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1.
Gene ; 814: 146134, 2022 Mar 10.
Article in English | MEDLINE | ID: covidwho-1587736

ABSTRACT

Coronavirus-related Severe Acute Respiratory Syndrome (SARS-CoV) in 2002/2003, Middle-East Respiratory Syndrome (MERS-CoV) in 2012/2013, and especially the current 2019/2021 Severe Acute Respiratory Syndrome-2 (SARS-CoV-2) affected negatively the national health systems' endurance worldwide. SARS-Cov-2 virus belongs to lineage b of beta-CoVs demonstrating a strong phylogenetic similarity with BatCoVRaTG13 type. Spike (S) glycoprotein projections -consisting of two subunits S1/S2- provide a unique crown-like formation (corona) on virion's surface. Concerning their functional role, S1 represents the main receptor-binding domain (RBD), whereas S2 is involved in the virus-cell membrane fusion mechanism. On Nov 26th 2021, WHO designated the new SARS-CoV-2 strain - named Omicron, from letter ''όµÎ¹κρον'' in the Greek alphabet - as a variant of concern (B.1.1529 variant). Potentially this new variant is associated with high transmissibility leading to elevated infectivity and probably increased re-infection rates. Its impact on morbidity/mortality remains under investigation. In the current paper, analyzing and comparing the alterations of SARS-CoV-2 S RNA sequences in the defined variants (Alpha to Omicron), we observed some interesting findings regarding the S1-RBD/S2 mutation/deletion equilibrium that maybe affect and modify its activity.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , COVID-19/transmission , Genome, Viral , Humans , Mutation , RNA, Viral , SARS-CoV-2/pathogenicity , Sequence Deletion
3.
Sci Rep ; 11(1): 22372, 2021 11 16.
Article in English | MEDLINE | ID: covidwho-1521767

ABSTRACT

More than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.


Subject(s)
Base Sequence , Fabry Disease/genetics , Mutation, Missense , Polymorphism, Single Nucleotide , Sequence Deletion , alpha-Galactosidase/genetics , Adult , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged
4.
Transbound Emerg Dis ; 68(6): 3075-3082, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1526429

ABSTRACT

The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.


Subject(s)
COVID-19 , SARS-CoV-2 , Sequence Deletion , COVID-19/virology , Disease Outbreaks , Genome, Viral , Humans , Phylogeny , SARS-CoV-2/genetics , Uruguay/epidemiology
5.
Exp Biol Med (Maywood) ; 246(21): 2332-2337, 2021 11.
Article in English | MEDLINE | ID: covidwho-1507096

ABSTRACT

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence/genetics , Amino Acid Substitution/genetics , Brazil/epidemiology , Humans , Polymorphism, Genetic/genetics , Sequence Deletion/genetics
6.
Viruses ; 13(9)2021 09 18.
Article in English | MEDLINE | ID: covidwho-1430978

ABSTRACT

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.


Subject(s)
COVID-19/virology , Genes, Viral , SARS-CoV-2/genetics , Sequence Deletion , Genetic Variation , Humans , Nanopore Sequencing , Open Reading Frames , Sequence Analysis, RNA , Whole Genome Sequencing
7.
Virus Genes ; 57(6): 556-560, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1375671

ABSTRACT

SARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged independently in at least six lineages of the virus (namely, B.1.1.7, B.1.1.298, B.1.160, B.1.177, B.1.258, B.1.375). We analyzed SARS-CoV-2 samples collected from various regions of Slovakia between November and December 2020 that were presumed to contain B.1.1.7 variant due to drop-out of the Spike gene target in an RT-qPCR test caused by this deletion. Sequencing of these samples revealed that although in some cases the samples were indeed confirmed as B.1.1.7, a substantial fraction of samples contained another ∆H69/∆V70 carrying mutant belonging to the lineage B.1.258, which has been circulating in Central Europe since August 2020, long before the import of B.1.1.7. Phylogenetic analysis shows that the early sublineage of B.1.258 acquired the N439K substitution in the receptor-binding domain (RBD) of the Spike protein and, later on, also the deletion ∆H69/∆V70 in the Spike N-terminal domain (NTD). This variant was particularly common in several European countries including the Czech Republic and Slovakia but has been quickly replaced by B.1.1.7 early in 2021.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , Phylogeny , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sequence Deletion , Spike Glycoprotein, Coronavirus/genetics , Europe/epidemiology , Humans , SARS-CoV-2/classification , Time Factors
8.
Cell ; 184(11): 2939-2954.e9, 2021 05 27.
Article in English | MEDLINE | ID: covidwho-1343152

