Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles.

Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics.

IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

[Exosomes' role in intercellular interactions in different variants of lung injury in fatal cases of COVID-19]. / Rol' ekzosom v mezhkletochnykh vzaimodeistviyakh pri razlichnykh variantakh porazheniya legkikh v letal'nykh sluchayakh COVID-19.

BACKGROUND: Extracellular vesicles are surrounded by a phospholipid bilayer, carrying various active biomolecules and participating in many physiological and pathological processes, including infectious ones. OBJECTIVE: To research the role of exosomes in intercellular interactions in the pathogenesis of various types of lung damage in fatal cases of COVID-19. MATERIAL AND METHODS: We conducted a clinical and morphological analysis of 118 fatal cases caused by coronavirus infection in Moscow. We selected 32 cases with morphological signs of various types of lung lesions for immunohistochemical reaction (IHC) with antibodies against tetraspanin proteins (CD63, CD81), which are involved in the assembly of exosomes, as well as with antibodies against viral proteins: nucleocapsid and spike protein. We determined the main producing cells of extracellular vesicles and cells containing viral proteins, carried out their comparison and quantitative analysis. RESULTS: IHC reaction with antibodies against CD63 showed cytoplasmic granular uniform and subapical staining of cells, as well as granular extracellular staining. We determined similar staining using antibodies against viral proteins. Extracellular vesicles were found in the same cells as viral proteins. The main producing cells of vesicles and cells containing viral proteins were found to be macrophages, type II pneumocytes, and endothelial cells. CONCLUSION: Taking into account the results of the literature, the localization of viral proteins and extracellular vesicles in the same cells indicates the key role of vesicles in the pathogenesis of various forms of lung damage by the SARS-CoV-2 virus, in the dissemination of the pathogen in the organism, which leads to interaction with the adaptive immune system and the formation of immunity.

The sliding motility of the bacilliform virions of Influenza A viruses.

Influenza A virus (IAV) infection relies on the action of the hemagglutinin (HA) and neuraminidase (NA) membrane proteins. The HA ligands anchor the IAV virion to the cell's surface by binding the sialic acid (SA) present on the host's receptors while NA is an enzyme capable of cleaving the SA from the extracellular environment. It is believed that the activity of NA ligands increases the motility of the virions favoring the propagation of the infection. In this work, we develop a numerical framework to study the dynamics of a virion moving across the cell surface for timescales much bigger than the typical ligand-receptor reaction times. We find that the rates controlling the ligand-receptor reactions and the maximal distance at which a pair of ligand-receptor molecules can interact greatly affect the motility of the virions. We also report on how different ways of organizing the two types of ligands on the virions' surface result in different types of motion that we rationalize using general principles. In particular, we show how the emerging motility of the virion is less sensitive to the rate controlling the enzymatic activity when NA ligands are clustered.

Sixteen capillary electrophoresis applications for viral vaccine analysis.

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.

Application of spectral library prediction for parallel reaction monitoring of viral peptides.

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.

Proteomics in the COVID-19 Battlefield: First Semester Check-Up.

Proteomics offers a wide collection of methodologies to study biological systems at the finest granularity. Faced with COVID-19, the most worrying pandemic in a century, proteomics researchers have made significant progress in understanding how the causative virus hijacks the host's cellular machinery and multiplies exponentially, how the disease can be diagnosed, and how it develops, as well as its severity predicted. Numerous cellular targets of potential interest for the development of new antiviral drugs have been documented. Here, the most striking results obtained in the proteomics field over this first semester of the pandemic are presented. The molecular machinery of SARS-CoV-2 is much more complex than initially believed, as many post-translational modifications can occur, leading to a myriad of proteoforms and a broad heterogeneity of viral particles. The interplay of protein-protein interactions, protein abundances, and post-translational modifications has yet to be fully documented to provide a full picture of this intriguing but lethal biological threat. Proteomics has the potential to provide rapid detection of the SARS-CoV-2 virus by mass spectrometry proteotyping, and to further increase the knowledge of severe respiratory syndrome COVID-19 and its long-term health consequences.

Composition of HBsAg is predictive of HBsAg loss during treatment in patients with HBeAg-positive chronic hepatitis B.

