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1.
Sci Rep ; 11(1): 22164, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1514425

ABSTRACT

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.


Subject(s)
Influenza A virus/immunology , Influenza Vaccines/therapeutic use , Interferon Type I/immunology , Orthomyxoviridae Infections/prevention & control , Vaccines, Attenuated/therapeutic use , Viral Nonstructural Proteins/immunology , Adaptive Immunity , Animals , COVID-19/immunology , COVID-19/prevention & control , Chickens , Gene Deletion , Humans , Influenza A virus/genetics , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/prevention & control , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral Nonstructural Proteins/genetics
2.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1481965

ABSTRACT

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.


Subject(s)
Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , RNA, Viral/administration & dosage , Replicon , Viral Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Defective Viruses/genetics , Defective Viruses/immunology , Female , Gene Deletion , Genes, env , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral/genetics , RNA, Viral/immunology , Vaccines, DNA , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics , Virulence/immunology
3.
Front Immunol ; 11: 604759, 2020.
Article in English | MEDLINE | ID: covidwho-1389169

ABSTRACT

Objective: To first describe and estimate the potential pathogenic role of Ig4 autoantibodies in complement-mediated thrombotic microangiopathy (TMA) in a patient with IgG4-related disease (IgG4-RD). Methods: This study is a case report presenting a retrospective review of the patient's medical chart. Plasma complement C3 and C4 levels, immunoglobulin isotypes and subclasses were determined by nephelometry, the complement pathways' activity (CH50, AP50, MBL) using WIESLAB® Complement System assays. Human complement factor H levels, anti-complement factor H auto-antibodies were analyzed by ELISA, using HRP-labeled secondary antibodies specific for human IgG, IgG4, and IgA, respectively. Genetic analyses were performed by exome sequencing of 14 gens implicated in complement disorders, as well as multiplex ligation-dependent probe amplification looking specifically for CFH, CFHR1-2-3, and 5. Results: Our brief report presents the first case of IgG4-RD with complement-mediated TMA originating from both pathogenic CFHR 1 and CFHR 4 genes deletions, and inhibitory anti-complement factor H autoantibodies of the IgG4 subclass. Remission was achieved with plasmaphereses, corticosteroids, and cyclophosphamide. Following remission, the patient was diagnosed with lymphocytic meningitis and SARS-CoV-2 pneumonia with an uneventful recovery. Conclusion: IgG4-RD can be associated with pathogenic IgG4 autoantibodies. Genetic predisposition such as CFHR1 and CFHR4 gene deletions enhance the susceptibility to the formation of inhibitory anti-Factor H IgG4 antibodies.


Subject(s)
Apolipoproteins/genetics , Atypical Hemolytic Uremic Syndrome/genetics , Autoantibodies/immunology , Complement C3b Inactivator Proteins/genetics , Complement Factor H/immunology , Immunoglobulin G4-Related Disease/genetics , Atypical Hemolytic Uremic Syndrome/immunology , Atypical Hemolytic Uremic Syndrome/pathology , Female , Gene Deletion , Genetic Predisposition to Disease/genetics , Humans , Immunoglobulin G/immunology , Immunoglobulin G4-Related Disease/immunology , Immunoglobulin G4-Related Disease/pathology , Middle Aged , Thrombotic Microangiopathies/immunology , Thrombotic Microangiopathies/pathology
4.
Int J Infect Dis ; 108: 137-144, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1272471

ABSTRACT

OBJECTIVES: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). METHODS: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. RESULTS: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. CONCLUSIONS: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.


Subject(s)
Malaria, Falciparum , Parasites , Animals , Antigens, Protozoan/genetics , Diagnostic Tests, Routine , Gene Deletion , Humans , Ireland/epidemiology , Malaria, Falciparum/diagnosis , Malaria, Falciparum/epidemiology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , United Kingdom/epidemiology
5.
J Infect Dis ; 221(4): 534-543, 2020 02 03.
Article in English | MEDLINE | ID: covidwho-1207300

