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1.
Proc Natl Acad Sci U S A ; 121(24): e2400163121, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38830098

RESUMEN

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.


Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Phlebovirus , Animales , Anticuerpos Biespecíficos/inmunología , Ratones , Anticuerpos Neutralizantes/inmunología , Phlebovirus/inmunología , Humanos , Anticuerpos Antivirales/inmunología , Glicoproteínas/inmunología , Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Modelos Animales de Enfermedad , Síndrome de Trombocitopenia Febril Grave/inmunología , Síndrome de Trombocitopenia Febril Grave/prevención & control
2.
Proc Natl Acad Sci U S A ; 121(44): e2412653121, 2024 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-39441632

RESUMEN

EfpA, the first major facilitator superfamily (MFS) protein identified in Mycobacterium tuberculosis (Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA's role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.


Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Transporte Biológico
3.
Proc Natl Acad Sci U S A ; 119(42): e2210724119, 2022 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-36191180

RESUMEN

Influenza virus neuraminidase (NA) is an important target for antiviral development because it plays a crucial role in releasing newly assembled viruses. Two unique influenza-like virus genomes were recently reported in the Wuhan Asiatic toad and Wuhan spiny eel. Their NA genes appear to be highly divergent from all known influenza NAs, raising key questions as to whether the Asiatic toad influenza-like virus NA (tNA) and spiny eel NA (eNA) have canonical NA activities and structures and whether they show sensitivity to NA inhibitors (NAIs). Here, we found that both tNA and eNA have neuraminidase activities. A detailed structural analysis revealed that tNA and eNA present similar overall structures to currently known NAs, with a conserved calcium binding site. Inhibition assays indicated that tNA is resistant to NAIs, while eNA is still sensitive to NAIs. E119 is conserved in canonical NAs. The P119E substitution in tNA can restore sensitivity to NAIs, and, in contrast, the E119P substitution in eNA decreased its sensitivity to NAIs. The structures of NA-inhibitor complexes further provide a detailed insight into NA-inhibitor interactions at the atomic level. Moreover, tNA and eNA have unique N-glycosylation sites compared with canonical NAs. Collectively, the structural features, NA activities, and sensitivities to NAIs suggest that fish- and amphibian-derived influenza-like viruses may circulate in these vertebrates. More attention should be paid to these influenza-like viruses because their NA molecules may play roles in the emergence of NAI resistance.


Asunto(s)
Gripe Humana , Orthomyxoviridae , Animales , Antivirales/farmacología , Calcio , Farmacorresistencia Viral/genética , Anguilas/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Neuraminidasa/química , Neuraminidasa/genética , Orthomyxoviridae/metabolismo
4.
Biotechnol Appl Biochem ; 70(5): 1731-1740, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37096330

RESUMEN

Interleukin-31 (IL-31), belonging to the IL-6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors' progression. Here, we reported the expression and purification of recombinant human IL-31 (rhIL-31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size-exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL-31 was mainly composed of alpha-helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL-31 exhibited a good binding ability to the recombinant hIL-31 receptor alpha fused with human Fc fragment (rhIL-31RA-hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL-31 was able to bind to hIL-31RA or hOSMRß expressed on the cell surface, independently. Furthermore, rhIL-31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL-31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL-31-related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL-31.


Asunto(s)
Interleucinas , Humanos , Ensayo de Inmunoadsorción Enzimática , Interleucinas/genética , Proteínas Recombinantes
5.
Anim Biotechnol ; 34(7): 2724-2735, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36007548

RESUMEN

Donkey milk has high nutritional and medicinal value, but there are few researches in donkey milk traits, especially on genome. The whole lactation of 89 donkeys was recorded and it was found that Xinjiang donkey had good lactation performance while great differences among individuals. In our previous study, four genes including LGALS2, NUMB, ADCY8 and CA8 were identified as milk-associated with Chinese Kazakh house, based on Equine 670k Chip genomic analysis. And then 15 SNPs of the four key genes were conducted for genotyping in Xinjiang donkey in this study, one of Chinese indigenous breed, 14 SNPs were successful classified. And those SNPs were correlation analysis with milk yield of Xinjiang donkeys. The results showed that NUMB g.46709914T > G was significantly correlated with daily milk yield of Xinjiang donkey in the early, middle, and late periods, while ADCY8 g.48366302T > C, CA8 g.89567442T > G and CA8 g.89598328T > A were significantly correlated with lactation in the late periods. These results indicate that NUMB g.46709914T > G can be as markers of candidate genes for lactating traits in donkeys, SNPs of ADCY8 and CA8 as potential. Our findings will not only help confirm key genes for donkey milk traits, but also provide future for genomic selection in donkeys.


