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1.
Nat Commun ; 12(1): 6304, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728625

RESUMO

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Epitopos , Humanos , Camundongos , Camundongos Transgênicos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Carga Viral/efeitos dos fármacos , Perda de Peso/efeitos dos fármacos
2.
Hepatology ; 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34779008

RESUMO

BACKGROUND AND AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability. APPROACHES AND RESULTS: By using HBV infection cell models, in vitro and in vivo recombinant cccDNA (rcccDNA) and the HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of APOBEC3A- and CRISPR/Cas9-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and HBx deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. The HBV cccDNA levels remained stable in non-dividing hepatocytes, however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally-active and transcriptionally-repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA in low transcriptional activity exhibited an epigenetic inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A. CONCLUSIONS: HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division, but significantly differ in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.

3.
J Virol ; : JVI0149221, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34668773

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021), with varying incidences and mortalities among regions/ethnicities. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike proteins and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity. IMPORTANCE There is considerable variation in disease severity among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19). Human genetic variation can affect disease outcome, and the coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 utilize human angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We found that several missense ACE2 SNVs that showed significantly altered binding with the spike proteins of SARS-CoV, SARS-CoV-2 and NL63-HCoV. We identified an ACE2 SNP D355N that restricts the spike protein-ACE2 interaction and consequently have the potential to protect individuals against SARS-CoV-2 infection. Our study highlights ACE2 polymorphisms could impact human susceptibility to SARS-CoV-2, which may contribute to ethnic and geographical differences in SARS-CoV-2 spread and pathogenicity.

4.
Protein Cell ; 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34554412

RESUMO

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes ß-coronavirus lineage B (ß-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-ß-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against ß-CoV-B and newly emerging SARS-CoV-2 variants of concern.

5.
PLoS Pathog ; 17(8): e1009838, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34370796

RESUMO

Through evolution, Hepatitis B Virus (HBV) developed highly intricate mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. Yet a clear picture of viral hitchhiking of cellular processes with spatial resolution is still largely unsolved. Here, by leveraging bDNA-based fluorescence in situ hybridization (FISH) combined with immunofluorescence, we developed a microscopic approach for multiplex detection of viral nucleic acids and proteins, which enabled us to probe some of the key aspects of HBV life cycle. We confirmed the slow kinetics and revealed the high variability of viral replication at single-cell level. We directly visualized HBV minichromosome in contact with acetylated histone 3 and RNA polymerase II and observed HBV-induced degradation of Smc5/6 complex only in primary hepatocytes. We quantified the frequency of HBV pregenomic RNAs occupied by translating ribosome or capsids. Statistics at molecular level suggested a rapid translation phase followed by a slow encapsidation and maturation phase. Finally, the roles of microtubules (MTs) on nucleocapsid assembly and virion morphogenesis were analyzed. Disruption of MTs resulted in the perinuclear retention of nucleocapsid. Meanwhile, large multivesicular body (MVB) formation was significantly disturbed as evidenced by the increase in number and decrease in volume of CD63+ vesicles, thus inhibiting mature virion secretion. In conclusion, these data provided spatially resolved molecular snapshots in the context of specific subcellular activities. The heterogeneity observed at single-cell level afforded valuable molecular insights which are otherwise unavailable from bulk measurements.

6.
Emerg Microbes Infect ; 10(1): 1609-1625, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340648

RESUMO

Flavivirus genus includes numerous arthropod-borne human pathogens that are clinically important. Flaviviruses are notorious for their ability to antagonize host interferon (IFN) induced anti-viral signalling. It has been documented that NS5s of flaviviruses mediate proteasome degradation of STAT2 to evade IFN signalling. Deciphering the molecular mechanism of the IFN antagonism by the viruses and reversing this antagonism may dictate anti-viral responses and provide novel antiviral approaches. In this report, by using Zika virus (ZIKV) as a model, we first demonstrated that ZIKV antagonized interferon signalling in an infectious dose-dependent manner; in other words, the virus antagonized interferon signalling at a high multiple of infection (MOI) and was sensitive to interferon signalling at a low MOI. Mechanistically, we found that ZIKV infection triggered degradation of ubiquitinated STAT2 and host short-lived proteins while didn't affect the proteasome activity per se. ZIKV infection resulted in suppression of host de novo protein synthesis. Overexpression of NS5 alone only marginally reduced STAT2 and had no effect on the host de novo protein synthesis. Ectopically expressed murine STAT2 that was resistant to NS5- and ZIKV-induced ablation exaggerated the IFN-induced anti-viral signalling. These data favour a new model of the innate immune evasion of ZIKV in which the viral infection triggers suppression of host de novo protein synthesis to accelerate the degradation of short-lived, ubiquitinated STAT2. As flaviviruses share a very conserved replication strategy, the mechanisms of IFN antagonism elucidated here might also be employed by other flaviviruses.

