RESUMO
Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
Assuntos
Pirazinas/química , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Recombinação Genética , Ribonucleotídeos/química , Animais , Antivirais , Catálise , Células Cultivadas , Técnicas Genéticas , Genoma , Genoma Viral , Recombinação Homóloga , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Mutagênese , Nucleotídeos/genética , Conformação Proteica , RNA/química , RNA Polimerase Dependente de RNA/metabolismo , RNA-Seq , Transgenes , VirulênciaRESUMO
Since April 2022, waves of SARS-CoV-2 Omicron variant cases have surfaced in Taiwan and spread throughout the island. Using high-throughput sequencing of the SARS-CoV-2 genome, we analyzed 2,405 PCR-positive swab samples from 2,339 persons and identified the Omicron BA.2.3.7 variant as a major lineage within recent community outbreaks in Taiwan.
Assuntos
COVID-19 , Humanos , Taiwan/epidemiologia , COVID-19/epidemiologia , SARS-CoV-2/genética , Surtos de DoençasRESUMO
BACKGROUND: The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene. METHODS: We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance. RESULTS: M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels. CONCLUSIONS: The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Animais , Camundongos , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Filogenia , Camundongos Endogâmicos C57BL , Nucleotídeos , Influenza Humana/tratamento farmacológico , Influenza Humana/genéticaRESUMO
There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.
Assuntos
Northern Blotting/métodos , Enterovirus Humano A/genética , Genoma Viral , RNA Viral/análise , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação Viral da Expressão Gênica , Humanos , Sítios Internos de Entrada Ribossomal/genética , Camundongos , Interferência de RNA , RNA Interferente Pequeno , RNA Viral/metabolismo , Ribonuclease III , Análise de Sequência de RNA/métodos , Replicação Viral/genéticaRESUMO
Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.
Assuntos
Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Cultura de Vírus/métodos , Betacoronavirus/genética , COVID-19 , Teste para COVID-19 , Vacinas contra COVID-19 , Correlação de Dados , Humanos , Nasofaringe/virologia , Pandemias , Faringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2RESUMO
Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.
Assuntos
Enterovirus Humano A/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Surtos de Doenças , Enterovirus/genética , Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Genoma Viral/genética , Humanos , Mutação , Filogenia , Poliomielite/epidemiologia , Poliomielite/virologia , Estudos Retrospectivos , VirulênciaRESUMO
Enteroviruses, which may cause neurological complications, have become a public health threat worldwide in recent years. Interactions between cellular proteins and enteroviral proteins could interfere with cellular biological processes to facilitate viral replication in infected cells. Enteroviral RNA-dependent RNA polymerase (RdRP), known as 3D protein, mainly functions as a replicase for viral RNA synthesis in infected cells. However, the 3D protein encoded by enterovirus A71 (EV-A71) could also interact with several cellular proteins to regulate cellular events and responses during infection. To globally investigate the functions of the EV-A71 3D protein in regulating biological processes in host cells, we performed immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify host proteins that may associate with the 3D protein. We found that the 3D protein interacts with factors involved in translation-related biological processes, including ribosomal proteins. In addition, polysome profiling analysis showed that the 3D protein cosediments with small and large subunits of ribosomes. We further discovered that the EV-A71 3D protein could enhance EV-A71 internal ribosome entry site (IRES)-dependent translation as well as cap-dependent translation. Collectively, this research demonstrated that the RNA polymerase encoded by EV-A71 could join a functional ribosomal complex and positively regulate viral and host translation.
Assuntos
Enterovirus Humano A/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Linhagem Celular , Cromatografia Líquida , Células HEK293 , Células HeLa , Humanos , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismoRESUMO
The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Enterovirus Humano A , Regulação Viral da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Sítios Internos de Entrada Ribossomal/fisiologia , Proteínas Virais/metabolismo , Regiões 5' não Traduzidas/fisiologia , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Immunoblotting , Imunoprecipitação , Sítios Internos de Entrada Ribossomal/genética , Biossíntese de Proteínas/fisiologia , Proteínas de Ligação a RNARESUMO
Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis.