BACKGROUND & AIMS: During treatment of chronic HBV infections, loss or seroconversion of the HBV surface antigen (HBsAg) is considered a functional cure. HBsAg consists of the large (LHBs), middle (MHBs), and small surface protein (SHBs) and their relative proportions correlate strongly with disease stage. Our aim was to assess the association between HBsAg composition and functional cure during treatment. METHODS: A total of 83 patients were retrospectively analyzed. HBsAg loss was achieved by 17/64 patients during nucleos(t)ide analogue (NA) treatment and 3/19 patients following treatment with pegylated interferon-alfa2a (PEG-IFN) for 48 weeks. Sixty-three patients without HBsAg loss were matched as controls. LHBs, MHBs and SHBs were quantified in sera collected before and during treatment. RESULTS: Before treatment, median MHBs levels were significantly lower in patients with subsequent HBsAg loss than in those without (p = 0.005). During treatment, MHBs and LHBs proportions showed a fast decline in patients with HBsAg loss, but not in patients with HBV e antigen seroconversion only or patients without serologic response. MHBs became undetectable by month 6 of NA treatment in all patients with HBsAg loss, which occurred on average 12.8 ± 8.7 (0-52) months before loss of total HBsAg. Receiver-operating characteristic analyses revealed that the proportion of MHBs was the best early predictor of HBsAg loss before NA treatment (AUC = 0.726, p = 0.019). In patients achieving HBsAg loss with PEG-IFN, the proportions of MHBs and LHBs showed similar kinetics. CONCLUSION: Quantification of HBsAg proteins shows promise as a novel tool to predict early treatment response. These assessments may help optimize individual antiviral treatment, increasing the rates of functional cure in chronically HBV-infected patients. LAY SUMMARY: The hepatitis B surface antigen (HBsAg) is a key serum marker for viral replication. Loss of HBsAg is considered stable remission, which can be achieved with antiviral treatments. We have investigated whether the ratios of the different components of HBsAg, namely the large (LHBs) and medium (MHBs) HBsAg during different treatments are associated with the occurrence of HBsAg loss. We found that LHBs and MHBs decrease earlier than total HBsAg before HBsAg loss and we propose LHBs and MHBs as promising novel biomarker candidates for predicting cure of HBV infection.

ICTV Virus Taxonomy Profile: Portogloboviridae.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae.

MASS SPECTROMETRY IN VIROLOGICAL SCIENCES.

Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro- and eukaryotes and viruses, which delivered "a piece of bad news wrapped in protein" as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro- and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Design of Neuraminidase-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis.

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.

Rhomboidal Pt(II) Metallacycle-Based Hybrid Viral Nanoparticles for Cell Imaging.

Supramolecular coordination complexes (SCCs) have emerged as anticancer agents. Tracking the movement of these metallic anticancer agents plays an important role in the field of biomedicines. Herein, we describe a method for tracking the movement of a rhomboidal Pt(II) metallacycle agent using the quantum dots encapsidation in vitro self-assembly system of viral proteins. When incubated with living Vero cells, self-assembly of hybrid viral nanoparticles were employed for simultaneous cell imaging and visual transmission of the Pt(II) metallacycle agent. Considering these results, we believe that the multifunctional biomaterials consisting of a supramolecular coordination complex and quantum dots provide a new alternative for probing of the delivery of Pt(II) metallacycle drugs.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Integrated analysis of viral miRNAs, mRNA and protein in the caudal fin cells of C. auratus gibelio with cyprinid herpesvirus 2 infection.

Cyprinid herpesvirus 2 (CyHV-2), a member of the genus Cyprinivirus in the family Alloherpesviridae, has attracted worldwide attention because it causes severe disease and high mortality in crucian carp and goldfish. In this study, we focus on mRNA, protein and viral miRNA expression profiles in C. auratus gibelio caudal fin (GiCF) cells infected with CyHV-2, using high-throughput sequence techniques and TMT-labelled analyses. The results revealed that 156 virus genes were differentially expressed during the infection. Among these differentially expressed genes, 7 viral genes were significantly up-regulated and 28 were significantly down-regulated at 96 hpi (hours post-infection) vs 48 hpi. Besides, a total of 78 viral proteins, including a large number of membrane proteins and capsid proteins associated with the viral assembly, were successfully detected by using proteome analysis. Furthermore, a total of 225,143,474 raw reads were generated from cDNA library of CyHV-2-infected GiCF cells using high-throughput sequencing technology. Following annotation and secondary structure prediction, 10 viral miRNAs were found as significantly modulated in CyHV-2-infected GiCF cells (2 down-regulated and 8 up-regulated). Finally, the CyHV-2 genes (orf19, orf23, orf118, orf121, orf127) targeted by the viral miRNA CyHV-2-KT-635 identified in this study, were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the regulation of CyHV-2-KT-635 on orf121 protein expression was verified by western blotting assay. Taken together, this study provides a valuable basis for further research on the expression of virus genes during CyHV-2 replication and the molecular mechanisms by which miRNA may regulate CyHV-2 virus.

Clinical testing for COVID-19.

As the novel coronavirus severe acute respiratory syndrome coronavirus 2 caused coronavirus disease 2019 cases in the United States, the initial test was developed and performed at the Centers for Disease Control and Prevention. As the number of cases increased, the demand for tests multiplied, leading the Centers for Disease Control and Prevention to use the Emergency Utilization Authorization to allow clinical and commercial laboratories to develop tests to detect the presence of the virus. Many nucleic acid tests based on RT-PCR were developed, each with different techniques, specifications, and turnaround time. As the illnesses turned into a pandemic, testing became more crucial. The test supply became inadequate to meet the need and so it had to be prioritized according to guidance. For surveillance, the need for serologic tests emerged. Here, we review the timeline of test development, the turnaround times, and the various approved tests, and compare them as regards the genes they detect. We concentrate on the point-of-care tests and discuss the basis for new serologic tests. We discuss the testing guidance for prioritization and their application in a hospital setting.

Shortlisting SARS-CoV-2 Peptides for Targeted Studies from Experimental Data-Dependent Acquisition Tandem Mass Spectrometry Data.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial tool for fighting the COVID-19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS-CoV-2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data-dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass-spectrometry method development and diagnostic of the new SARS-CoV-2 is proposed and the best candidates are commented.