7.
Nat Commun ; 12(1): 5026, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408158

RESUMO

Nationwide prospective surveillance of all-age patients with acute respiratory infections was conducted in China between 2009‒2019. Here we report the etiological and epidemiological features of the 231,107 eligible patients enrolled in this analysis. Children <5 years old and school-age children have the highest viral positivity rate (46.9%) and bacterial positivity rate (30.9%). Influenza virus, respiratory syncytial virus and human rhinovirus are the three leading viral pathogens with proportions of 28.5%, 16.8% and 16.7%, and Streptococcus pneumoniae, Mycoplasma pneumoniae and Klebsiella pneumoniae are the three leading bacterial pathogens (29.9%, 18.6% and 15.8%). Negative interactions between viruses and positive interactions between viral and bacterial pathogens are common. A Join-Point analysis reveals the age-specific positivity rate and how this varied for individual pathogens. These data indicate that differential priorities for diagnosis, prevention and control should be highlighted in terms of acute respiratory tract infection patients' demography, geographic locations and season of illness in China.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Viroses/virologia , Vírus/isolamento & purificação , Adolescente , Adulto , Bactérias/classificação , Bactérias/genética , Infecções Bacterianas/epidemiologia , Criança , Pré-Escolar , China/epidemiologia , Feminino , Humanos , Lactente , Masculino , Estudos Prospectivos , Infecções Respiratórias/epidemiologia , Estações do Ano , Viroses/epidemiologia , Vírus/classificação , Vírus/genética , Adulto Jovem
8.
Front Microbiol ; 12: 715450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335553

RESUMO

Hepatitis B virus (HBV) infection is a global public health problem that plagues approximately 240 million people. Chronic hepatitis B (CHB) often leads to liver inflammation and aberrant repair which results in diseases ranging from liver fibrosis, cirrhosis, to hepatocellular carcinoma. Despite its narrow species tropism, researchers have established various in vivo models for HBV or its related viruses which have provided a wealth of knowledge on viral lifecycle, pathogenesis, and immunity. Here we briefly revisit over five decades of endeavor in animal model development for HBV and summarize their advantages and limitations. We also suggest directions for further improvements that are crucial for elucidation of the viral immune-evasion strategies and for development of novel therapeutics for a functional cure.

9.
Signal Transduct Target Ther ; 6(1): 288, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326308

RESUMO

The COVID-19 pandemic poses a global threat to public health and economy. The continuously emerging SARS-CoV-2 variants present a major challenge to the development of antiviral agents and vaccines. In this study, we identified that EK1 and cholesterol-coupled derivative of EK1, EK1C4, as pan-CoV fusion inhibitors, exhibit potent antiviral activity against SARS-CoV-2 infection in both lung- and intestine-derived cell lines (Calu-3 and Caco2, respectively). They are also effective against infection of pseudotyped SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.1.248 (Gamma) as well as those with mutations in S protein, including N417T, E484K, N501Y, and D614G, which are common in South African and Brazilian variants. Crystal structure revealed that EK1 targets the HR1 domain in the SARS-CoV-2 S protein to block virus-cell fusion and provide mechanistic insights into its broad and effective antiviral activity. Nasal administration of EK1 peptides to hACE2 transgenic mice significantly reduced viral titers in lung and intestinal tissues. EK1 showed good safety profiles in various animal models, supporting further clinical development of EK1-based pan-CoV fusion inhibitors against SARS-CoV-2 and its variants.


Assuntos
Antivirais , COVID-19/tratamento farmacológico , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Células CACO-2 , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos Transgênicos , Domínios Proteicos , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Cell Chem Biol ; 28(9): 1366-1378.e4, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-33798447

RESUMO

Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly.

11.
Nat Commun ; 12(1): 2464, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33927201

RESUMO

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged <5 years had higher overall positive rate of viral pathogens, while bacterial pathogens were more common in patients aged 18‒45 years. A joinpoint analysis revealed the age-specific positivity rate and how this varied for individual pathogens. Our findings fill crucial gaps of how the distributions of enteropathogens change across China in patients with diarrhea. This allows enhanced identification of the predominant diarrheal pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures.


Assuntos
Diarreia/epidemiologia , Diarreia/patologia , Gastroenterite/epidemiologia , Gastroenterite/patologia , Adolescente , Adulto , Fatores Etários , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/patologia , Criança , Pré-Escolar , China/epidemiologia , Diarreia/microbiologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/patologia , Gastroenterite/microbiologia , Humanos , Pessoa de Meia-Idade , Norovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/patologia , Salmonella/isolamento & purificação , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/patologia , Shigella/isolamento & purificação , Vibrioses/epidemiologia , Vibrioses/patologia , Vibrio parahaemolyticus/isolamento & purificação , Adulto Jovem
12.
Nat Commun ; 12(1): 2114, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837182

RESUMO

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.