Asunto(s)
Equidae , Leche , Femenino , Caballos , Animales , Equidae/genética , Lactancia/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética
6.
Biochem Biophys Res Commun ; 614: 114-119, 2022 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-35576682

RESUMEN

Oncostatin M receptor beta (OSMRß) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRß might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRß (cOSMRß) was experimentally expressed as a native antigen to develop an effective cOSMRß-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRß expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRß with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRß (fOSMRß) and human OSMRß (hOSMRß). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623-734) of cOSMRß. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.


Asunto(s)
Subunidad beta del Receptor de Oncostatina M , Transducción de Señal , Animales , Anticuerpos Monoclonales , Gatos , Perros , Inflamación , Ratones , Oncostatina M/metabolismo , Subunidad beta del Receptor de Oncostatina M/metabolismo , Prurito
7.
JAMA ; 323(16): 1582-1589, 2020 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-32219428

RESUMEN

Importance: Coronavirus disease 2019 (COVID-19) is a pandemic with no specific therapeutic agents and substantial mortality. It is critical to find new treatments. Objective: To determine whether convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Design, Setting, and Participants: Case series of 5 critically ill patients with laboratory-confirmed COVID-19 and acute respiratory distress syndrome (ARDS) who met the following criteria: severe pneumonia with rapid progression and continuously high viral load despite antiviral treatment; Pao2/Fio2 <300; and mechanical ventilation. All 5 were treated with convalescent plasma transfusion. The study was conducted at the infectious disease department, Shenzhen Third People's Hospital in Shenzhen, China, from January 20, 2020, to March 25, 2020; final date of follow-up was March 25, 2020. Clinical outcomes were compared before and after convalescent plasma transfusion. Exposures: Patients received transfusion with convalescent plasma with a SARS-CoV-2-specific antibody (IgG) binding titer greater than 1:1000 (end point dilution titer, by enzyme-linked immunosorbent assay [ELISA]) and a neutralization titer greater than 40 (end point dilution titer) that had been obtained from 5 patients who recovered from COVID-19. Convalescent plasma was administered between 10 and 22 days after admission. Main Outcomes and Measures: Changes of body temperature, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), Pao2/Fio2, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilatory and extracorporeal membrane oxygenation (ECMO) supports before and after convalescent plasma transfusion. Results: All 5 patients (age range, 36-65 years; 2 women) were receiving mechanical ventilation at the time of treatment and all had received antiviral agents and methylprednisolone. Following plasma transfusion, body temperature normalized within 3 days in 4 of 5 patients, the SOFA score decreased, and Pao2/Fio2 increased within 12 days (range, 172-276 before and 284-366 after). Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2-specific ELISA and neutralizing antibody titers increased following the transfusion (range, 40-60 before and 80-320 on day 7). ARDS resolved in 4 patients at 12 days after transfusion, and 3 patients were weaned from mechanical ventilation within 2 weeks of treatment. Of the 5 patients, 3 have been discharged from the hospital (length of stay: 53, 51, and 55 days), and 2 are in stable condition at 37 days after transfusion. Conclusions and Relevance: In this preliminary uncontrolled case series of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these observations require evaluation in clinical trials.


Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Betacoronavirus/inmunología , Infecciones por Coronavirus/terapia , Neumonía Viral/terapia , Síndrome de Dificultad Respiratoria/terapia , Adulto , Anciano , Anticuerpos Antivirales/sangre , Antivirales/uso terapéutico , Donantes de Sangre , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/fisiopatología , Enfermedad Crítica , Femenino , Glucocorticoides/uso terapéutico , Humanos , Inmunización Pasiva , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Puntuaciones en la Disfunción de Órganos , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/fisiopatología , SARS-CoV-2 , Sueroterapia para COVID-19
8.
Nature ; 497(7449): 392-6, 2013 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-23615615

RESUMEN

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.


