SARS-CoV-2 antibodies, serum inflammatory biomarkers and clinical severity of hospitalized COVID-19 patients
Background The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. Patients and methods This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers wer…
Globally defining the effects of mutations in a picornavirus capsid
The capsids of non-enveloped viruses are highly multimeric and multifunctional protein assemblies that play key roles in viral biology and pathogenesis. Despite their importance, a comprehensive understanding of how mutations affect viral fitness across different structural and functional attributes of the capsid is lacking. To address this limitation, we globally define the effects of mutations across the capsid of a human picornavirus. Using this resource, we identify structural and sequence determinants that accurately predict mutational fitness effects, refine evolutionary analyses, and define the sequence specificity of key capsid-encoded motifs. Furthermore, capitalizing on the derive…
Single-Cell Analysis of RNA Virus Infection Identifies Multiple Genetically Diverse Viral Genomes within Single Infectious Units
Summary Genetic diversity enables a virus to colonize novel hosts, evade immunity, and evolve drug resistance. However, viral diversity is typically assessed at the population level. Given the existence of cell-to-cell variation, it is critical to understand viral genetic structure at the single-cell level. By combining single-cell isolation with ultra-deep sequencing, we characterized the genetic structure and diversity of a RNA virus shortly after single-cell bottlenecks. Full-length sequences from 881 viral plaques derived from 90 individual cells reveal that sequence variants pre-existing in different viral genomes can be co-transmitted within the same infectious unit to individual cell…
Increased RNA virus population diversity improves adaptability
The replication machinery of most RNA viruses lacks proofreading mechanisms. As a result, RNA virus populations harbor a large amount of genetic diversity that confers them the ability to rapidly adapt to changes in their environment. In this work, we investigate whether further increasing the initial population diversity of a model RNA virus can improve adaptation to a single selection pressure, thermal inactivation. For this, we experimentally increased the diversity of coxsackievirus B3 (CVB3) populations across the capsid region. We then compared the ability of these high diversity CVB3 populations to achieve resistance to thermal inactivation relative to standard CVB3 populations in an…
Chaperoning the Mononegavirales: Current Knowledge and Future Directions
This article belongs to the Special Issue Breakthroughs in Viral Replication.
Evolutionary and phenotypic characterization of spike mutations in a new SARS-CoV-2 Lineage reveals two Variants of Interest
Molecular epidemiology of SARS-CoV-2 aims to monitor the appearance of new variants with the potential to change the virulence or transmissibility of the virus. During the first year of SARS-CoV-2 evolution, numerous variants with possible public health impact have emerged. We have detected two mutations in the Spike protein at amino acid positions 1163 and 1167 that have appeared independently multiple times in different genetic backgrounds, indicating they may increase viral fitness. Interestingly, the majority of these sequences appear in transmission clusters, with the genotype encoding mutations at both positions increasing in frequency more than single-site mutants. This genetic outco…
Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays
medRxiv: https://doi.org/10.1101/2020.09.07.20188151
Extremely High Mutation Rate of HIV-1 In Vivo.
Rates of spontaneous mutation critically determine the genetic diversity and evolution of RNA viruses. Although these rates have been characterized in vitro and in cell culture models, they have seldom been determined in vivo for human viruses. Here, we use the intrapatient frequency of premature stop codons to quantify the HIV-1 genome-wide rate of spontaneous mutation in DNA sequences from peripheral blood mononuclear cells. This reveals an extremely high mutation rate of (4.1 ± 1.7) × 10−3 per base per cell, the highest reported for any biological entity. Sequencing of plasma-derived sequences yielded a mutation frequency 44 times lower, indicating that a large fraction of viral genomes …
The external domains of the HIV-1 envelope are a mutational cold spot
In RNA viruses, mutations occur fast and have large fitness effects. While this affords remarkable adaptability, it can also endanger viral survival due to the accumulation of deleterious mutations. How RNA viruses reconcile these two opposed facets of mutation is still unknown. Here we show that, in human immunodeficiency virus (HIV-1), spontaneous mutations are not randomly located along the viral genome. We find that the viral mutation rate experiences a threefold reduction in the region encoding the most external domains of the viral envelope, which are strongly targeted by neutralizing antibodies. This contrasts with the hypermutation mechanisms deployed by other, more slowly mutating …
Highly heterogeneous mutation rates in the hepatitis C virus genome.
Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters a…
Chaperoning the <em>Mononegavirales</em>: Current Knowledge and Future Directions
The order Mononegavirales harbors numerous viruses of significant relevance for human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins…
Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.
Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N -(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) an…
Potential Influence of Helminth Molecules on COVID-19 Pathology
In recent months, the parasitology research community has been tasked with investigation of the influence of parasite coinfection on coronavirus disease 2019 (COVID-19) outcomes. Herein, we share our approach to analyze the effect of the trematode Fasciola hepatica as a modulator of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and of COVID-19 pathology.
Suitability of two rapid lateral flow immunochromatographic assays for predicting SARS‐CoV‐2 neutralizing activity of sera
Purpose: Assessment of commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially-available lateral flow immunochromatographic assays (LFIC) (Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for COVID-19 diagnosis in hospitalized patients, and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Patients and Methods: Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP)…
Author response: Globally defining the effects of mutations in a picornavirus capsid
Membrane-Associated Enteroviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes
Summary Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection (MOI), this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We find that infected cells release membranous structures (including vesicles) that contain 8–21 infectious particles on average. However, in most cases (62%–93%), these structures do not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, …
Neutralizing antibodies against SARS-CoV-2 variants of concern elicited by the comirnaty COVID-19 vaccine in nursing home residents.
Immunosenescence may impact the functionality and breadth of vaccine-elicited humoral immune responses. The ability of sera to neutralize the SARS-CoV-2 spike protein (S) from Beta, Gamma, Delta, and Epsilon variants of concern (VOCs) relative to the ancestral Wuhan-Hu-1 strain was compared in Comirnaty COVID-19-vaccinated elderly nursing home residents, either SARS-CoV-2 naïve (n = 22) or experienced (n = 8), or SARS-CoV-2 naïve younger individuals (n = 18) and non-vaccinated individuals who recovered from severe COVID-19 (n = 19). In all groups, except that including SARS-CoV-2-experienced nursing home residents, some participants lacked NtAb against one or more VOCs, mainly the Beta vari…
Adoptive transfer of ex vivo expanded SARS‐CoV‐2‐specific cytotoxic lymphocytes: A viable strategy for COVID‐19 immunosuppressed patients?
Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections is on focus of research. We evaluate herein the feasibility of expanding virus‐specific T cells (VST) against SARS‐CoV‐2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS‐CoV‐2 asymptomatic infection/negative serology, (b) SARS‐CoV‐2 symptomatic infection/positive serology, and (c) no history of SARS‐CoV‐2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 6…
Comprehensive profiling of polyclonal sera targeting a non-enveloped viral capsid
AbstractDespite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predic…
Hsp90 dictates viral sequence space by balancing the evolutionary tradeoffs between protein stability, aggregation and translation rate
AbstractAcquisition of mutations is central to evolution but the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, are proposed to promote sequence diversification by buffering destabilizing mutations. However, whether and how chaperones directly control protein evolution remains poorly understood. Here, we examine the effect of reducing the activity of the key eukaryotic chaperone Hsp90 on poliovirus evolution. Contrary to predictions of a buffering model, inhibiting Hsp90 increases population sequence diversity and promotes accumulation of mutations reducin…