ABSTRACT

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations, including P.1 from Brazil, B.1.351 from South Africa, and B.1.1.7 from the UK (12, 10, and 9 changes in the spike, respectively). All have mutations in the ACE2 binding site, with P.1 and B.1.351 having a virtually identical triplet (E484K, K417N/T, and N501Y), which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine-induced antibody responses than B.1.351, suggesting that changes outside the receptor-binding domain (RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Binding Sites , COVID-19/therapy , COVID-19/virology , Cell Line , Humans , Immune Evasion , Immunization, Passive , Mutation , Protein Binding , Protein Domains , SARS-CoV-2/genetics , Sequence Deletion , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Vaccines/immunology
9.
Ann Saudi Med ; 41(3): 141-146, 2021.
Article in English | MEDLINE | ID: covidwho-1261414

ABSTRACT

BACKGROUND: Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism may play a role in the pathogenesis of coronavirus-19 disease (COVID-19). OBJECTIVES: Investigate the relationship between ACE I/D polymorphism and the clinical severity of COVID-19. DESIGN: Prospective cohort study. SETTING: Tertiary care hospital. PATIENTS AND METHODS: The study included COVID-19 patients with asymptomatic, mild, and severe disease with clinical data and whole blood samples collected from 1 April 2020 to 1 July 2020. ACE I/D genotypes were determined by polymerase chain reaction and agarose gel electrophoresis. MAIN OUTCOME MEASURE: ACE DD, DI and II genotypes frequencies. SAMPLE SIZE: 90 cases, 30 in each disease severity group. RESULTS: Age and the frequency of general comorbidity increased significantly from the asymptomatic disease group to the severe disease group. Advanced age, diabetes mellitus and presence of ischemic heart disease were independent risk factors for severe COVID-19 [OR and 95 % CI: 1.052 (1.021-1.083), 5.204 (1.006-26.892) and 5.922 (1.109-31.633), respectively]. The ACE II genotype was the dominant genotype (50%) in asymptomatic patients, while the DD genotype was the dominant genotype (63.3 %) in severe disease. The ACE II geno-type was protective against severe COVID-19 [OR and 95% CI: .323 (.112-.929)]. All nine patients (8.9%) who died had severe disease. CONCLUSIONS: The clinical severity of COVID-19 infection may be associated with the ACE I/D polymorphism. LIMITATIONS: Small sample size and single center. CONFLICT OF INTEREST: None.


Subject(s)
COVID-19/genetics , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Severity of Illness Index , Adult , Aged , Base Sequence , COVID-19/diagnosis , Female , Follow-Up Studies , Genetic Markers , Genotype , Genotyping Techniques , Humans , Male , Middle Aged , Mutagenesis, Insertional , Prospective Studies , Sequence Deletion
10.
Bioessays ; 43(7): e2100015, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1245362

ABSTRACT

RaTG13, MP789, and RmYN02 are the strains closest to SARS-CoV-2, and their existence came to light only after the start of the pandemic. Their genomes have been used to support a natural origin of SARS-CoV-2 but after a close examination all of them exhibit several issues. We specifically address the presence in RmYN02 and closely related RacCSxxx strains of a claimed natural PAA/PVA amino acid insertion at the S1/S2 junction of their spike protein at the same position where the PRRA insertion in SARS-CoV-2 has created a polybasic furin cleavage site. We show that RmYN02/RacCSxxx instead of the claimed insertion carry a 6-nucleotide deletion in the region and that the 12-nucleotide insertion in SARS-CoV-2 remains unique among Sarbecoviruses. Also, our analysis of RaTG13 and RmYN02's metagenomic datasets found unexpected reads which could indicate possible contamination. Because of their importance to inferring SARS-CoV-2's origin, we call for a careful reevaluation of RaTG13, MP789 and RmYN02 sequencing records and assembly methods.