Assuntos
Substituição de Aminoácidos/efeitos dos fármacos , COVID-19/prevenção & controle , Catequina/análogos & derivados , Proteínas do Nucleocapsídeo/genética , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , COVID-19/virologia , Catequina/farmacologia , Genoma Viral/genética , Humanos , Extração Líquido-Líquido , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação Viral/genética
13.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33658332

RESUMO

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs-including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria-could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/veterinária , Receptores Virais/genética , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Especificidade de Hospedeiro , Humanos , Pandemias/prevenção & controle , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Filogenia , Ligação Proteica , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Zoonoses Virais/genética , Zoonoses Virais/prevenção & controle , Zoonoses Virais/virologia , Ligação Viral , Internalização do Vírus
14.
PLoS Pathog ; 17(3): e1009392, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33760889

RESUMO

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.


Assuntos
COVID-19/enzimologia , COVID-19/genética , Peptidil Dipeptidase A/genética , Receptores Virais/genética , SARS-CoV-2/fisiologia , Animais , Sequência de Bases , COVID-19/virologia , Especificidade de Hospedeiro , Humanos , Camundongos/genética , Camundongos/virologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Phascolarctidae/genética , Phascolarctidae/virologia , Receptores Virais/metabolismo , SARS-CoV-2/genética , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
15.
Sci Rep ; 11(1): 4405, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623127

RESUMO

Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

17.
Nat Commun ; 12(1): 961, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574281

RESUMO

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Assuntos
COVID-19/virologia , Sistemas CRISPR-Cas , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Internalização do Vírus , Células A549 , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Endossomos/virologia , Células HeLa , Humanos , Mesocricetus , Serina Endopeptidases , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
18.
Cell Rep ; 34(5): 108699, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33485405

RESUMO

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Epitopos/imunologia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/uso terapêutico , Reações Antígeno-Anticorpo , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Criança , Análise por Conglomerados , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
19.
Hepatology ; 73(2): 486-502, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32333814

RESUMO

BACKGROUND AND AIMS: Interferon (IFN)-α, composed of numerous subtypes, plays a crucial role in immune defense. As the most studied subtype, IFN-α2 has been used for treating chronic hepatitis B virus (HBV) infection, with advantages of finite treatment duration and sustained virologic response, but its efficacy remains relatively low. This study aimed to screen for IFN-α subtypes with the highest anti-HBV potency and to characterize mechanisms of IFN-α-mediated HBV restriction. APPROACH AND RESULTS: Using cell culture-based HBV infection systems and a human-liver chimeric mouse model, IFN-α subtype-mediated antiviral response and signaling activation were comprehensively analyzed. IFN-α14 was identified as the most effective subtype in suppression of HBV covalently closed circular DNA transcription and HBV e antigen/HBV surface antigen production, with median inhibitory concentration values approximately 100-fold lower than those of the conventional IFN-α2. IFN-α14 alone elicited IFN-α and IFN-γ signaling crosstalk in a manner similar to the combined use of IFN-α2 and IFN-γ, inducing multiple potent antiviral effectors, which synergistically restricted HBV replication. Guanylate binding protein 5, one of the most differentially expressed genes between IFN-α14-treated and IFN-α2-treated liver cells, was identified as an HBV restriction factor. A strong IFN-α-IFN-α receptor subunit 1 interaction determines the anti-HBV activity of IFN-α. The in vivo anti-HBV activity of IFN-α14 and treatment-related transcriptional patterns were further confirmed, and few adverse effects were observed. CONCLUSIONS: A concerted IFN-α and IFN-γ response in liver, which could be efficiently elicited by IFN-α subtype 14, is associated with potent HBV suppression. These data deepen the understanding of the divergent activities of IFN-α subtypes and the mechanism underlying the synergism between IFN-α and IFN-γ signaling, with implications for improved IFN therapy and HBV curative strategies.


Assuntos
Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/farmacologia , Interferon gama/metabolismo , Animais , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/transplante , Humanos , Interferon-alfa/genética , Interferon-alfa/uso terapêutico , Camundongos , Camundongos Knockout , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resposta Viral Sustentada , Quimeras de Transplante , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
20.
Antiviral Res ; 185: 104974, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33217430

RESUMO

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Cromossomos Artificiais Bacterianos , Replicon/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , COVID-19/tratamento farmacológico , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Interferon-alfa/farmacologia , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Sofosbuvir/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacos
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