Asunto(s)
Hurones/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Especificidad del Huésped , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Receptores Virales/metabolismo , Animales , Aves/metabolismo , Aves/virología , Embrión de Pollo , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Modelos Biológicos , Modelos Moleculares , Mutación , Conformación Proteica , Especificidad de la Especie
9.
J Virol ; 90(4): 2142-9, 2016 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-26656694

RESUMEN

The neuraminidase stalk of the newly emerged H7N9 influenza virus possesses a 5-amino-acid deletion. This study focuses on characterizing the biological functions of H7N9 with varied neuraminidase stalk lengths. Results indicate that the 5-amino-acid deletion had no impact on virus infectivity or replication in vitro or in vivo compared to that of a virus with a full-length stalk, but enhanced virulence in mice was observed for H7N9 encoding a 19- to 20-amino-acid deletion, suggesting that N9 stalk length impacts virulence in mammals, as N1 stalk length does.


Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/virología , Proteínas Virales/genética , Factores de Virulencia/genética , Animales , Peso Corporal , Citocinas/análisis , Histocitoquímica , Pulmón/patología , Pulmón/virología , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/patología , Eliminación de Secuencia , Carga Viral , Virulencia
10.
J Virol ; 90(20): 9263-84, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27489273

RESUMEN

UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.


Asunto(s)
Especificidad del Huésped/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Mamíferos/virología , Virulencia/genética , Células A549 , Alelos , Animales , Aves/virología , Línea Celular , Línea Celular Tumoral , Perros , Células HEK293 , Humanos , Gripe Aviar/virología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Filogenia , Virus Reordenados/genética , Proteínas Virales/genética , Replicación Viral/genética
11.
J Infect Dis ; 214(12): 1937-1946, 2016 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-27738054

RESUMEN

BACKGROUND: The emergence of infections by the novel avian influenza A(H7N9) virus has posed a threat to human health. Cross-immunity between A(H7N9) and other heterosubtypic influenza viruses affected by antigenicity-dependent substitutions needs to be investigated. METHODS: We investigated the cellular and humoral immune responses against A(H7N9) and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), by serological and T-cell-specific assays, in a healthy population. The molecular bases of the cellular and humoral antigenic variability of A(H7N9) were illuminated by structural determination. RESULTS: We not only found that antibodies against A(H7N9) were lacking in the studied population, but also revealed that both CD4+ and CD8+ T cells that cross-reacted with A(H7N9) were at significantly lower levels than those against the A(H1N1)pdm09 peptides with substitutions. Moreover, individual peptides for A(H7N9) with low cross-reactivity were identified. Structural determination indicated that substitutions within these peptides influence the antigenic variability of A(H7N9) through both major histocompatibility complex (MHC) binding and T-cell receptor docking. CONCLUSIONS: The impact of antigenicity-dependent substitutions on cross-reactivity of T-cell immunity against the novel influenza virus A(H7N9) in the healthy population benefits the understanding of immune evasion of influenza viruses and provides a useful reference for universal vaccine development.


Asunto(s)
Reacciones Cruzadas , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Adaptación Biológica , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Donantes de Sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Estudios de Cohortes , Flujo Genético , Voluntarios Sanos , Humanos
12.
J Gen Virol ; 97(8): 1797-1806, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27217257

RESUMEN

Influenza A viruses have the potential to cause pandemics due to the introduction of novel subtypes against which human hosts have little or no preexisting immunity. Such viruses may result from reassortment between human and animal influenza viruses. Recently, new influenza-like viruses were identified in bats, raising the concern for a new reservoir of potentially harmful influenza viruses that could form reassortants with categorized human influenza A viruses. However, until now, it has not been possible to generate a recombinant reassortant virus containing a single functional gene or domain from H17N10 that could propagate. Here, we demonstrate that a recombinant A/Puerto Rico/8/1934 (H1N1) virus with NS1 gene from H17N10 influenza-like virus can be successfully rescued. We used luciferase reporter assays and quantitative reverse transcriptase PCR to show that the NS1 protein from H17N10 inhibited Sendai-virus (SeV)-induced activation of IFN-ß expression with an efficiency similar to NS1 from an H5N1 strain. Moreover, the crystal structure of the NS1 (H17N10) RNA-binding domain is also similar to that of other NS1s. These results demonstrate that H17N10 influenza-like virus indeed contains functional genes that are compatible with categorized influenza A viruses. Although the chance of this particular event occurring in nature seems negligible, further research is needed to address the possibility of the natural formation of reassortants.


Asunto(s)
Quirópteros/virología , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/veterinaria , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Modelos Moleculares , Infecciones por Orthomyxoviridae/virología , Conformación Proteica , Virus Reordenados/genética , Virus Reordenados/fisiología , Genética Inversa , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
13.
J Virol ; 89(1): 2-13, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25320305

RESUMEN

UNLABELLED: The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans. IMPORTANCE: To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.


Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Animales , Línea Celular , Embrión de Pollo , Modelos Animales de Enfermedad , Células Epiteliales/virología , Femenino , Humanos , Subtipo H7N9 del Virus de la Influenza A/fisiología , Subtipo H9N2 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Subtipo H9N2 del Virus de la Influenza A/fisiología , Ratones Endogámicos BALB C , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Reordenados/genética , Virus Reordenados/patogenicidad , Genética Inversa , Virulencia , Replicación Viral
14.
Virol J ; 12: 63, 2015 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-25880060

RESUMEN

BACKGROUND: The plateau pika (Ochotona curzoniae) is a small rabbit-like mammal that lives at high altitudes in the Qinghai-Tibet plateau and is in close contact with birds. Following the outbreak of highly pathogenic avian influenza (HPAI) H5N1 during 2005 in the migratory birds of Qinghai Lake, two clades of H5N1 have been found in pikas. However, the influenza virus receptor distribution in different tissues of this animal and its susceptibility to influenza A viruses have remained unclear. METHODS: The sialic acid receptor distribution tropism in pika was investigated using fluorescent Sambucus nigra and biotinylated Maackia amurensis I and II. Furthermore, the replication of three influenza A viruses H1N1, H3N2, and H5N1 in this animal was examined by immunohistochemistry and RT-PCR. Morphological and histopathological changes caused by infection were also analyzed with hematoxylin and eosin (H & E) staining. RESULTS: Human influenza virus-recognizing SAα2,6Gal receptors are widely expressed in the lung, kidney, liver, spleen, duodenum, ileum, rectum, and heart, whereas avian influenza virus-recognizing SAα2,3Gal receptors are strongly expressed in the trachea and lung of pika. M1 could be detected in the lungs of pikas infected with H1N1, H3N2, and H5N1 by either immunostaining or RT-PCR, and in the brain of H5N1-infected pikas. Additionally, three subtypes of influenza A viruses were able to infect pika and caused varying degrees of pneumonia with epithelial desquamation and alveolar inflammatory cell infiltration. Slight pathological changes were observed in H1N1-infected lungs. A few small bronchi and terminal bronchioles were infiltrated by lymphocytic cells in H3N2-infected lungs. In contrast, serious lung damage, such as alveolar capillary hyperemia, edema, alveolar collapse, and lymphocytic infiltrations was observed in H5N1-infected group. Furthermore, neural system changes were present in the brains of H5N1-infected pikas. CONCLUSIONS: SAα2,6Gal receptors are extensively present in many of the tissues and organs in wild plateau pika, whereas SA2,3Gal-linked receptors are dominant on the tracheal epithelial cells. H1N1, H3N2, and H5N1 were able to infect pika and caused different degrees of pathogenic changes in the lungs. Altogether, these results suggest that wild pika has the potential to be a host for different subtypes of influenza A viruses.


Asunto(s)
Enfermedades de los Animales/metabolismo , Enfermedades de los Animales/virología , Virus de la Influenza A , Lagomorpha/virología , Infecciones por Orthomyxoviridae/veterinaria , Receptores de Superficie Celular/metabolismo , Enfermedades de los Animales/genética , Enfermedades de los Animales/patología , Animales , Femenino , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Especificidad de Órganos , Receptores de Superficie Celular/genética
15.
Lancet ; 381(9881): 1926-32, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23643111

RESUMEN

BACKGROUND: On March 30, 2013, a novel avian influenza A H7N9 virus that infects human beings was identified. This virus had been detected in six provinces and municipal cities in China as of April 18, 2013. We correlated genomic sequences from avian influenza viruses with ecological information and did phylogenetic and coalescent analyses to extrapolate the potential origins of the virus and possible routes of reassortment events. METHODS: We downloaded H7N9 virus genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) database and public sequences used from the Influenza Virus Resource. We constructed phylogenetic trees and did 1000 bootstrap replicates for each tree. Two rounds of phylogenetic analyses were done. We used at least 100 closely related sequences for each gene to infer the overall topology, removed suspicious sequences from the trees, and focused on the closest clades to the novel H7N9 viruses. We compared our tree topologies with those from a bayesian evolutionary analysis by sampling trees (BEAST) analysis. We used the bayesian Markov chain Monte Carlo method to jointly estimate phylogenies, divergence times, and other evolutionary parameters for all eight gene fragments. We used sequence alignment and homology-modelling methods to study specific mutations regarding phenotypes, specifically addressing the human receptor binding properties. FINDINGS: The novel avian influenza A H7N9 virus originated from multiple reassortment events. The HA gene might have originated from avian influenza viruses of duck origin, and the NA gene might have transferred from migratory birds infected with avian influenza viruses along the east Asian flyway. The six internal genes of this virus probably originated from two different groups of H9N2 avian influenza viruses, which were isolated from chickens. Detailed analyses also showed that ducks and chickens probably acted as the intermediate hosts leading to the emergence of this virulent H7N9 virus. Genotypic and potential phenotypic differences imply that the isolates causing this outbreak form two separate subclades. INTERPRETATION: The novel avian influenza A H7N9 virus might have evolved from at least four origins. Diversity among isolates implies that the H7N9 virus has evolved into at least two different lineages. Unknown intermediate hosts involved might be implicated, extensive global surveillance is needed, and domestic-poultry-to-person transmission should be closely watched in the future. FUNDING: China Ministry of Science and Technology Project 973, National Natural Science Foundation of China, China Health and Family Planning Commission, Chinese Academy of Sciences.


