RESUMO
Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Animais , Camundongos , Humanos , Vacinas contra COVID-19 , Anticorpos Antivirais , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinação/métodos , Vírion , Imunogenicidade da VacinaRESUMO
BACKGROUND: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. METHODS: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. FINDINGS: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. INTERPRETATION: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. FUNDING: Funding bodies are described in the Acknowledgments section.
Assuntos
COVID-19 , Humanos , Animais , Cricetinae , Tratamento Farmacológico da COVID-19 , Atraso no Tratamento , SARS-CoV-2 , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Corticosteroides , Antivirais/farmacologia , Antivirais/uso terapêuticoRESUMO
Thrombosis is the major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the pathology of vascular endothelial cells (ECs) has received much attention. Although there is evidence of the infection of ECs in human autopsy tissues, their detailed pathophysiology remains unclear due to the lack of animal model to study it. We used a mouse-adapted SARS-CoV-2 virus strain in young and mid-aged mice. Only mid-aged mice developed fatal pneumonia with thrombosis. Pulmonary ECs were isolated from these infected mice and RNA-Seq was performed. The pulmonary EC transcriptome revealed that significantly higher levels of viral genes were detected in ECs from mid-aged mice with upregulation of viral response genes such as DDX58 and IRF7. In addition, the thrombogenesis-related genes encoding PLAT, PF4, F3 PAI-1, and P-selectin were upregulated. In addition, the inflammation-related molecules such as CXCL2 and CXCL10 were upregulated in the mid-aged ECs upon viral infection. Our mouse model demonstrated that SARS-CoV-2 virus entry into aged vascular ECs upregulated thrombogenesis and inflammation-related genes and led to fatal pneumonia with thrombosis. Current results of EC transcriptome showed that EC uptake virus and become thrombogenic by activating neutrophils and platelets in the aged mice, suggesting age-associated EC response as a novel finding in human severe COVID-19.
Assuntos
COVID-19 , Pneumonia , Trombose , Humanos , Camundongos , Animais , Pessoa de Meia-Idade , Idoso , SARS-CoV-2 , Células Endoteliais , Pulmão/patologia , Inflamação/patologia , Pneumonia/patologia , Trombose/patologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.
Assuntos
Nucleosídeos , Vírus de RNA de Cadeia Positiva , Animais , Camundongos , Nucleosídeos/farmacologia , Antivirais/farmacologia , SARS-CoV-2 , Replicação Viral , RNARESUMO
Bunyaviruses, including the Lassa virus (LASV), are known to cause hemorrhagic fever and have a high fatality rate among hospitalized patients, as there are few effective treatments. We focused on the fact that bunyaviruses use cap-dependent endonuclease (CEN) for viral replication, which is similar to influenza viruses. This led us to screen carbamoyl pyridone bicycle (CAB) compounds, which compose a series of baloxavir acid (BXA) derivatives, against lymphocytic choriomeningitis virus (LCMV) and Junin virus (JUNV) among the bunyaviruses. This led to the discovery of 1c, which has potent anti-bunyaviral activities. In SAR studies, we found that a large lipophilic side chain is preferred for the 1-position of the CAB scaffold, similar to the influenza CEN inhibitor, and that a small alkyl group for the 3-position shows high activity. Moreover, the 7carboxyl group of the scaffold is essential for anti-bunyaviral activities, and the antiviral activity is reduced by conversion to various carboxylic acid bioisosteres. The SAR results are discussed using a binding model of 9d in the active center of the known LCMV CEN crystal structure. These compounds show promise as broad-spectrum anti-bunyavirus therapeutics, given their relatively favorable metabolic stability and PK profiles.
Assuntos
Influenza Humana , Orthomyxoviridae , Humanos , Relação Estrutura-Atividade , Antivirais/farmacologia , Antivirais/química , Endonucleases/metabolismoRESUMO
Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.
Assuntos
Quirópteros , Murinae , Infecções por Rotavirus , Rotavirus , Animais , Quirópteros/virologia , Diarreia/veterinária , Diarreia/virologia , Genoma Viral , Genótipo , Quênia , Filogenia , Rotavirus/genética , Rotavirus/isolamento & purificação , Infecções por Rotavirus/veterinária , Murinae/virologiaRESUMO
In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.
Assuntos
COVID-19 , Humanos , Cricetinae , SARS-CoV-2 , Carga Viral , Estudos Prospectivos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/metabolismoRESUMO
The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/química , Cisteína , Cisteína Endopeptidases/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Peptídeos/químicaRESUMO
OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. METHODS: Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24â h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT-qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. RESULTS: Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. CONCLUSIONS: This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Animais , Feminino , Camundongos , Antivirais/uso terapêutico , Antivirais/farmacologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão , SARS-CoV-2 , Redução de PesoRESUMO
Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.
