scholarly journals The study of antibody responses to influenza neuraminidase using a lentiviral pseudotype based ELLA

2017 ◽  
Author(s):  
Fabrizio Biuso ◽  
George Carnell ◽  
Emanuele Montomoli ◽  
Nigel Temperton
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Virus Entry ◽  
Antibody Responses ◽  
Surface Glycoprotein ◽  
Wild Type Virus ◽  
Target Cells ◽  
Type Virus ◽  
Wild Type ◽  
Reassortant Viruses

AbstractInfluenza pseudotypes represent an alternative to wild type virus for serological assays. To date, pseudotypes (PV) have predominantly been used as surrogates for wild type viruses in microneutralisation assays, where the surface glycoprotein of interest and a reporter gene (such as Luciferase) are used to assess if virus entry into target cells could be inhibited by serum antibodies. The influenza neuraminidase (NA) has the ability to bud and release new virions with or without the contribution of Haemagglutinin (HA). Influenza pseudotypes expressing NA alone, or with HA, were produced to evaluate the antibody response against NA using the enzyme-linked lectin assay (ELLA). The expression of an avian HA with human NAs has enabled the detection of specific antibody reponses against the human circulating subtypes of NA. Within this study a PV-based ELLA assay has been investigated with a pilot panel of sera prepared for an international CONSISE study. Preliminary results have confirmed that the assay is sensitive and could potentially represent a valid alternative to the classical ELLA assay, which requires the employment of reassortant viruses.

scholarly journals Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

2016 ◽  
Vol 90 (6) ◽  
pp. 3229-3242 ◽  
Author(s):  
Young-Eui Kim ◽  
Se Eun Oh ◽  
Ki Mun Kwon ◽  
Chan Hee Lee ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Terminal Region ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Viral Growth ◽  
Internal Region ◽  
Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

scholarly journals A Double Mutation in Glycoprotein gB Compensates for Ineffective gD-Dependent Initiation of Herpes Simplex Virus Type 1 Infection

2010 ◽  
Vol 84 (23) ◽  
pp. 12200-12209 ◽  
Author(s):  
Hiroaki Uchida ◽  
Janet Chan ◽  
William F. Goins ◽  
Paola Grandi ◽  
Izumi Kumagai ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Entry ◽  
Growth Factor Receptor ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Double Mutation ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.

scholarly journals Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001384
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Philip Meade ◽  
Nicholas Dambrauskas ◽  
Barbara Mühlemann ◽  
...  
Keyword(s):  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Fold Reduction ◽  
Reactive Antibody

Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model.

scholarly journals Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Jennifer Stencel-Baerenwald ◽  
Kerstin Reiss ◽  
Bärbel S. Blaum ◽  
Daniel Colvin ◽  
Xiao-Nan Li ◽  
...  
Keyword(s):  
Cell Surface ◽  
Viral Disease ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Ependymal Cells ◽  
Target Cells ◽  
Type Virus ◽  
Wild Type ◽  
Newborn Mice ◽  
Serotype 1

ABSTRACTReceptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease.IMPORTANCEReceptor utilization strongly influences viral disease, often dictating host range and target cell selection. Different reovirus serotypes bind to different glycans, but a precise function for these molecules in pathogenesis is unknown. We used type 1 (T1) reovirus deficient in binding the GM2 glycan and mice lacking GM2 to pinpoint a role for glycan engagement in hydrocephalus caused by T1 reovirus. This work indicates that engagement of a specific glycan can lead to infection of specific cells in the host and consequent disease at that site. Since reovirus is being developed as a vaccine vector and oncolytic agent, understanding reovirus-glycan interactions may allow manipulation of reovirus glycan-binding properties for therapeutic applications.

scholarly journals Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

2020 ◽  
Author(s):  
Pui Wang ◽  
Siu-Ying Lau ◽  
Shaofeng Deng ◽  
Pin Chen ◽  
Bobo Wing-Yee Mok ◽  
...  
Keyword(s):  
Antibody Response ◽  
Proinflammatory Cytokines ◽  
Neutralizing Antibody ◽  
Spike Protein ◽  
Parental Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Cleavage Motif ◽  
Sterilizing Immunity

AbstractSARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.

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