Subject(s)
COVID-19/virology , Chiroptera/virology , Pangolins/virology , SARS Virus/genetics , SARS-CoV-2/classification , SARS-CoV-2/genetics , Uncertainty , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/epidemiology , COVID-19/transmission , Datasets as Topic , Furin/metabolism , Humans , Pandemics , Phylogeny , SARS Virus/classification , SARS Virus/isolation & purification , SARS-CoV-2/isolation & purification , Sequence Deletion/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Zoonoses/transmission , Viral Zoonoses/virology
11.
mBio ; 12(3)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1225698

ABSTRACT

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.


Subject(s)
Bronchi/virology , Furin/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Transcriptome , Animals , Bronchi/cytology , Cells, Cultured , Chlorocebus aethiops , Epithelial Cells/virology , Humans , RNA-Seq , Respiratory Mucosa/cytology , Sequence Deletion , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
12.
Biochim Biophys Acta Mol Basis Dis ; 1867(8): 166154, 2021 08 01.
Article in English | MEDLINE | ID: covidwho-1209165

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome-2 (SARS-CoV-2) exhibits a broad spectrum of clinical manifestations. Despite the fact that SARS-CoV-2 has slower evolutionary rate than other coronaviruses, different mutational hotspots have been identified along the SARS-CoV-2 genome. METHODS: We performed whole-genome high throughput sequencing on isolates from 50 Egyptian patients to see if the variation in clinical symptoms was related to mutations in the SARS-CoV-2 genome. Then, we investigated the relationship between the observed mutations and the clinical characteristics of the patients. RESULTS: Among the 36 most common mutations, we found two frameshift deletions linked to an increased risk of shortness of breath, a V6 deletion in the spike glycoprotein's signal peptide region linked to an increased risk of fever, longer fever duration and nasal congestion, and L3606-nsp6 deletion linked to a higher prevalence of cough and conjunctival congestion. S5398L nsp13-helicase was linked to an increased risk of fever duration and progression. The most common mutations (241, 3037, 14,408, and 23,403) were not linked to clinical variability. However, the E3909G-nsp7 variant was more common in children (2-13 years old) and was associated with a shorter duration of symptoms. The duration of fever was significantly reduced with E1363D-nsp3 and E3073A-nsp4. CONCLUSIONS: The most common mutations, D614G/spike-glycoprotein and P4715L/RNA-dependent-RNA-polymerase, were linked to transmissibility regardless of symptom variability. E3909G-nsp7 could explain why children recover so quickly. Nsp6-L3606fs, spike-glycoprotein-V6fs, and nsp13-S5398L variants may be linked to clinical symptom worsening. These variations related to host-virus interactions might open new therapeutic avenues for symptom relief and disease containment.


Subject(s)
COVID-19/virology , Mutation , SARS-CoV-2/genetics , Adolescent , Adult , COVID-19/epidemiology , COVID-19/pathology , Child , Child, Preschool , Egypt/epidemiology , Female , Frameshift Mutation , Genome, Viral , Humans , Male , Middle Aged , Sequence Deletion , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics , Young Adult
13.
J Med Virol ; 93(3): 1732-1738, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196496

ABSTRACT

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Inactivated/immunology , Animals , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/genetics , Humans , Mice , Mice, Inbred BALB C , Phosphoproteins/genetics , Phosphoproteins/immunology , Rabbits , SARS-CoV-2/genetics , Sequence Deletion/genetics , Sequence Deletion/immunology , Spike Glycoprotein, Coronavirus/genetics
15.
Int J Infect Dis ; 105: 653-655, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1120241

