RESUMO
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/química , Betacoronavirus/genética , COVID-19 , Linhagem Celular , Coronavirus/química , Coronavirus/genética , Coronavirus/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Desenvolvimento de Medicamentos , Ésteres , Gabexato/análogos & derivados , Gabexato/farmacologia , Guanidinas , Humanos , Imunização Passiva , Leucina/análogos & derivados , Leucina/farmacologia , Pandemias , Peptidil Dipeptidase A/química , Receptores Virais/química , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vesiculovirus/genética , Soroterapia para COVID-19RESUMO
Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.
Assuntos
Síndrome Hemolítico-Urêmica , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Humanos , Pré-Escolar , Criança , Adolescente , Células Epiteliais , Pulmão , Influenza Humana/epidemiologiaRESUMO
Canine distemper virus (CDV), a morbillivirus within the family Paramyxoviridae, is a highly contagious infectious agent causing a multisystemic, devastating disease in a broad range of host species, characterized by severe immunosuppression, encephalitis and pneumonia. The present study aimed at investigating pulmonary immune responses of CDV-infected dogs in situ using immunohistochemistry and whole transcriptome analyses by bulk RNA sequencing. Spatiotemporal analysis of phenotypic changes revealed pulmonary immune responses primarily driven by MHC-II+, Iba-1+ and CD204+ innate immune cells during acute and subacute infection phases, which paralleled pathologic lesion development and coincided with high viral loads in CDV-infected lungs. CD20+ B cell numbers initially declined, followed by lymphoid repopulation in the advanced disease phase. Transcriptome analysis demonstrated an increased expression of transcripts related to innate immunity, antiviral defense mechanisms, type I interferon responses and regulation of cell death in the lung of CDV-infected dogs. Molecular analyses also revealed disturbed cytokine responses with a pro-inflammatory M1 macrophage polarization and impaired mucociliary defense in CDV-infected lungs. The exploratory study provides detailed data on CDV-related pulmonary immune responses, expanding the list of immunologic parameters potentially leading to viral elimination and virus-induced pulmonary immunopathology in canine distemper.
Assuntos
Vírus da Cinomose Canina , Cinomose , Animais , Citocinas/genética , Citocinas/metabolismo , Vírus da Cinomose Canina/genética , Cães , Imunidade , Pulmão/patologiaRESUMO
Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air-liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.
Assuntos
Complexo Respiratório Bovino/microbiologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/fisiologia , Sistema Respiratório/microbiologia , Animais , Bovinos , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Infecções por Pasteurella/microbiologiaRESUMO
We analyzed the virulence of pandemic H1N1 2009 influenza A viruses in vivo and in vitro. Selected viruses isolated in 2009, 2010, 2014, and 2015 were assessed using an aerosol-mediated high-dose infection model for pigs as well as air-liquid interface cultures of differentiated airway epithelial cells. Using a dyspnea score, rectal temperature, lung lesions, and viral load in the lung as parameters, the strains from 2014-2015 were significantly less virulent than the strains isolated in 2009-2010. In vitro, the viruses from 2009-2010 also differed from the 2014-2015 viruses by increased release of infectious virus, a more pronounced loss of ciliated cells, and a reduced thickness of the epithelial cell layer. Our in vivo and in vitro results reveal an evolution of A(H1N1)pdm09 viruses toward lower virulence. Our in vitro culture system can be used to predict the virulence of influenza viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Pulmão/virologia , Infecções por Orthomyxoviridae/veterinária , Virulência , Animais , Células Cultivadas , Células Epiteliais/virologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Sus scrofa , Carga Viral/veterináriaRESUMO
Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.
Assuntos
Aderência Bacteriana/fisiologia , Coinfecção/microbiologia , Proteínas Hemolisinas/fisiologia , Pulmão/microbiologia , Infecções por Orthomyxoviridae/microbiologia , Streptococcus suis/patogenicidade , Animais , Células Cultivadas , Cães , Células Epiteliais/microbiologia , SuínosRESUMO
We detected a highly pathogenic avian influenza A(H5N8) virus in lung samples of 2 gray seals (Halichoerus grypus) stranded on the Baltic coast of Poland in 2016 and 2017. This virus, clade 2.3.4.4 B, was closely related to avian H5N8 viruses circulating in Europe at the time.
Assuntos
Vírus da Influenza A Subtipo H5N8 , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Focas Verdadeiras/virologia , Animais , Países Bálticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H5N8/classificação , Vírus da Influenza A Subtipo H5N8/genética , Vírus da Influenza A Subtipo H5N8/isolamento & purificação , Masculino , Oceanos e Mares , Filogenia , PolôniaRESUMO
Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3-linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3-linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co-sedimentation assay revealed that viruses with a preference for α2,3-linked sialic acids bind to GBS in a sialic acid-dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination-inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre-infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co-infected by influenza viruses. This co-infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3-linked sialic acids and human viruses binding to this linkage type.