ABSTRACT

BACKGROUND: The safety and immunogenicity of live respiratory syncytial virus (RSV) candidate vaccine, LID/ΔM2-2/1030s, with deletion of RSV ribonucleic acid synthesis regulatory protein M2-2 and genetically stabilized temperature-sensitivity mutation 1030s in the RSV polymerase protein was evaluated in RSV-seronegative children. METHODS: Respiratory syncytial virus-seronegative children ages 6-24 months received 1 intranasal dose of 105 plaque-forming units (PFU) of LID/ΔM2-2/1030s (n = 21) or placebo (n = 11). The RSV serum antibodies, vaccine shedding, and reactogenicity were assessed. During the following RSV season, medically attended acute respiratory illness (MAARI) and pre- and postsurveillance serum antibody titers were monitored. RESULTS: Eighty-five percent of vaccinees shed LID/ΔM2-2/1030s vaccine (median peak nasal wash titers: 3.1 log10 PFU/mL by immunoplaque assay; 5.1 log10 copies/mL by reverse-transcription quantitative polymerase chain reaction) and had ≥4-fold rise in serum-neutralizing antibodies. Respiratory symptoms and fever were common (60% vaccinees and 27% placebo recipients). One vaccinee had grade 2 wheezing with rhinovirus but without concurrent LID/ΔM2-2/1030s shedding. Five of 19 vaccinees had ≥4-fold increases in antibody titers postsurveillance without RSV-MAARI, indicating anamnestic responses without significant illness after infection with community-acquired RSV. CONCLUSIONS: LID/ΔM2-2/1030s had excellent infectivity without evidence of genetic instability, induced durable immunity, and primed for anamnestic antibody responses, making it an attractive candidate for further evaluation.


Subject(s)
Gene Deletion , RNA-Dependent RNA Polymerase/genetics , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/immunology , Vaccination , Viral Proteins/genetics , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Body Temperature , Double-Blind Method , Female , Humans , Immunogenicity, Vaccine , Infant , Male , Point Mutation , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus Vaccines/adverse effects , Respiratory Syncytial Virus, Human/genetics , Vaccines, Attenuated , Virus Replication/genetics
6.
PLoS One ; 16(3): e0248371, 2021.
Article in English | MEDLINE | ID: covidwho-1147457

ABSTRACT

Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.


Subject(s)
Genetic Variation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Aged, 80 and over , COVID-19/pathology , COVID-19/virology , Female , Gene Deletion , Humans , Male , Middle Aged , Mutation, Missense , Oropharynx/virology , Pakistan , Phylogeny , RNA, Viral/chemistry , RNA, Viral/isolation & purification , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Whole Genome Sequencing , Young Adult
7.
Biochem Biophys Res Commun ; 553: 25-29, 2021 05 14.
Article in English | MEDLINE | ID: covidwho-1147359

ABSTRACT

The current COVID-19 pandemic is caused by infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A sex-bias has been observed, with increased susceptibility and mortality in male compared to female patients. The gene for the SARS-CoV-2 receptor ACE2 is located on the X chromosome. We previously generated TP53 mutant pigs that exhibit a sex-specific patho-phenotype due to altered regulation of numerous X chromosome genes. In this study, we explored the effect of p53 deficiency on ACE2 expression in pigs. First, we identified the p53 binding site in the ACE2 promoter and could show its regulatory effect on ACE2 expression by luciferase assay in porcine primary kidney fibroblast cells. Later, quantitative PCR and western blot showed tissue- and gender-specific expression changes of ACE2 and its truncated isoform in p53-deficient pigs. We believe these findings will broaden the knowledge on ACE2 regulation and COVID-19 susceptibility.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Gene Expression Regulation , Organ Specificity , Sex Characteristics , Sus scrofa/metabolism , Tumor Suppressor Protein p53/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Base Sequence , Binding Sites , COVID-19/metabolism , COVID-19/virology , Disease Models, Animal , Female , Fibroblasts , Gene Deletion , Male , Promoter Regions, Genetic/genetics , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , X Chromosome/genetics
8.
Biochem Biophys Res Commun ; 550: 8-14, 2021 04 23.
Article in English | MEDLINE | ID: covidwho-1101113

ABSTRACT

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.


Subject(s)
COVID-19/transmission , COVID-19/virology , Epidemiological Monitoring , Gene Deletion , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics , COVID-19/epidemiology , Codon, Nonsense , Codon, Terminator/genetics , Evolution, Molecular , Genes, Viral/genetics , Humans , Phylogeny , SARS-CoV-2/pathogenicity , Selection, Genetic , Time Factors , United Kingdom/epidemiology
9.
Cell Host Microbe ; 29(3): 489-502.e8, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1064930

ABSTRACT

The SARS-CoV-2 virus, the causative agent of COVID-19, is undergoing constant mutation. Here, we utilized an integrative approach combining epidemiology, virus genome sequencing, clinical phenotyping, and experimental validation to locate mutations of clinical importance. We identified 35 recurrent variants, some of which are associated with clinical phenotypes related to severity. One variant, containing a deletion in the Nsp1-coding region (Δ500-532), was found in more than 20% of our sequenced samples and associates with higher RT-PCR cycle thresholds and lower serum IFN-ß levels of infected patients. Deletion variants in this locus were found in 37 countries worldwide, and viruses isolated from clinical samples or engineered by reverse genetics with related deletions in Nsp1 also induce lower IFN-ß responses in infected Calu-3 cells. Taken together, our virologic surveillance characterizes recurrent genetic diversity and identified mutations in Nsp1 of biological and clinical importance, which collectively may aid molecular diagnostics and drug design.


