scholarly journals Construction of eGFP-Tagged Senecavirus A for Facilitating Virus Neutralization Test and Antiviral Assay

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 283 ◽  
Author(s):  
Fuxiao Liu ◽  
Yilan Huang ◽  
Qianqian Wang ◽  
Hu Shan
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Neutralization Test ◽  
High Capacity ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Serum Samples ◽  
Neutralization Titer ◽  
Porcine Serum

Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus Senecavirus in the family Picornaviridae. The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.

scholarly journals SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus)

2021 ◽  
Vol 118 (47) ◽  
pp. e2114828118
Author(s):  
Jeffrey C. Chandler ◽  
Sarah N. Bevins ◽  
Jeremy W. Ellis ◽  
Timothy J. Linder ◽  
Rachel M. Tell ◽  
...  
Keyword(s):  
Odocoileus Virginianus ◽  
Neutralization Test ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Serum Samples ◽  
Urban Centers ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Shedding ◽  
High Concordance ◽  
Wild Deer

Widespread human SARS-CoV-2 infections combined with human–wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have angiotensin-converting enzyme 2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, exhibit social behavior, and can be abundant near urban centers. We evaluated 624 prepandemic and postpandemic serum samples from wild deer from four US states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples tested with a SARS-CoV-2 virus neutralization test showed high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

scholarly journals A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-19 Patients

2021 ◽  
Author(s):  
Yiqi Ruben Luo ◽  
Cassandra Yun ◽  
Indrani Chakraborty ◽  
Alan H.B. Wu ◽  
Kara L. Lynch
Keyword(s):  
Symptom Onset ◽  
Neutralization Test ◽  
Neutralizing Antibody ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Label Free ◽  
Serum Samples ◽  
Initial Rise ◽  
Igg Avidity ◽  
Antibody Titers

AbstractBackgroundThe laboratory-based methods to measure the SARS-CoV-2 humoral response include virus neutralization tests (VNTs) to determine antibody neutralization potency. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed.MethodsA surrogate virus neutralization test established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of RBD to ACE2 after neutralizing RBD with antibodies in serum.ResultsThe LF-sVNT neutralizing antibody titers (IC50) were determined from serum samples (n=246) from COVID-19 patients (n=113), as well as the IgG concentrations and the IgG avidity indices. Although there is variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there is an initial rise, plateau and then in some cases a gradual decline at later timepoints after 40 days post-symptom onset. The IgG avidity indices, in the same cases, plateau after the initial rise and did not show a decline.ConclusionsThe LF-sVNT can be a valuable tool in clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau and then remains constant up to 8 months post-infection. The decline of antibody neutralization potency can be attributed to the reduction in antibody quantity rather than the deterioration of antibody avidity, a measure of antibody quality.SummaryA surrogate virus neutralization test established on a label-free immunoassay platform (LF-sVNT). Using the LF-sVNT and other assays, 246 serum samples from 113 COVID-19 patients were measured. We observed the time course of antibody characteristics beyond 200 days post-symptom onset.

scholarly journals Development of a Fluorescent-Microsphere Immunoassay for Detection of Antibodies Specific to Equine Arteritis Virus and Comparison with the Virus Neutralization Test

2007 ◽  
Vol 15 (1) ◽  
pp. 76-87 ◽  
Author(s):  
Yun Young Go ◽  
Susan J. Wong ◽  
Adam J. Branscum ◽  
Valerie L. Demarest ◽  
Kathleen M. Shuck ◽  
...  
Keyword(s):  
Neutralization Test ◽  
Equine Arteritis Virus ◽  
Virus Neutralization Test ◽  
Cloning And Expression ◽  
Structural Proteins ◽  
Virus Neutralization ◽  
Serum Samples ◽  
Expression Of Genes ◽  
Confirmatory Assay ◽  
Microsphere Immunoassay

ABSTRACT The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP51-255], M1-162, and N1-110), as well as partial sequences of these structural proteins (GP51-116, GP575-112, GP555-98, M88-162, and N1-69) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP555-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP555-98 MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP555-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

scholarly journals SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus)

2021 ◽  
Author(s):  
Jeffrey C Chandler ◽  
Sarah N Bevins ◽  
Jeremy W Ellis ◽  
Timothy J Linder ◽  
Rachel M Tell ◽  
...  
Keyword(s):  
Odocoileus Virginianus ◽  
Neutralization Test ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Serum Samples ◽  
Urban Centers ◽  
Viral Shedding ◽  
High Affinity ◽  
High Concordance ◽  
Wild Deer

Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted whitetailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have ACE2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, and can be abundant near urban centers. We evaluated 624 pre- and post-pandemic serum samples from wild deer from four U.S. states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples was tested using a SARS-CoV-2 virus neutralization test with high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

scholarly journals Predicting the presence and titer of rabies virus neutralizing antibodies from low-volume serum samples in low-containment facilities

2020 ◽  
Author(s):  
Diana K. Meza ◽  
Alice Broos ◽  
Daniel J. Becker ◽  
Abdelkader Behdenna ◽  
Brian J. Willett ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Neutralization Test ◽  
Leukemia Virus ◽  
Fluorescent Antibody ◽  
Neutralizing Antibody ◽  
High Specificity ◽  
Virus Neutralization ◽  
Serum Samples

SummarySerology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing on Rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein–tagged murine leukemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. We further used statistical analysis to generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titer predictions were correlated across tests repeated on different dates both for dog samples (r = 0.93), and for a second dataset of sera from wild common vampire bats (r = 0.72, N = 41), indicating repeatability. Our test uses a starting volume of 3.5 μL of serum, estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high-throughput detection of rabies virus neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

scholarly journals Five Commercial Immunoassays for SARS-CoV-2 Antibody Determination and Their Comparison and Correlation with the Virus Neutralization Test

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 593
Author(s):  
Václav Šimánek ◽  
Ladislav Pecen ◽  
Zuzana Krátká ◽  
Tomáš Fürst ◽  
Hana Řezáčková ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Young Patients ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Age Dependent ◽  
Previous Contact ◽  
Dependent Elderly ◽  
Protein Assays ◽  
Antibody Determination

There is an ongoing debate as to whether SARS-CoV-2 antibodies can be found in patients who have recovered from COVID-19 disease. Currently, there is no consensus on whether the antibodies, if present, are protective. Our regular measurements of SARS-CoV-2 antibodies, starting in July 2020, have provided us with the opportunity of becoming acquainted with the five different immunoassays. A total of 149 patients were enrolled in our study. We measured the samples using each immunoassay, then performing a virus neutralization test and comparing the results of SARS-CoV-2 antibodies with this test. We observed that the production of neutralizing antibodies is age-dependent. Elderly patients have a higher proportion of high neutralizing titers than young patients. Based on our results, and in combination with the literature findings, we can conclude that the serological SARS-CoV-2 antibody measurement is a helpful tool in the fight against COVID-19. The assays can provide information about the patient’s previous contact with the virus. Anti-spike protein assays correlate well with the virus neutralization test and can be used in the screening of potential convalescent plasma donors.

scholarly journals Evaluation of six commercial SARS-CoV-2 serology assays detecting different antibodies for clinical testing and serosurveillance

2021 ◽  
Author(s):  
Suellen Nicholson ◽  
Theo Karapanagiotidis ◽  
Arseniy Khvorov ◽  
Celia Douros ◽  
Francesca Mordant ◽  
...  
Keyword(s):  
Cell Culture ◽  
Neutralization Test ◽  
Serological Test ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Binding Assays ◽  
Humoral Immune ◽  
A Cell

Abstract Background Serological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. Methods We evaluated the performance of six commercially available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared to a cell culture-based microneutralisation (MN) assay. We tested sera from patients with prior RT-PCR confirmed SARS-CoV-2 infection, pre-pandemic sera and potential cross-reactive sera from patients with other non-COVID-19 acute infections. Results For sera collected &gt; 14 days post-symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA and 83.3% for Wantai IgM. Specificity for the best performing assay was 99.5% for the Wantai total Ab and for the lowest performing assay was 97.1% for sVNT (as per IFU). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG and sVNT (as per IFU) with (97%, 97% and 95% respectively) and Wantai IgM having the poorest agreement at 93%. Conclusion Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture-based neutralization assay showed good result correlation between all assays. However correlation between the cell-based neutralization test and some assays detecting Ab’s not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimise serological test algorithms for assessing antibody responses post SARS-CoV-2 infection or vaccination.

scholarly journals Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

2007 ◽  
Vol 81 (17) ◽  
pp. 9490-9501 ◽  
Author(s):  
Christine D. Krempl ◽  
Anna Wnekowicz ◽  
Elaine W. Lamirande ◽  
Giw Nayebagha ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Virulent Strain ◽  
Consensus Sequence ◽  
Cytoplasmic Tail ◽  
Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

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