Assuntos
Vírus da Influenza A/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos Bacterianos/metabolismo , Streptococcus agalactiae/metabolismo , Coinfecção , Humanos , Influenza Humana/complicações , Infecções Estreptocócicas/complicaçõesRESUMO
Porcine precision-cut lung slices (PCLS) were used to analyze the effect of the ciliary activity on infection of airway epithelial cells by influenza viruses. Treatment of slices with 2% NaCl for 30 min resulted in reversible ciliostasis. When PCLS were infected by a swine influenza virus of the H3N2 subtype under ciliostatic conditions, the viral yield was about twofold or threefold higher at 24 or 48 h post-infection, respectively, as compared to slices with ciliary activity. Therefore, the cilia beating not only transports the mucus out of the airways, it also impedes virus infection.
Assuntos
Pulmão/fisiopatologia , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Cílios/patologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Pulmão/virologia , Infecções por Orthomyxoviridae/fisiopatologia , Infecções por Orthomyxoviridae/virologia , Suínos , Doenças dos Suínos/fisiopatologiaRESUMO
In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Interferons/imunologia , Mycoplasma hyopneumoniae/fisiologia , Infecções por Orthomyxoviridae/veterinária , Pneumonia Suína Micoplasmática/microbiologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Animais , Suscetibilidade a Doenças , Vírus da Influenza A Subtipo H1N1/genética , Interferons/genética , Interleucina-6/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/virologia , Macrófagos/imunologia , Mycoplasma hyopneumoniae/genética , Infiltração de Neutrófilos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Suína Micoplasmática/imunologia , Suínos , Doenças dos Suínos/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.
Assuntos
Encéfalo/virologia , Quirópteros/virologia , Proteína HN/imunologia , Vírus da Caxumba/imunologia , Caxumba/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/imunologia , Encéfalo/imunologia , Feminino , Expressão Gênica , Proteína HN/administração & dosagem , Proteína HN/genética , Humanos , Masculino , Caxumba/prevenção & controle , Caxumba/virologia , Vírus da Caxumba/classificação , Vírus da Caxumba/genética , Vírus da Caxumba/patogenicidade , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , VirulênciaRESUMO
UNLABELLED: A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE: The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.
Assuntos
Quirópteros/virologia , Regulação Viral da Expressão Gênica/genética , Proteína HN/genética , Vírus da Caxumba/enzimologia , Proteínas Virais de Fusão/genética , Animais , Anticorpos Antivirais/imunologia , Sequência de Bases , Chlorocebus aethiops , Primers do DNA/genética , Citometria de Fluxo , Células Gigantes/metabolismo , Proteína HN/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Vírus da Caxumba/genética , Plasmídeos/genética , Análise de Sequência de DNA , Homologia de Sequência , Células Vero , Proteínas Virais de Fusão/metabolismoRESUMO
Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.
Assuntos
Ebolavirus/metabolismo , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Macrófagos/virologia , Fatores de Virulência/metabolismo , Linhagem Celular , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Lectinas/metabolismo , Internalização do VírusRESUMO
Bacterial co-infections are a major complication in influenza-virus-induced disease in both humans and animals. Either of the pathogens may induce a host response that affects the infection by the other pathogen. A unique feature in the co-infection by swine influenza viruses (SIV) and Streptococcus suis serotype 2 is the direct interaction between the two pathogens. It is mediated by the haemagglutinin of SIV that recognizes the α2,6-linked sialic acid present in the capsular polysaccharide of Streptococcus suis. In the present study, this interaction was demonstrated for SIV of both H1N1 and H3N2 subtypes as well as for human influenza viruses that recognize α2,6-linked sialic acid. Binding of SIV to Streptococcus suis resulted in co-sedimentation of virus with bacteria during low-speed centrifugation. Viruses bound to bacteria retained infectivity but induced only tiny plaques compared with control virus. Infection of porcine tracheal cells by SIV facilitated adherence of Streptococcus suis, which was evident by co-staining of bacterial and viral antigen. Sialic-acid-dependent binding of Streptococcus suis was already detectable after incubation for 30âmin. By contrast, bacterial co-infection had a negative effect on the replication of SIV as indicated by lower virus titres in the supernatant and a delay in the kinetics of virus release.
Assuntos
Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Infecções por Orthomyxoviridae/veterinária , Infecções Estreptocócicas/microbiologia , Streptococcus suis/metabolismo , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Animais , Coinfecção/microbiologia , Coinfecção/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Infecções por Orthomyxoviridae/virologia , Ligação Proteica , Suínos , Traqueia/microbiologia , Traqueia/virologiaRESUMO
Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1) but not in nonchiropteran BHK-21 and Vero76 cells. Here, we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon coexpression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the nonchiropteran cells was detectable only in a fraction of cells. In contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and nonchiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the endoplasmic reticulum (ER), as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type, as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems, with larger amounts of viral glycoprotein present on chiropteran cells than on nonchiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses, which is necessary to assess the risk of zoonotic virus transmission. Importance: Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a zoonotic potential as high as that of their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein expression of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in nonchiropteran cells than it is in chiropteran cells, explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.