Assuntos
Antivirais , Endonucleases , Orthobunyavirus , Animais , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Endonucleases/antagonistas & inibidores , Humanos , Camundongos , Orthobunyavirus/efeitos dos fármacos , Orthobunyavirus/genética , Orthobunyavirus/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV.
Assuntos
Culicidae , Flavivirus , Sequência de Aminoácidos , Animais , Insetos , FilogeniaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Vacinas contra COVID-19 , Proteases 3C de Coronavírus , Humanos , Camundongos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêuticoRESUMO
Human infections caused by the H5 highly pathogenic avian influenza virus (HPAIV) sporadically threaten public health. The susceptibility of HPAIVs to baloxavir acid (BXA), a new class of inhibitors for the influenza virus cap-dependent endonuclease, has been confirmed in vitro, but it has not yet been fully characterized. Here, the efficacy of BXA against HPAIVs, including recent H5N8 variants, was assessed in vitro. The antiviral efficacy of baloxavir marboxil (BXM) in H5N1 virus-infected mice was also investigated. BXA exhibited similar in vitro activities against H5N1, H5N6, and H5N8 variants tested in comparison with seasonal and other zoonotic strains. Compared with oseltamivir phosphate (OSP), BXM monotherapy in mice infected with the H5N1 HPAIV clinical isolate, the A/Hong Kong/483/1997 strain, also caused a significant reduction in viral titers in the lungs, brains, and kidneys, thereby preventing acute lung inflammation and reducing mortality. Furthermore, compared with BXM or OSP monotherapy, combination treatments with BXM and OSP using a 48-h delayed treatment model showed a more potent effect on viral replication in the organs, accompanied by improved survival. In conclusion, BXM has a potent antiviral efficacy against H5 HPAIV infections.
Assuntos
Dibenzotiepinas/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Morfolinas/farmacologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Piridonas/farmacologia , Triazinas/farmacologia , Células A549 , Animais , Antivirais/farmacologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Quimioterapia Combinada , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Oseltamivir/farmacologia , Pneumonia/tratamento farmacológico , Análise de Sequência , Replicação Viral/efeitos dos fármacosRESUMO
Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.
RESUMO
The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.
Assuntos
Células Epiteliais Alveolares/virologia , COVID-19/virologia , Técnicas de Cultura de Células/métodos , SARS-CoV-2/fisiologia , Células A549 , Células Epiteliais Alveolares/patologia , Células Cultivadas , Suscetibilidade a Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Regulação para Cima/genéticaRESUMO
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/patologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas/imunologia , Furina/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Vacinas Atenuadas/imunologia , Células Vero , Virulência/genéticaRESUMO
Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Engenharia Celular , Descoberta de Drogas , Modelos Biológicos , SARS-CoV-2/fisiologia , Animais , Antivirais/uso terapêutico , Linhagem Celular , Humanos , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.
Assuntos
SARS-CoV-2/genética , Serina Endopeptidases/deficiência , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Mutação , SARS-CoV-2/classificação , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/fisiologia , Alinhamento de Sequência , Inoculações Seriadas , Células Vero , Tropismo ViralRESUMO
Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC50s of Compound-X and Compound-Y against DENV2 were 3.9 µM and 9.2 µM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Quinolonas/farmacologia , Replicação Viral/efeitos dos fármacos , Células A549 , Substituição de Aminoácidos , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Metiltransferases/efeitos dos fármacos , Mutação , Ácido Micofenólico/farmacologia , RNA Viral , Ribavirina/farmacologiaRESUMO
The pharmacokinetic (PK) and pharmacodynamic (PD) parameters which correlated with the in vivo efficacy of cefiderocol were evaluated using neutropenic murine thigh and lung infection models in which the infections were caused by a variety of Gram-negative bacilli. The dose fractionation study using the thigh infection model in which the infection was caused by Pseudomonas aeruginosa showed that the cumulative percentage of a 24-h period that the free drug concentration in plasma exceeds the MIC (%fT>MIC) rather than the free peak level divided by the MIC (fCmax/MIC) and the area under the free concentration-time curve over 24 h divided by the MIC (fAUC/MIC) was the PK/PD parameter that best correlated with efficacy. The study with multiple carbapenem-resistant strains revealed that the %fT>MIC determined in iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) better reflected the in vivo efficacy of cefiderocol than the %fT>MIC determined in cation-adjusted Mueller-Hinton broth (CAMHB). The mean %fT>MIC of cefiderocol required for a 1-log10 reduction against 10 strains of Enterobacteriaceae and 3 strains of Pseudomonas aeruginosa in the thigh infection models were 73.3% and 77.2%, respectively. The mean %fT>MIC for Enterobacteriaceae, P. aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia in the lung infection model were 64.4%, 70.3%, 88.1%, and 53.9%, respectively. These results indicate that cefiderocol has potent efficacy against Gram-negative bacilli, including carbapenem-resistant strains, irrespective of the bacterial species, in neutropenic thigh and lung infection models and that the in vivo efficacy correlated with the in vitro MIC under iron-deficient conditions.