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1.
Front Microbiol ; 13: 735363, 2022.
Article in English | MEDLINE | ID: covidwho-1809432

ABSTRACT

Objective: We aimed to evaluate the performance of nanopore amplicon sequencing detection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples. Method: We carried out a single-center, prospective cohort study in a Wuhan hospital and collected a total of 86 clinical samples, including 54 pharyngeal swabs, 31 sputum samples, and 1 fecal sample, from 86 patients with coronavirus disease 2019 (COVID-19) from Feb 20 to May 15, 2020. We performed parallel detection with nanopore-based genome amplification and sequencing (NAS) on the Oxford Nanopore Technologies (ONT) minION platform and routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). In addition, 27 negative control samples were detected using the two methods. The sensitivity and specificity of NAS were evaluated and compared with those of RT-qPCR. Results: The viral read number and reference genome coverage were both significantly different between the two groups of samples, and the latter was a better indicator for SARS-CoV-2 detection. Based on the reference genome coverage, NAS revealed both high sensitivity (96.5%) and specificity (100%) compared with RT-qPCR (80.2 and 96.3%, respectively), although the samples had been stored for half a year before the detection. The total time cost was less than 15 h, which was acceptable compared with that of RT-qPCR (∼2.5 h). In addition, the reference genome coverage of the viral reads was in line with the cycle threshold value of RT-qPCR, indicating that this number could also be used as an indicator of the viral load in a sample. The viral load in sputum might be related to the severity of the infection, particularly in patients within 4 weeks after onset of clinical manifestations, which could be used to evaluate the infection. Conclusion: Our results showed the high sensitivity and specificity of the NAS method for SARS-CoV-2 detection compared with RT-qPCR. The sequencing results were also used as an indicator of the viral load to display the viral dynamics during infection. This study proved the wide application prospect of nanopore sequencing detection for SARS-CoV-2 and may more knowledge about the clinical characteristics of COVID-19.

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-315201

ABSTRACT

The mutations make uncertain to SARS-CoV-2 disease control and vaccine development. At population-level, single nucleotide polymorphism (SNPs) have displayed mutations for illustrating epidemiology, transmission, and pathogenesis of COVID-19. These mutations are to be expected by the analysis of intra-host level, which presented as intra-host variations (iSNVs). Here, we performed spatio-temporal analysis on iSNVs in 402 clinical samples from 170 patients, and observed an increase of genetic diversity along the day post symptom onset within individual patient and among subpopulations divided by gender, age, illness severity and viral shedding time, suggested a positive selection at intra-host level. The comparison of iSNVs and SNPs displayed that most of nonsynonymous mutations were not fixed suggested a purifying selection. This two-step fitness selection enforced iSNVs containing more nonsynonymous mutations, that highlight the potential characters of SARS-CoV-2 for viral infections and global transmissions.

3.
Cell Rep ; 38(2): 110205, 2022 01 11.
Article in English | MEDLINE | ID: covidwho-1588142

ABSTRACT

Spontaneous mutations introduce uncertainty into coronavirus disease 2019 (COVID-19) control procedures and vaccine development. Here, we perform a spatiotemporal analysis on intra-host single-nucleotide variants (iSNVs) in 402 clinical samples from 170 affected individuals, which reveals an increase in genetic diversity over time after symptom onset in individuals. Nonsynonymous mutations are overrepresented in the pool of iSNVs but underrepresented at the single-nucleotide polymorphism (SNP) level, suggesting a two-step fitness selection process: a large number of nonsynonymous substitutions are generated in the host (positive selection), and these substitutions tend to be unfixed as SNPs in the population (negative selection). Dynamic iSNV changes in subpopulations with different gender, age, illness severity, and viral shedding time displayed a varied fitness selection process among populations. Our study highlights that iSNVs provide a mutational pool shaping the rapid global evolution of the virus.


Subject(s)
COVID-19/virology , Host-Pathogen Interactions/genetics , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Genome, Viral/genetics , Humans , Infant , Infant, Newborn , Male , Middle Aged , Mutation/genetics , Phylogeny , Polymorphism, Single Nucleotide/genetics , Spike Glycoprotein, Coronavirus/genetics , Young Adult
4.
Clin Infect Dis ; 73(9): e2814-e2817, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1501023

ABSTRACT

Intrahost analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic sequences identified 2 viral haplotypes comprised of 3 genetically linked mutations from the respiratory and intestinal tracts of a patient with coronavirus disease 2019. Spatiotemporal data suggest that this patient initially had dual infection of 2 SARS-CoV-2 variants, which subsequently redistributed into the 2 systems.


Subject(s)
COVID-19 , SARS-CoV-2 , Genomics , Humans , Respiratory System
5.
BMC Public Health ; 21(1): 1096, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262502

