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1.
Genome Med ; 13(1): 30, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-1097198

ABSTRACT

BACKGROUND: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. METHODS: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). RESULTS: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. CONCLUSIONS: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.

2.
Micro. Res. Ann ; 11(9)20200312.
Article in English | ELSEVIER | ID: covidwho-1043951

ABSTRACT

A complete genome sequence was obtained for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab specimen of a Nepalese patient with coronavirus disease 2019 (COVID-19), who had returned to Nepal after traveling to Wuhan, China.

3.
Clin Chem ; 66(4): 549-555, 2020 04 01.
Article in English | MEDLINE | ID: covidwho-1024088

ABSTRACT

BACKGROUND: A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. METHODS: We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. RESULTS: Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10-4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. CONCLUSIONS: The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Disease Outbreaks , Humans , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction
4.
Nat Commun ; 12(1): 63, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-1007631

ABSTRACT

The SARS-CoV-2 pandemic poses the greatest global public health challenge in a century. Neutralizing antibody is a correlate of protection and data on kinetics of virus neutralizing antibody responses are needed. We tested 293 sera from an observational cohort of 195 reverse transcription polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections collected from 0 to 209 days after onset of symptoms. Of 115 sera collected ≥61 days after onset of illness tested using plaque reduction neutralization (PRNT) assays, 99.1% remained seropositive for both 90% (PRNT90) and 50% (PRNT50) neutralization endpoints. We estimate that it takes at least 372, 416 and 133 days for PRNT50 titres to drop to the detection limit of a titre of 1:10 for severe, mild and asymptomatic patients, respectively. At day 90 after onset of symptoms (or initial RT-PCR detection in asymptomatic infections), it took 69, 87 and 31 days for PRNT50 antibody titres to decrease by half (T1/2) in severe, mild and asymptomatic infections, respectively. Patients with severe disease had higher peak PRNT90 and PRNT50 antibody titres than patients with mild or asymptomatic infections. Age did not appear to compromise antibody responses, even after accounting for severity. We conclude that SARS-CoV-2 infection elicits robust neutralizing antibody titres in most individuals.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , /immunology , Adolescent , Adult , Animals , Antibodies, Viral/blood , /epidemiology , Chlorocebus aethiops , Cohort Studies , Female , Hong Kong/epidemiology , Humans , Male , Middle Aged , Neutralization Tests , Pandemics , Vero Cells , Young Adult
5.
Euro Surveill ; 25(3)2020 01.
Article in English | MEDLINE | ID: covidwho-1004613

ABSTRACT

BACKGROUND: The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. AIM: We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. METHODS: Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. RESULTS: The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project. CONCLUSION: The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.


Subject(s)
Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Coronavirus/classification , Coronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus/isolation & purification , Disease Outbreaks , Humans , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity
6.
Emerg Infect Dis ; 26(1): 173-176, 2020 01.
Article in English | MEDLINE | ID: covidwho-966221

ABSTRACT

We examined nasal swabs and serum samples acquired from dromedary camels in Nigeria and Ethiopia during 2015-2017 for evidence of influenza virus infection. We detected antibodies against influenza A(H1N1) and A(H3N2) viruses and isolated an influenza A(H1N1)pdm09-like virus from a camel in Nigeria. Influenza surveillance in dromedary camels is needed.

8.
J Clin Microbiol ; 2020 Nov 02.
Article in English | MEDLINE | ID: covidwho-901259

ABSTRACT

Surrogate neutralization assays for SARS-CoV-2 that can be done without biosafety-level-3 containment and in multiple species are desirable. We evaluate a recently developed surrogate virus neutralization test (sVNT) in comparison to 90% plaque reduction neutralization tests (PRNT90) in human, canine, cat and hamster sera. With PRNT90 as reference, sVNT had sensitivity of 98.9% and specificity of 98.8% respectively. Using a panel of immune sera to other coronaviruses, we confirm the lack of cross reactivity to other coronaviruses in SARS-CoV-2 sVNT and PRNT90 assays, except for cross-reactivity to SARS-CoV-1 in sVNT.

9.
Emerg Infect Dis ; 26(12): 3076-3078, 2020 12.
Article in English | MEDLINE | ID: covidwho-890312

ABSTRACT

In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.

11.
Emerg Infect Dis ; 26(11): 2713-2716, 2020 11.
Article in English | MEDLINE | ID: covidwho-878004

ABSTRACT

Four persons with severe acute respiratory syndrome coronavirus 2 infection had traveled on the same flight from Boston, Massachusetts, USA, to Hong Kong, China. Their virus genetic sequences are identical, unique, and belong to a clade not previously identified in Hong Kong, which strongly suggests that the virus can be transmitted during air travel.


Subject(s)
Air Travel , Betacoronavirus , Coronavirus Infections/transmission , Disease Transmission, Infectious/statistics & numerical data , Pneumonia, Viral/transmission , Travel-Related Illness , Adult , Aged , Boston/epidemiology , Coronavirus Infections/epidemiology , Female , Hong Kong/epidemiology , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/epidemiology
12.
Lancet Infect Dis ; 2020 Oct 06.
Article in English | MEDLINE | ID: covidwho-817086

