome (Ensembl Version 58) by mapping the reads to the sequences flanking the NlaIII restriction sites on both the coding and non-coding strands. SARS-CoV-2 Spike-pseudovirus Based Neutralization Assay SARS-CoV-2 has four structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). 31, 3802–3819. Nevertheless, one significant characteristic of hantaviruses is that their replication in mammalian cell culture tends to be slow and non-lytic (McCaughey et al., 1999). Nucleic Acids Res. Funding: This project was funded with Federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority, under Contract No. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 dpi (time) or 0.01 (MOI). 25, 387–393. HEK293 cells were seeded into 24-well plates and transfected with the different luciferase plasmids (100 ng) and pRL-TK (10 ng). Infect. The Z-RVP-384 assay is applicable to measuring serum antibodies in humans and multiple other species. 2005 Apr;125(1):35-40. doi: 10.1016/j.jviromet.2004.12.003. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. All samples on a failed plate must be retested. The improved plaque formation test is dependent on the low pH-induced cytopathic effects of hantavirus but is time-consuming and has low reproducibility. 298, 552–558. To investigate the roles of IFITM1, IFITM2, IFITM3, Mx1, and Mx2 in HTNV infection, these proteins were ectopically expressed in HUVECs, which were then infected with HTNV at an MOI of 0.1. The classification of HFRS severity and the exclusion criteria were previously described (Ma et al., 2012; Yi et al., 2013). DDX3 Interacts with Influenza A Virus NS1 and NP proteins and exerts antiviral function through regulation of stress granule formation. The principle of limiting the infection to a single round increases the precision of the assay measurements. The stock virus 50% endpoint must be between 100 and 500 TCID 50 /0.1 mL for a test to be valid. https://doi.org/10.1371/journal.pone.0250516.g002. The matrix effect serves as the LLOQ. (2016). Each serum dilution was titered six separate times on 4 occasions, generating 24 measurements per dilution resulting in average log10EC50 neutralizing antibody titers that ranged from 1.54 to 4.26. 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. (C, D) Two positive cynomolgus macaque sera were diluted in negative cynomolgus macaque serum. All cDNAs were subsequently subcloned into the pLVX-IRES-ZsGreen vector (Clontech, Mountain View, CA, USA) using the Xho I and BamH I restriction sites. The assay was linear and accurate in human serum over a serum dilution range resulting in neutralizing titers between ~3.5 log10EC50 to ~2 log10EC50 (Fig 5A) and ~3 log10EC50 to ~2 log10EC50 (Fig 5B, Table 2). Any sample > ULOQ is re-tested using a pre-dilution of 1:200 or greater. Figure 2. RNA was extracted at 24 hpi as described above, and the quality was analyzed using the FastQC software by Beijing Genomics Institute (BGI, Shenzhen, China). Equivalent readers may be used. (A) Differentially expressed DExD/H box helicases after HTNV infection shown by heat map from DGE analysis. Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives. Euthanasia (carbon dioxide asphyxiation) was performed in accordance with accepted America Veterinary Medical Association (AVMA) guideline. Biophys. Testing of sera with a potency > 5.0 log10 required an additional predilution step of the serum (e.g. https://doi.org/10.1371/journal.pone.0250516.g008. doi: 10.1016/j.virol.2012.09.041, Chen, G., Liu, C. H., Zhou, L., and Krug, R. M. (2014). Actually, neutralization and binding were not carried out under the same conditions in the latter study : virus and NAb were incubated for 3 h at 37°C and then for an additional 21 h at 4°C before the infectivity assay. The GO analysis suggested that increased ribonucleoprotein complex expression (Figure 6A), enhanced biological process of RNA binding (Figure 6B) and elevated molecular function RNA processing (Figure 6C) occurred after HTNV infection. The structure, antigenicity, stability and reproducibility of the Z-RVP reagent has been described [32]. This product is intended for research use … All data were expressed as the mean ± SD. Data are presented as the mean ± SD. MI. doi: 10.1016/j.jviromet.2010.08.005, Wang, K., Singh, D., Zeng, Z., Coleman, S. J., Huang, Y., Savich, G. L., et al. Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3' UTR structures. Analyte sensitivity is defined by the LLOQ, as such the sensitivity of the assay is restricted to analyte values at or above the evaluated matrix effect. Figure 3. This book comprises an overview about the generation of antibody diversity and essential techniques in antibody engineering: construction of immune, naive and synthetic libraries, all available in vitro display methods, humanization by ... At 48 h post-infection, the ICW assay were performed to detect the amount of HTNV NP; positive/negative (P/N) responses >2.1 were considered significant. (2017). Discover a faster, simpler path to publishing in a high-quality journal. 