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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

S -Adenosylhomocysteine Analogs Selectively Suppress Pan-Coronavirus Replication by Inhibition of nsp14 Methyltransferase

To address the ongoing threat of SARS-CoV-2 and potential emergence of novel coronaviruses, we employed a comprehensive strategy to identify and synthesize inhibitors of coronavirus methyltransferases with chemical analogs of S-adenosylhomocysteine (SAH). Two analogs, designated 4h and 4p, inhibit both mouse hepatitis virus and SARS-CoV-2 replication. Compound 4p was the most potent with half-maximal inhibition of biochemical activity at 0.2 μM and antiviral activity at ∼20 μM. This compound also has low cytotoxicity and preferentially inhibits nsp14 over nsp16 and human methyltransferases. Furthermore, molecular docking based on a newly determined crystal structure of the apo nsp16−nsp10 complex predicts that 4p occupies both the Sadenosylmethione and Gppp binding pockets of nsp14 and nsp16. Selectivity of 4p for nsp14 is likely due to the enhanced structural stability of the nsp14 binding pocket relative to nsp16. These findings highlight SAH analogs as scaffolds for pan-coronavirus therapeutics and underscore the value of structure-guided design in antiviral drug discovery.

Coronavirus↗
DOE OSTI2024

Tertiary folds of the SL5 RNA from the 5 ' proximal region of SARS-CoV-2 and related coronaviruses

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T’s “arms.” Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Atypical Divergence of SARS-CoV-2 Orf8 from Orf7a within the Coronavirus Lineage Suggests Potential Stealthy Viral Strategies in Immune Evasion

Orf8, one of the most puzzling genes in the SARS lineage of coronaviruses, marks a unique and striking difference in genome organization between SARS-CoV-2 and SARS-CoV-1. Here, using sequence comparisons, we unequivocally reveal the distant sequence similarities between SARS-CoV-2 Orf8 with its SARS-CoV-1 counterparts and the X4-like genes of coronaviruses, including its highly divergent “paralog” gene Orf7a, whose product is a potential immune antagonist of known structure. Supervised sequence space walks unravel identity levels that drop below 10% and yet exhibit subtle conservation patterns in this novel superfamily, characterized by an immunoglobulin-like beta sandwich topology. We document the high accuracy of the sequence space walk process in detail and characterize the subgroups of the superfamily in sequence space by systematic annotation of gene and taxon groups. While SARS-CoV-1 Orf7a and Orf8 genes are most similar to bat virus sequences, their SARS-CoV-2 counterparts are closer to pangolin virus homologs, reflecting the fine structure of conservation patterns within the SARS-CoV-2 genomes. The divergence between Orf7a and Orf8 is exceptionally idiosyncratic, since Orf7a is more constrained, whereas Orf8 is subject to rampant change, a peculiar feature that may be related to hitherto-unknown viral infection strategies. Despite their common origin, the Orf7a and Orf8 protein families exhibit different modes of evolutionary trajectories within the coronavirus lineage, which might be partly attributable to their complex interactions with the mammalian host cell, reflected by a multitude of functional associations of Orf8 in SARS-CoV-2 compared to a very small number of interactions discovered for Orf7a.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Metatranscriptomics analysis reveals a novel transcriptional and translational landscape during Middle East respiratory syndrome coronavirus infection

Among all RNA viruses, coronavirus RNA transcription is the most complex and involves a process termed “discontinuous transcription” that results in the production of a set of 3'-nested, co-terminal genomic and subgenomic RNAs during infection. While the expression of the classic canonical set of subgenomic RNAs depends on the recognition of a 6- to 7-nt transcription regulatory core sequence (TRS), here, we use deep sequence and metagenomics analysis strategies and show that the coronavirus transcriptome is even more vast and more complex than previously appreciated and involves the production of leader-containing transcripts that have canonical and noncanonical leader-body junctions. Moreover, by ribosome protection and proteomics analyses, we show that both positive- and negative-sense transcripts are translationally active. The data support the hypothesis that the coronavirus proteome is much vaster than previously noted in the literature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Antiviral Effect of Antimicrobial Peptoid TM9 and Murine Model of Respiratory Coronavirus Infection

New antiviral agents are essential to improving treatment and control of SARS-CoV-2 infections that can lead to the disease COVID-19. Antimicrobial peptoids are sequence-specific oligo-N-substituted glycine peptidomimetics that emulate the structure and function of natural antimicrobial peptides but are resistant to proteases. We demonstrate antiviral activity of a new peptoid (TM9) against the coronavirus, murine hepatitis virus (MHV), as a closely related model for the structure and antiviral susceptibility profile of SARS-CoV-2. This peptoid mimics the human cathelicidin LL-37, which has also been shown to have antimicrobial and antiviral activity. In this study, TM9 was effective against three murine coronavirus strains, demonstrating that the therapeutic window is large enough to allow the use of TM9 for treatment. All three isolates of MHV generated infection in mice after 15 min of exposure by aerosol using the Madison aerosol chamber, and all three viral strains could be isolated from the lungs throughout the 5-day observation period post-infection, with the peak titers on day 2. MHV-A59 and MHV-A59-GFP were also isolated from the liver, heart, spleen, olfactory bulbs, and brain. These data demonstrate that MHV serves as a valuable natural murine model of coronavirus pathogenesis in multiple organs, including the brain.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells

The main protease enzyme (Mpro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that Mpro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported. To enable work on Mpro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) Mpro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing Mpro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with Mpro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that requires conserved residues within the dimerization interface. Proteolytic activity is dispensable for dimerization and, uniquely, a C145A catalytically inactive mutant exhibits enhanced dimerization signal likely due to lower cytotoxicity. Mpro enzymes from multiple different coronaviruses also dimerize in this system, indicating mechanistic conservation. Interestingly, this dimerization biosensor also provides a quantitative read-out of inhibitor-facilitated dimerization. Covalent SARS2 Mpro inhibitors such as nirmatrelvir cause a 3- to 5-fold increase in luciferase activity. Together with corroborating structural, biophysical, and molecular dynamics experiments, our studies support a model in which covalent Mpro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.

SARS-CoV-2 main protease (Mpro/3CLpro)↗
DOE OSTI2023

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV)

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results—but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A 3.4-Å cryo-electron microscopy structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles

Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV-1 and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other sites, as well as different glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. Overall, this structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the coronavirus particles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparison of the Antiviral Activity of Remdesivir, Chloroquine, and Interferon-β as Single or Dual Agents Against the Human Beta-Coronavirus OC43

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-β, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-β exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-β plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-β alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-β plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Quantitative Fit Evaluation of N95 Filtering Facepiece Respirators and Coronavirus Inactivation Following Heat Treatment

Abstract Reuse of filtering facepiece respirators (FFRs, commonly referred to as N95s) normally meant for single use has become common in healthcare facilities due to shortages caused by the COVID-19 pandemic. Here, we report that murine hepatitis coronavirus initially seeded on FFR filter material is inactivated (6 order of magnitude reduction as measured by median tissue culture infective dose, TCID50) after dry heating at 75°C for 30 min. We also find that the quantitative fit of FFRs after heat treatment at this temperature, under dry conditions or at 90% relative humidity, is not affected by single or 10 heating cycles. Previous studies have reported that the filtration efficiency of FFRs is not negatively impacted by these heating conditions. These results suggest that thermal inactivation of coronaviruses is a potentially rapid and widely deployable method to reuse N95 FFRs in emergency situations where reusing FFRs is a necessity and broad-spectrum sterilization is unavailable. However, we also observe that a radiative heat source (e.g. an exposed heating element) results in rapid qualitative degradation of the FFR. Finally, we discuss differences in the results reported here and other recent studies investigating heat as a means to recycle FFRs. These differences suggest that while our repeated decontamination cycles do not affect FFR fit, overall wear time and the number of donning/doffing cycles are important factors that likely degrade FFR fit and must be investigated further.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure and epitope of a neutralizing monoclonal antibody that targets the stem helix of β coronaviruses

Monoclonal antibodies that retain neutralizing activity against multiple coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV), and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1 Å resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743–749, which interacts with the N-terminal end of the SH helix in the S post-fusion conformation. The conformation of 1249A8-bound SH is distinct from the SH conformation observed in the post-fusion SARS-CoV-2 S structure, suggesting 1249A8 disrupts the secondary structure and refolding events required for CoV post-fusion S to initiate membrane fusion and ultimately infection. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Dysregulation of lung epithelial cell homeostasis and immunity contributes to Middle East respiratory syndrome coronavirus disease severity

Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.

MERS-CoV↗
DOE OSTI2021

Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. Finally, as vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Abstract Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Integrative illustration for coronavirus outreach

Two illustrations integrate current knowledge about severe acute respiratory syndrome (SARS) coronaviruses and their life cycle. They have been widely used in education and outreach through free distribution as part of a coronavirus-related resource at Protein Data Bank (PDB)-101, the education portal of the RCSB PDB. Scientific sources for creation of the illustrations and examples of dissemination and response are presented.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

High Contagiousness and Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2

Severe acute respiratory syndrome coronavirus 2 is the causative agent of the ongoing coronavirus disease pandemic. Initial estimates of the early dynamics of the outbreak in Wuhan, China, suggested a doubling time of the number of infected persons of 6–7 days and a basic reproductive number (R 0 ) of 2.2–2.7. We collected extensive individual case reports across China and estimated key epidemiologic parameters, including the incubation period (4.2 days). We then designed 2 mathematical modeling approaches to infer the outbreak dynamics in Wuhan by using high-resolution domestic travel and infection data. Results show that the doubling time early in the epidemic in Wuhan was 2.3–3.3 days. Assuming a serial interval of 6–9 days, we calculated a median R0 value of 5.7 (95% CI 3.8–8.9). Here, we further show that active surveillance, contact tracing, quarantine, and early strong social distancing efforts are needed to stop transmission of the virus.

60 APPLIED LIFE SCIENCES↗