Assuntos
Mutação Puntual , Biossíntese de Proteínas/fisiologia , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Substituição de Aminoácidos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Capuzes de RNA/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication.
Assuntos
Enzimas Reparadoras do DNA/fisiologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/química , Proteínas Nucleares/fisiologia , Proteômica/métodos , Fatores de Processamento de RNA/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Cromatografia Líquida , Humanos , Imunoprecipitação , Vírus da Influenza A Subtipo H1N1/fisiologia , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/análiseRESUMO
The spliceosomal factor TRAP150 is essential for pre-mRNA splicing in vivo and, when overexpressed, it enhances splicing efficiency. In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5' splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3' splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression.
Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/metabolismo , Éxons , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Células HEK293 , Células HeLa , Humanos , Poliadenilação , Sítios de Splice de RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismoRESUMO
It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression.
Assuntos
RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Cromatina/metabolismo , Epigênese Genética , RNA Polimerase II/química , Precursores de RNA/genética , Estabilidade de RNARESUMO
Since the COVID-19 pandemic was declared, vaccines against SARS-CoV-2 have been urgently developed around the world. On the basis of the mRNA vaccine technology developed previously, COVID-19 mRNA vaccines were promptly tested in animals, advanced to clinical trials, and then authorized for emergency use in humans. The administration of COVID-19 mRNA vaccines has successfully reduced the hospitalization and mortality caused by the viral infection, although the virus continuously evolves with its transmission. Therefore, the development of mRNA vaccine technology, including RNA modification and delivery systems, is well recognized for its contribution to moderating the harms caused by the COVID-19 pandemic. The scientists who developed these technologies, Katalin Karikó, Drew Weissman, and Pieter Cullis, were awarded the 2022 Tang Prize in Biopharmaceutical Science. In this review, we summarize the principles, safety and efficacy of as well as the immune response to COVID-19 mRNA vaccines. Since mRNA vaccine approaches could be practical for the prevention of infectious diseases, we also briefly describe mRNA vaccines against other human viral pathogens in clinical trials.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Pandemias/prevenção & controle , Vacinas de mRNARESUMO
The mechanisms underlying tissue-specific replication of enteroviruses are currently unclear. Although enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are both common pathogens that cause hand-foot-mouth disease, they display quite different neurotropic properties. Herein, we characterized the role of the internal ribosome entry site (IRES) in determining neurovirulence using an oral inoculation model of EV-A71. The receptor transgenic (hSCARB2-Tg) mice developed neurological symptoms after being infected with a mouse-adapted EV-A71 strain (MP4) via different administrative routes. Intragastric administration of the MP4 strain caused pathological changes such as neuronal loss and neuropil vacuolation, whereas replacing EV-A71 IRES with CV-A16 abolished the neuropathological phenotypes. The attenuated neurotropic potential of IRES-swapped EV-A71 was observed even in mice that received intraperitoneal and intracerebral inoculations. Fewer chimeric MP4 viral RNAs and proteins were detected in the mouse tissues, regardless of the inoculation route. Tissue-specific replication can be reflected in cell-based characterization. While chimeric MP4 virus replicated poorly in human intestinal C2BBe1 and neuroblastoma SH-SY5Y cells, its replication in susceptible rhabdomyosarcoma cells was not affected. Overall, our results demonstrated that the IRES determined the neurotropic potential of EV-A71 and CV-A16, emphasizing the importance of the IRES in tissue tropism, along with the host receptors.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Doença de Mão, Pé e Boca , Neuroblastoma , Humanos , Animais , Camundongos , Enterovirus/genética , Enterovirus Humano A/genética , Sítios Internos de Entrada Ribossomal , Antígenos ViraisRESUMO