2021 Apr 10;17(6):1574-1580. doi: 10.7150/ijbs.59184. AVG (min) was the average taken over the three minimum signal plate averages, and AVG (max) was the average taken over the three maximum signal plate averages [35]. doi: 10.18632/oncotarget.11329, Zhang, Z., Kim, T., Bao, M., Facchinetti, V., Jung, S. Y., Ghaffari, A. 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. Then, the cells were washed with DPBS (HyClone) two times and incubated with DMEM with 10% FBS. The 2019-nCoV Neutralization ELISA Kit is an enzyme-linked immunosorbent assay for the detectionof antibodies or other proteins (molecular) that can block the binding of SARS-CoV-2 … (2012). About BioCentury. 2.2.5 Primary Antibody When indirect immunofluorescence (IFA) and not cytopathic effect (CPE) is used The hantaviral tripartite genome consists of the S, M, and L segments, which encode the nucleoprotein (NP), glycoprotein (GP, which is post-translationally cleaved into the N-terminal Gn and C-terminal Gc components), and viral RNA-dependent RNA polymerase (RdRp), respectively. A prototype of the Z-RVP-384 assay was used in the evaluation of this standard [42]. 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). Conceptualization, doi: 10.1111/j.1538-7836.2005.01781.x, Fullam, A., and Schroder, M. (2013). (2012). HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly. NIBSC standard 16/352 was reconstituted and aliquoted into single use aliquots for testing by 4 different operators on 9 separate occasions in the manual Z-RVP-384 assay. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. This is poured over a … 138, 32–39. 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). This publication is intended to contribute to prevention and control of the morbidity and mortality associated with dengue and to serve as an authoritative reference source for health workers and researchers. Pseudovirus Neutralization Assay in SARS-CoV-2 research. To date, neither effective therapeutic drugs nor FDA licensed prophylactic vaccines against HTNV infection are available. Microbiol. To estimate the upper limit of quantitation (ULOQ) of the Z-RVP-384 assay, a high titer rabbit hyperimmune serum (estimated titer of ~5 log10EC50) was pre-diluted 1:8, 1:25, 1:50, 1:200 and 1:400 in assay medium and titered in separate experiments (Fig 8A) the standard error and % Fit Error (FE) was stable from the 1:25–1:200 dilutions (Fig 8B). Manenti A, Maggetti M, Casa E, Martinuzzi D, Torelli A, Trombetta CM, et al. Zika virus (ZIKV), first identified in 1947, is a member of the Flaviviridae [1–3]. The plates were incubated under 5% CO2 for 5 days at 36°C ± 2°C, before visual observation of the cell monolayer under a microscope for the presence of CPE resulting from viral infection. 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. Please enable it to take advantage of the complete set of features! Heat-inactivated ZIKV hyperimmune rabbit serum was diluted 1:5 in assay media then serially diluted 5-fold ten times. The data indicated that the current inactivated bivalent vaccines in China still needed further improvements to efficiently activate host humoral immunity. The GENLISA™ SARS-CoV-2 (Covid-19) Surrogate Virus Neutralization Test (sVNT) ELISA kit is used as an analytical tool for the qualitative and quantitative detection all types of neutralizing antibodies against SARS-CoV-2 in serum or plasma. The log10EC50 values from each dilution were determined and plotted against the input dilutions (log10) of the serum. The SPH2015 strain of ZIKV was the first strain isolated from the outbreak in Brazil in 2015 and has a high degree of genetic similarity to other ZIKV strains from the same outbreak in South and Central America during 2015–16 and other recent Asian-lineage ZIKV strains [53]. Front Pharmacol. Prior to inclusion, all participants were informed of the study objectives and signed the consent form before medical records and blood samples were obtained. Oligonucleotide primers and sequences. These DExD/H box helicases were ectopically expressed in HUVECs with a lentivirus system labeled with ZsGreen (Figure 7C). 2018 Dec 14;12(12):e0007011. The DOE was then analyzed based on the EC50 titers and confidence limits, V-factor and dynamic range. The principle stages of the assay are serum heat inactivation and serial dilution, addition of Z-RVP and neutralization at 37°C for 1 hour, addition of Vero cells, incubation at 37°C, 5% CO2, for 72 hours, addition of Renilla substrate and detection of chemiluminescence, and calculation of neutralization titers. The midpoint of 2.5 μL was chosen as optimal for this Z-RVP lot and assay conditions. Sixteen healthy individuals aged 25–35 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). 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. The signals on the PVDF membrane were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).
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