ABSTRACT

OBJECTIVES: To determine the impact of the 32 bp deletion (CCR5Δ32) in the coding region of the C-C chemokine receptor 5 (CCR5) on the risk of contracting SARS-CoV-2 and severe COVID-19. METHODS: Cross-sectional study among stem cell donors registered with DKMS in Germany. Genetic information was linked to self-reported COVID-19 outcome data. Multivariable regression models were fitted to determine the risk of contracting SARS-CoV-2, severe respiratory tract infection (RTI) and respiratory hospitalization. RESULTS: CCR5 information was available for 110 544 donors who were tested at least once for SARS-CoV-2; 5536 reported SARS-CoV-2 infection. For 4758 donors, the COVID-19 disease course was fully evaluable; 498 reported no symptoms, 1227 described symptoms of severe respiratory tract infection, of whom 164 required respiratory hospitalization. The distribution of CCR5Δ32 genotypes (homozygous wild-type vs CCR5Δ32 present) did not differ significantly between individuals with or without SARS-CoV-2 infection (odds ratio (OR) 0.96, 95% CI 0.89-1.03, P = 0.21) nor between individuals with or without symptomatic infection (OR 1.13, 95% CI 0.88-1.45, P = 0.32), severe RTI (OR 1.03, 95% CI 0.88-1.22, P = 0.68) or respiratory hospitalization (OR 1.16, 95% CI 0.79-1.69, P = 0.45). CONCLUSIONS: Our data implicate that CCR5Δ32 mutations do not determine the risk of SARS-CoV-2 infections nor the disease course. TRIAL REGISTRATION: We registered the study with the German Center for Infection Research (https://dzif.clinicalsite.org/de/cat/2099/trial/4361).


Subject(s)
COVID-19/genetics , Receptors, CCR5/genetics , Sequence Deletion , Adolescent , Adult , COVID-19/physiopathology , Cross-Sectional Studies , Disease Progression , Female , Genotype , Germany , Hospitalization , Humans , Male , Middle Aged , Odds Ratio , Risk Factors , SARS-CoV-2 , Self Report , Young Adult
16.
EMBO J ; 40(6): e105543, 2021 03 15.
Article in English | MEDLINE | ID: covidwho-1084490

ABSTRACT

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.


Subject(s)
Autophagy-Related Proteins/genetics , Influenza A virus/pathogenicity , Microtubule-Associated Proteins/metabolism , Orthomyxoviridae Infections/genetics , Sequence Deletion , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Animals , Autophagy , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Chick Embryo , Cytokines/metabolism , Dogs , Madin Darby Canine Kidney Cells , Mice , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/mortality , Protein Domains , Virus Replication
17.
Science ; 371(6534): 1139-1142, 2021 03 12.
Article in English | MEDLINE | ID: covidwho-1063045

ABSTRACT

Zoonotic pandemics, such as that caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can follow the spillover of animal viruses into highly susceptible human populations. The descendants of these viruses have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses. In the spike glycoprotein, we found that recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/genetics , COVID-19/virology , Immune Evasion , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Amino Acid Substitution , Antigens, Viral/chemistry , Evolution, Molecular , Genetic Drift , Humans , Protein Conformation , Sequence Deletion , Spike Glycoprotein, Coronavirus/chemistry
18.
Genes (Basel) ; 12(2)2021 01 29.
Article in English | MEDLINE | ID: covidwho-1055035

ABSTRACT

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , COVID-19/metabolism , COVID-19/virology , Cell Line , Chlorocebus aethiops , Computer Simulation , Humans , Pandemics , Protein Binding , Protein Domains , Sequence Deletion , Vero Cells
19.
Euro Surveill ; 26(3)2021 01.
Article in English | MEDLINE | ID: covidwho-1041125

ABSTRACT

We report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC.


Subject(s)
Base Sequence , COVID-19/epidemiology , Genome, Viral , SARS-CoV-2/genetics , Sequence Deletion/genetics , Spike Glycoprotein, Coronavirus/genetics , France/epidemiology , Humans
20.
Emerg Microbes Infect ; 9(1): 2361-2367, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-894519

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39 µg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Envelope Proteins/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/epidemiology , Female , Humans , Immunization , Male , Mice , Mice, Inbred BALB C , Pandemics , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Sequence Deletion , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/immunology , Virulence
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