ABSTRACT

BACKGROUND: Middle East respiratory syndrome (MERS) remains of global public health concern. Dromedary camels are the source of zoonotic infection. Over 70% of MERS coronavirus (MERS-CoV)-infected dromedaries are found in Africa but no zoonotic disease has been reported in Africa. We aimed to understand whether individuals with exposure to dromedaries in Africa had been infected by MERS-CoV. METHODS: Workers slaughtering dromedaries in an abattoir in Kano, Nigeria, were compared with abattoir workers without direct dromedary contact, non-abattoir workers from Kano, and controls from Guangzhou, China. Exposure to dromedaries was ascertained using a questionnaire. Serum and peripheral blood mononuclear cells (PBMCs) were tested for MERS-CoV specific neutralising antibody and T-cell responses. FINDINGS: None of the participants from Nigeria or Guangdong were MERS-CoV seropositive. 18 (30%) of 61 abattoir workers with exposure to dromedaries, but none of 20 abattoir workers without exposure (p=0·0042), ten non-abattoir workers or 24 controls from Guangzhou (p=0·0002) had evidence of MERS-CoV-specific CD4+ or CD8+ T cells in PBMC. T-cell responses to other endemic human coronaviruses (229E, OC43, HKU-1, and NL-63) were observed in all groups with no association with dromedary exposure. Drinking both unpasteurised camel milk and camel urine was significantly and negatively associated with T-cell positivity (odds ratio 0·07, 95% CI 0·01-0·54). INTERPRETATION: Zoonotic infection of dromedary-exposed individuals is taking place in Nigeria and suggests that the extent of MERS-CoV infections in Africa is underestimated. MERS-CoV could therefore adapt to human transmission in Africa rather than the Arabian Peninsula, where attention is currently focused. FUNDING: The National Science and Technology Major Project, National Institutes of Health.

13.
Preprint | SSRN | ID: ppcovidwho-677

ABSTRACT

Background: A novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, to cause a respiratory disease (CO

14.
Emerg Infect Dis ; 26(11): 2713-2716, 2020 11.
Article in English | MEDLINE | ID: covidwho-781930

ABSTRACT

Four persons with severe acute respiratory syndrome coronavirus 2 infection had traveled on the same flight from Boston, Massachusetts, USA, to Hong Kong, China. Their virus genetic sequences are identical, unique, and belong to a clade not previously identified in Hong Kong, which strongly suggests that the virus can be transmitted during air travel.


Subject(s)
Air Travel , Betacoronavirus , Coronavirus Infections/transmission , Disease Transmission, Infectious/statistics & numerical data , Pneumonia, Viral/transmission , Travel-Related Illness , Adult , Aged , Boston/epidemiology , Coronavirus Infections/epidemiology , Female , Hong Kong/epidemiology , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/epidemiology
16.
Emerg Infect Dis ; 26(12): 3071-3074, 2020 12.
Article in English | MEDLINE | ID: covidwho-771556

ABSTRACT

We tested 50 cats from coronavirus disease households or close contacts in Hong Kong, China, for severe acute respiratory syndrome coronavirus 2 RNA in respiratory and fecal samples. We found 6 cases of apparent human-to-feline transmission involving healthy cats. Virus genomes sequenced from 1 cat and its owner were identical.

17.
J Virol ; 94(15)2020 07 16.
Article in English | MEDLINE | ID: covidwho-762192

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.


Subject(s)
Coronavirus Infections/virology , Genotype , Host-Pathogen Interactions , Middle East Respiratory Syndrome Coronavirus/physiology , Adult , Animals , Disease Models, Animal , Genome, Viral , Host-Pathogen Interactions/immunology , Humans , Interferon Type I/pharmacology , Male , Mice , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Phylogeny , RNA, Viral , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Virulence , Virus Replication/drug effects , Virus Replication/genetics , Whole Genome Sequencing
18.
Science ; 369(6508): 1210-1220, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-704393

ABSTRACT

Coronavirus disease 2019 (COVID-19) represents a global crisis, yet major knowledge gaps remain about human immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed immune responses in 76 COVID-19 patients and 69 healthy individuals from Hong Kong and Atlanta, Georgia, United States. In the peripheral blood mononuclear cells (PBMCs) of COVID-19 patients, we observed reduced expression of human leukocyte antigen class DR (HLA-DR) and proinflammatory cytokines by myeloid cells as well as impaired mammalian target of rapamycin (mTOR) signaling and interferon-α (IFN-α) production by plasmacytoid dendritic cells. By contrast, we detected enhanced plasma levels of inflammatory mediators-including EN-RAGE, TNFSF14, and oncostatin M-which correlated with disease severity and increased bacterial products in plasma. Single-cell transcriptomics revealed a lack of type I IFNs, reduced HLA-DR in the myeloid cells of patients with severe COVID-19, and transient expression of IFN-stimulated genes. This was consistent with bulk PBMC transcriptomics and transient, low IFN-α levels in plasma during infection. These results reveal mechanisms and potential therapeutic targets for COVID-19.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Cytokines/blood , DNA, Bacterial/blood , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Flow Cytometry , HLA-DR Antigens/analysis , Humans , Immunity , Immunity, Innate , Immunoglobulins/blood , Immunoglobulins/immunology , Inflammation Mediators/blood , Interferon Type I/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/blood , Male , Myeloid Cells/immunology , Myeloid Cells/metabolism , Pandemics , Signal Transduction , Single-Cell Analysis , Systems Biology , TOR Serine-Threonine Kinases/metabolism , Transcription, Genetic , Transcriptome
19.
Emerg Infect Dis ; 26(11): 2701-2704, 2020 11.
Article in English | MEDLINE | ID: covidwho-694508

ABSTRACT

We investigated 68 respiratory specimens from 35 coronavirus disease patients in Hong Kong, of whom 32 had mild disease. We found that severe acute respiratory syndrome coronavirus 2 and subgenomic RNA were rarely detectable beyond 8 days after onset of illness. However, virus RNA was detectable for many weeks by reverse transcription PCR.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA, Viral/analysis , Respiratory System/virology , Severity of Illness Index , Adult , Aged , Female , Hong Kong , Humans , Male , Middle Aged , Pandemics , Reverse Transcriptase Polymerase Chain Reaction
20.
Clin Chem ; 66(10): 1349-1350, 2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-676579
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