Subject(s)
COVID-19/immunology , COVID-19/virology , Interferon Type I/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Viral Nonstructural Proteins/genetics , A549 Cells , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Base Sequence , COVID-19/blood , Cell Line , Child , Child, Preschool , Chlorocebus aethiops , Female , Gene Deletion , Genomics , HEK293 Cells , Humans , Infant , Interferon Type I/blood , Interferon-beta/blood , Interferon-beta/metabolism , Male , Middle Aged , Molecular Epidemiology , Reverse Genetics , Vero Cells , Viral Nonstructural Proteins/immunology , Young Adult
10.
Emerg Microbes Infect ; 9(1): 2685-2696, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-933805

ABSTRACT

The newly emerged betacoronavirus, SARS-CoV-2, causes the COVID-19 pandemic since December 2019 with more than 35 million laboratory confirmed human infections and over one million deaths within nine months. The genome of SARS-CoV-2 continues to evolve during the global transmission with the notable emergence of the spike D614G substitution that enhances infectivity. Some of these viral adaptations may alter not only the infectivity but also viral pathogenesis. Continuous phylogenomic analysis of circulating viral strains and functional investigation of new non-synonymous substitutions may help to understand the evolution of virus, its virulence and transmissibility. Here we describe a loss of an accessory protein orf3b (57 amino acids) in current circulating SARS-CoV-2 strains, contributing around 24% of more than 100,000 complete viral genomes analysed. The loss of 3b is caused by the presence of an early stop codon which is created by an orf3a Q57H substitution. There is an increasing trend in the loss of orf3b which has reached 32% in May 2020. Geographically, loss of 3b is more prevalent in certain countries including Colombia (46%), USA (48%), South Korea (51%), France (66%), Saudi Arabia (72%), Finland (76%) and Egypt (77%). Interestingly, the loss of 3b coincides with the emergence of spike D614G substitution. In addition, we found that truncated orf3b has lost the interferon antagonism compared to the full-length orf3b, suggesting a loss of function by the newly adapted virus. Further investigation of orf3b deletion and spike D614G substitution on virulence and infectivity respectively will provide important insights into SARS-CoV-2 evolution.


Subject(s)
Gene Deletion , SARS-CoV-2/genetics , Viral Proteins/genetics , Amino Acid Sequence , Cells, Cultured , Humans , Interferons/antagonists & inhibitors , SARS-CoV-2/pathogenicity , Viral Proteins/chemistry , Viral Proteins/immunology
11.
Virus Res ; 291: 198222, 2021 01 02.
Article in English | MEDLINE | ID: covidwho-912664

ABSTRACT

The envelope glycoprotein (E) is the smallest structural component of SARS-CoVs; plays an essential role in the viral replication starting from envelope formation to assembly. The in silico analysis of 2086 whole genome sequences from India performed in this study provides the first observation on the extensive deletion of amino acid residues in the C-terminal region of the envelope glycoprotein in 34 Indian SARS-CoV-2 genomes. These amino acid deletions map to the homopentameric interface and PDZ binding motif (PBM) present in the C-terminal region of E protein as well as immediately after the reverse primer binding region as per Charité protocol in 26 of these genomes, hence, their detection through RT-qPCR may not be hampered and therefore E gene-based RT-qPCR would still detect these isolates. Eight genomes from the State of Odisha had deletion even in the primer binding site. It is possible that the deletions in the C-terminal region of E protein of these genomes are a result of adapting to a newer geographical area and host. The information on the clinical status was available only for 9 out of 34 cases and these were asymptomatic. However, further studies are indispensable to understand the functional consequences of amino acid deletion in the C terminal region of SARS-CoV-2 envelope protein in the viral pathogenesis and host adaptation.


Subject(s)
Coronavirus Envelope Proteins/genetics , SARS-CoV-2/genetics , Adult , Amino Acid Sequence , Computer Simulation , Coronavirus Envelope Proteins/immunology , Epitopes, B-Lymphocyte , Female , Gene Deletion , Genome, Viral , Humans , India , Male , Middle Aged , Real-Time Polymerase Chain Reaction , SARS-CoV-2/isolation & purification
12.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-840682

ABSTRACT

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.


Subject(s)
Pestivirus Infections/virology , Pestivirus/physiology , RNA, Viral , Viral Envelope Proteins/metabolism , Virus Replication , Gene Deletion , Gene Expression , Genes, Reporter , Genetic Engineering , Host-Pathogen Interactions , Pestivirus Infections/immunology , Replicon , Viral Envelope Proteins/genetics , Virion , Virus Assembly
13.
Lancet ; 396(10251): 603-611, 2020 08 29.
Article in English | MEDLINE | ID: covidwho-719049

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. METHODS: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. FINDINGS: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only. INTERPRETATION: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. FUNDING: National Medical Research Council Singapore.