ABSTRACT

BACKGROUND: With the accelerated aging of the Chinese population, elder abuse has become a serious social problem. As COVID-19 has had a very large impact on economic development and lifestyle in China, it has also affected elder abuse. The purpose of this study is to estimate the prevalence of elder abuse in China during the COVID-19 pandemic, and to identify changes in risk factors for elder abuse in the context of COVID-19. METHODS: We designed a cross-sectional study. In Hunan Province, a face-to-face questionnaire survey was conducted among elderly people over 65 years of age. To ensure the consistency of the measurement standards, we used the elder abuse questionnaire from the "Third Survey on Chinese Women's Social Status." According to related research, we selected 10 victim-related risk factors as independent variables. A logistic regression model was established to analyze the relationship between the independent variables and the four kinds of abuse. RESULTS: We collected 10,362 samples from Hunan Province. During the COVID-19 pandemic, the prevalence of financial abuse and neglect was significantly higher than that in 2010. Income had a significant impact on the four types of abuse. The lower the income was, the greater the risk of abuse. Moreover, factors such as an older age, being a woman, a lower cognitive ability, and not having a cohabiting spouse increased the possibility of abuse. The greater the number of children was, the greater the risks of physical abuse, financial abuse, and elder neglect. Seniors with higher education levels, those who frequently participated in social activities, and those with religious beliefs were less likely to suffer abuse. CONCLUSIONS: During the COVID-19 epidemic, the prevalence of elder abuse in China has increased, which may be related to economic instability and social distancing measures. Increasing the income of the elderly and giving them more social support are important measures to reduce the prevalence of elder abuse.


Subject(s)
COVID-19 , Elder Abuse , Aged , Child , China/epidemiology , Cross-Sectional Studies , Female , Humans , Pandemics , Prevalence , Risk Factors , SARS-CoV-2
6.
Mol Cell ; 80(6): 1123-1134.e4, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-939163

ABSTRACT

Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.


Subject(s)
COVID-19/genetics , Genome, Viral , High-Throughput Nucleotide Sequencing , RNA, Viral/genetics , SARS-CoV-2/genetics , Whole Genome Sequencing , Animals , Humans , Mice , NIH 3T3 Cells , RNA, Viral/metabolism , SARS-CoV-2/metabolism
7.
Nat Commun ; 11(1): 5503, 2020 10 30.
Article in English | MEDLINE | ID: covidwho-894393

ABSTRACT

The spread of SARS-CoV-2 in Beijing before May, 2020 resulted from transmission following both domestic and global importation of cases. Here we present genomic surveillance data on 102 imported cases, which account for 17.2% of the total cases in Beijing. Our data suggest that all of the cases in Beijing can be broadly classified into one of three groups: Wuhan exposure, local transmission and overseas imports. We classify all sequenced genomes into seven clusters based on representative high-frequency single nucleotide polymorphisms (SNPs). Genomic comparisons reveal higher genomic diversity in the imported group compared to both the Wuhan exposure and local transmission groups, indicating continuous genomic evolution during global transmission. The imported group show region-specific SNPs, while the intra-host single nucleotide variations present as random features, and show no significant differences among groups. Epidemiological data suggest that detection of cases at immigration with mandatory quarantine may be an effective way to prevent recurring outbreaks triggered by imported cases. Notably, we also identify a set of novel indels. Our data imply that SARS-CoV-2 genomes may have high mutational tolerance.


Subject(s)
Betacoronavirus/growth & development , Coronavirus Infections/virology , Pneumonia, Viral/virology , Adult , Beijing/epidemiology , COVID-19 , Coronavirus Infections/epidemiology , Female , Genome, Viral , Genomics , Genotype , Humans , Male , Middle Aged , Mutation , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Polymorphism, Single Nucleotide , SARS-CoV-2 , Travel , Young Adult
8.
J Virol ; 94(18)2020 08 31.
Article in English | MEDLINE | ID: covidwho-803471

ABSTRACT

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.


Subject(s)
Animal Diseases/metabolism , Animal Diseases/virology , Betacoronavirus/physiology , Coronavirus Infections/veterinary , Pandemics/veterinary , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/veterinary , Receptors, Virus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/classification , COVID-19 , Cell Line , Host Specificity , Humans , Models, Molecular , Mutation , Peptidyl-Dipeptidase A/chemistry , Phylogeny , Protein Binding , Protein Domains , Proteolysis , Receptors, Virus/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Viral Tropism , Virus Internalization
9.
Emerg Microbes Infect ; 9(1): 1567-1579, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-707709

ABSTRACT

Diverse SARS-like coronaviruses (SL-CoVs) have been identified from bats and other animal species. Like SARS-CoV, some bat SL-CoVs, such as WIV1, also use angiotensin converting enzyme 2 (ACE2) from human and bat as entry receptor. However, whether these viruses can also use the ACE2 of other animal species as their receptor remains to be determined. We report herein that WIV1 has a broader tropism to ACE2 orthologs than SARS-CoV isolate Tor2. Among the 9 ACE2 orthologs examined, human ACE2 exhibited the highest efficiency to mediate the infection of WIV1 pseudotyped virus. Our findings thus imply that WIV1 has the potential to infect a wide range of wild animals and may directly jump to humans. We also showed that cell entry of WIV1 could be restricted by interferon-induced transmembrane proteins (IFITMs). However, WIV1 could exploit the airway protease TMPRSS2 to partially evade the IFITM3 restriction. Interestingly, we also found that amphotericin B could enhance the infectious entry of SARS-CoVs and SL-CoVs by evading IFITM3-mediated restriction. Collectively, our findings further underscore the risk of exposure to animal SL-CoVs and highlight the vulnerability of patients who take amphotericin B to infection by SL-CoVs, including the most recently emerging (SARS-CoV-2).


Subject(s)
Betacoronavirus/physiology , Chiroptera/virology , Membrane Proteins/metabolism , Peptidyl-Dipeptidase A/metabolism , RNA-Binding Proteins/metabolism , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/classification , HEK293 Cells , Humans , Rats , Receptors, Coronavirus , SARS Virus/physiology , Viverridae
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