TRAP150 has been identified as a subunit of the transcription regulatory complex TRAP/Mediator, and also a component of the spliceosome. The exact function of TRAP150, however, remains unclear. We recently identified TRAP150 by its association with the mRNA export factor TAP. TRAP150 contains an arginine/serine-rich domain and has sequence similarity with the cell death-promoting transcriptional repressor BCLAF1. We found that TRAP150 co-localizes with splicing factors in nuclear speckles, and is required for pre-mRNA splicing and activates splicing in vivo. TRAP150 remains associated with the spliced mRNA after splicing, and accordingly, it interacts with the integral exon junction complex. Unexpectedly, when tethered to a precursor mRNA, TRAP150 can trigger mRNA degradation in the nucleus. However, unlike nonsense-mediated decay, TRAP150-mediated mRNA decay is irrespective of the presence of upstream stop codons and occurs in the nucleus. Moreover, TRAP150 activates pre-mRNA splicing and induces mRNA degradation by its separable functional domains. Therefore, TRAP150 represents a multi-functional protein involved in nuclear mRNA metabolism.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Códon sem Sentido , Humanos , Camundongos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
A novel coronavirus (CoV) that emerged in Wuhan, Hubei province in China, in December 2019, has rapidly spread worldwide. Named as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this virus has been responsible for infecting about 153 million people and causing 3 million deaths by May 2021. There is obvious interest in gaining novel insights into the epidemiologic evolution of this virus; however, inappropriate application and interpretation of genomic and phylogenetic analyses has led to dangerous outcomes and misunderstandings. This chapter focuses on not only introducing this virus, its genomic characteristics and molecular mechanisms but also describing the application and interpretation of phylogenetic tree analyses, in order to provide useful information to better understand the evolution and epidemiology of this virus. In addition, recombinant region and genetic ancestry of SARS-CoV-2 remain unknown. It is urgently required to collect samples and obtain related viral genetic data from animal sources for identifying the intermediate host of SARS-CoV-2 that is responsible for its cross-species transmission.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , COVID-19/epidemiologia , China/epidemiologia , Humanos , Filogenia , SARS-CoV-2/genéticaRESUMO
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The global COVID-19 pandemic continues to threaten the lives of hundreds of millions of people, with a severe negative impact on the global economy. Although several COVID-19 vaccines are currently being administered, none of them is 100% effective. Moreover, SARS-CoV-2 variants remain an important worldwide public health issue. Hence, the accelerated development of efficacious antiviral agents is urgently needed. Coronavirus depends on various host cell factors for replication. An ongoing research objective is the identification of host factors that could be exploited as targets for drugs and compounds effective against SARS-CoV-2. In the present review, we discuss the molecular mechanisms of SARS-CoV-2 and related coronaviruses, focusing on the host factors or pathways involved in SARS-CoV-2 replication that have been identified by genome-wide CRISPR screening.
Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Vacinas contra COVID-19 , Humanos , Pandemias/prevenção & controle , SARS-CoV-2/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (-20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at -20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at -20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant.IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at -20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and -20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.
Assuntos
COVID-19 , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Temperatura Baixa , Aptidão Genética/genética , Humanos , Mutação , Estabilidade ProteicaRESUMO
Enterovirus-induced infection of the central nervous system (CNS) results in acute inflammation of the brain (encephalitis) and constitutes a significant global burden to human health. These viruses are thought to be highly cytolytic, therefore normal brain function could be greatly compromised following enteroviral infection of the CNS. A further layer of complexity is added by evidence showing that some enteroviruses may establish a persistent infection within the CNS and eventually lead to pathogenesis of certain neurodegenerative disorders. Interestingly, enterovirus encephalitis is particularly common among young children, suggesting a potential causal link between the development of the neuroimmune system and enteroviral neuroinvasion. Although the CNS involvement in enterovirus infections is a relatively rare complication, it represents a serious underlying cause of mortality. Here we review a selection of enteroviruses that infect the CNS and discuss recent advances in the characterization of these enteroviruses with regard to their routes of CNS infection, tropism, virulence, and immune responses.
RESUMO
Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.