Subject(s)
Coronavirus Infections/virology , Gene Deletion , Genome, Viral/genetics , Pneumonia, Viral/virology , Adult , Aged , Betacoronavirus , COVID-19 , Coronavirus Infections/complications , Coronavirus Infections/epidemiology , Humans , Hypoxia/etiology , Hypoxia/therapy , Middle Aged , Open Reading Frames , Pandemics , Pneumonia, Viral/complications , Pneumonia, Viral/epidemiology , Prospective Studies , Respiratory Therapy , SARS-CoV-2 , Severity of Illness Index , Singapore/epidemiology , Virus Replication
14.
Emerg Microbes Infect ; 9(1): 1900-1911, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-695197

ABSTRACT

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site (PRRAR/S), generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We suggest that natural selection has favoured a "Don't burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Genome, Viral/genetics , Pneumonia, Viral/virology , Quasispecies/genetics , Respiratory Tract Infections/virology , Spike Glycoprotein, Coronavirus/genetics , Adult , Aged , COVID-19 , Computational Biology , Female , Gene Deletion , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Pandemics , RNA Cleavage , SARS-CoV-2 , Sequence Analysis, RNA
15.
Front Immunol ; 11: 939, 2020.
Article in English | MEDLINE | ID: covidwho-380929

ABSTRACT

Zoonotic infections are an imminent threat to human health. Pangolins were recently identified as carriers and intermediate hosts of coronaviruses. Previous research has shown that infection with coronaviruses activates an innate immune response upon sensing of viral RNA by interferon-induced with helicase C domain 1 (IFIH1), also known as MDA5. Here, we performed a comparative genomics study of RNA sensor genes in three species of pangolins. DDX58/RIG-I, a sensor of cytoplasmic viral RNA and toll-like receptors (TLR) 3, 7, and 8, which bind RNA in endosomes, are conserved in pangolins. By contrast, IFIH1 a sensor of intracellular double-stranded RNA, has been inactivated by mutations in pangolins. Likewise, Z-DNA-binding protein (ZBP1), which senses both Z-DNA and Z-RNA, has been lost during the evolution of pangolins. These results suggest that the innate immune response to viruses differs significantly between pangolins and other mammals, including humans. We put forward the hypothesis that loss of IFIH1 and ZBP1 provided an evolutionary advantage by reducing inflammation-induced damage to host tissues and thereby contributed to a switch from resistance to tolerance of viral infections in pangolins.


Subject(s)
Coronavirus Infections/immunology , Eutheria/virology , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , Animals , Coronavirus/immunology , DEAD Box Protein 58/genetics , Gene Deletion , Humans , Immunity, Innate/immunology , RNA, Viral/immunology , RNA-Binding Proteins/genetics , Zoonoses/virology
17.
Virus Res ; 278: 197869, 2020 03.
Article in English | MEDLINE | ID: covidwho-2388

ABSTRACT

Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV studies have been conducted in China, more sequence information is needed to understand the molecular characterization of PDCoV. In this study, the partial ORF1a, spike protein (S) and nucleocapsid protein (N) were sequenced from Shandong Province between 2017 and 2018. The sequencing results for the S protein from 10 PDCoV strains showed 96.7 %-99.7 % nucleotide sequence identity with the China lineage strains, while sharing a lower level of nucleotide sequence identity, ranging from 95.7 to 96.8%, with the Vietnam/Laos/Thailand lineage strains. N protein sequencing analysis showed that these strains showed nucleotide homologies of 97.3%-99.3% with the reference strains. Phylogenetic analyses based on S protein sequences showed that these PDCoV strains were classified into the China lineage. The discontinuous 2 + 3 aa deletions at 400-401 and 758-760 were found in the Nsp2 and Nsp3 coding region in five strains, respectively, with similar deletions having been identified in Vietnam, Thailand, and Laos. Three novel patterns of deletion were observed for the first time in the Nsp2 and Nsp3 regions. Importantly, those findings suggest that PDCoV may have undergone a high degree of variation since PDCoV was first detected in China.


Subject(s)
Coronavirus Infections/veterinary , Deltacoronavirus/classification , Deltacoronavirus/genetics , Genome, Viral , Phylogeny , Animals , China/epidemiology , Coronavirus Infections/epidemiology , Diarrhea/virology , Feces/virology , Gene Deletion , Prevalence , Swine , Swine Diseases/virology , Viral Proteins/genetics
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