Assuntos
Quirópteros/virologia , Henipavirus/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Compartimento Celular , Linhagem Celular , Cricetinae , Citometria de Fluxo , Células Gigantes , Humanos , Especificidade da EspécieRESUMO
Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry. These viruses include important human and animal pathogens, such as influenza, parainfluenza, mumps, corona, noro, rota, and DNA tumor viruses. Attachment to sialic acid is mediated by receptor binding proteins that are constituents of viral envelopes or exposed at the surface of non-enveloped viruses. Some of these viruses are also equipped with a neuraminidase or a sialyl-O-acetyl-esterase. These receptor-destroying enzymes promote virus release from infected cells and neutralize sialic acid-containing soluble proteins interfering with cell surface binding of the virus. Variations in the receptor specificity are important determinants for host range, tissue tropism, pathogenicity, and transmissibility of these viruses.
Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virais/metabolismo , Vírus/metabolismo , Acetilesterase/química , Acetilesterase/metabolismo , Animais , Membrana Celular/química , Membrana Celular/virologia , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Células Eucarióticas/virologia , Humanos , Neuraminidase/química , Neuraminidase/metabolismo , Ligação Proteica , Receptores de Superfície Celular/química , Receptores Virais/química , Ácidos Siálicos/química , Especificidade da Espécie , Proteínas Virais/química , Tropismo Viral/fisiologia , Internalização do Vírus , Vírus/químicaRESUMO
BACKGROUND: The lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (PEDV) pathogenesis. Vero cell, an African green monkey kidney cell line, was often used to isolate and propagate PEDV. Nonetheless, the target cells of PEDV in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. The immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. Type I interferon (IFN) plays an important role in antiviral immune response. Limited reports showed that PEDV could inhibit type I interferon production. In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-ß production. RESULTS: PEDV not only failed to induce IFN-ß expression, but also inhibited dsRNA-mediated IFN-ß production in IECs. As the key IFN-ß transcription factors, we found that dsRNA-induced activation of IFN regulatory factor 3 (IRF-3) was inhibited after PEDV infection, but not nuclear factor-kappaB (NF-κB). To identify the mechanism of PEDV intervention with dsRNA-mediated IFN-ß expression more accurately, the role of individual molecules of RIG-I signaling pathway were investigated. In the upstream of IRF-3, TANK-binding kinase 1 (TBK1)-or inhibitor of κB kinase-ε (IKKε)-mediated IFN-ß production was not blocked by PEDV, while RIG-I-and its adapter molecule IFN-ß promoter stimulator 1 (IPS-1)-mediated IFN-ß production were completely inhibited after PEDV infection. CONCLUSION: Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-ß production by blocking the activation of IPS-1 in RIG-I-mediated pathway. Our studies offered new visions in understanding of the interaction between PEDV and host innate immune system.
Assuntos
Interferon beta/biossíntese , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Vírus da Diarreia Epidêmica Suína/fisiologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais , Fator Regulador 3 de Interferon/metabolismo , NF-kappa B/metabolismo , Poli I-C/farmacologia , Suínos , Células VeroRESUMO
In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression.
Assuntos
Quirópteros/virologia , Glicoproteínas/metabolismo , Infecções por Henipavirus/veterinária , Henipavirus/isolamento & purificação , Henipavirus/metabolismo , Proteínas Virais/metabolismo , África , Animais , Quirópteros/metabolismo , Glicoproteínas/genética , Henipavirus/classificação , Henipavirus/genética , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Vírus Nipah/genética , Vírus Nipah/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Transmissible gastroenteritis virus (TGEV) is an enveloped (+) RNA virus belonging to the family Coronaviridae. Among the viral membrane proteins, the spike (S) protein mediates receptor recognition/attachment to the host cell and fusion of viral and cellular membranes. The cytoplasmic tail of the S protein contains a tyrosine-dependent sorting signal with the consensus sequence YXXΦ. In the context of the S protein of TGEV (1440YEPI1443), this motif acts as a retention signal, preventing surface expression of the protein. Here, we show that a chimeric S protein, containing the six C-terminal amino acids of the glycoprotein G of vesicular stomatitis virus (VSV) is no longer retained intracellularly, despite the presence of the tyrosine tetrapeptide motif. Following transport to the cell surface, the chimeric protein was rapidly endocytosed. Analysis of mutant proteins generated by site-directed mutagenesis revealed that a single amino acid exchange (1445K/M, position: +2 downstream of the tyrosine-based motif) was responsible for the altered sorting behavior.
Assuntos
Coronavirus/metabolismo , Lisina/metabolismo , Metionina/genética , Transporte Proteico/fisiologia , Endocitose , TransfecçãoRESUMO
Serological screening and detection of genomic RNA indicates that members of the genus Henipavirus are present not only in Southeast Asia but also in African fruit bats. We demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional similarities to glycoproteins of Nipah virus.