Asunto(s)
Genoma Viral/genética , Virus de la Influenza A/genética , Gripe Aviar/virología , Animales , Evolución Biológica , Patos , Cadenas de Markov , Fenotipo , Filogenia , Aves de Corral , Alineación de Secuencia/métodos , Homología de Secuencia
16.
J Virol ; 87(16): 9077-85, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23760233

RESUMEN

Interspecies transmission (host switching/jumping) of influenza viruses is a key scientific question that must be addressed. In addition to the vigorous research on highly pathogenic avian influenza viruses (HPAIVs), studies of the mechanism of interspecies transmission of low-pathogenic avian influenza viruses (LPAIVs) could also provide insights into host tropism and virulence evolution. Influenza A viruses harboring hemagglutinin (HA) H13 (e.g., H13N6) are LPAIVs. In this study, soluble H13 HA glycoprotein was purified, and its receptor binding activity was characterized. The results revealed that H13 exclusively binds the avian α2-3-linked sialic acid receptor; no binding to the mammalian α2-6-linked sialic acid receptor was detected. Furthermore, the molecular basis of the H13 receptor binding specificity was revealed by comparative analysis of the crystal structures of both receptor-bound H13 and H5 HAs, which might be contributed by the hydrophobic residue V186. Work with an H13N186 mutant confirmed the importance of V186 in the receptor binding specificity of H13 HA, which shows that the mutant protein reduced the binding of an avian receptor analog but increased the binding of a human receptor analog. Detailed structural analysis also demonstrated that the conserved binding sites of the recently well-studied broadly neutralizing human monoclonal antibodies targeting the HA2 domain are found in H13. Our results expand our understanding of virulence evolution, receptor binding preference, and species tropism of the LPAIVs and HPAIVs.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/metabolismo , Gripe Aviar/virología , Receptores Virales/metabolismo , Ácidos Siálicos/metabolismo , Animales , Aves , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/aislamiento & purificación , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/fisiología , Modelos Moleculares , Unión Proteica , Conformación Proteica , Tropismo Viral
17.
J Virol ; 87(24): 13134-40, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24067982

RESUMEN

Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.


Asunto(s)
Infecciones por Coronaviridae/virología , Coronavirus/metabolismo , Secuencias Repetitivas de Aminoácido , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Coronavirus/química , Coronavirus/genética , Cristalización , Células HEK293 , Humanos , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus/genética
18.
Int J Biol Macromol ; 279(Pt 4): 135678, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-39368892

RESUMEN

In this paper, a new organic-inorganic biomass composite aerogel was prepared by freeze-drying method with glucomannan, hydrophilic isocyanate, water-soluble flame retardant, and water glass as raw materials. Biomass Konjac glucose mannan (KGM) was used as the main network framework, KGM was chemically cross-linked and alkali-cross-linked with hydrophilic isocyanate and Na2SiO3 solution, and flame retardant modified with water-soluble flame retardant and water glass. The microstructure showed an obvious organic-inorganic interpenetrating network structure. The compressive strength of sample K2S4P2 was 4.751 ± 0.089 MPa, and the compression modulus of sample K2S4P1B modified by boric acid hydrolysis of Na2SiO3 was 63.76 ± 1.81 × 103 m2/s2. The introduction of boron ions contributes to the thermal stability of organic components. The peak and total heat release rates of sample K2S4P1A4 decreased by 80.3 % and 50.8 %, respectively. In addition, the thermal simulation calculation of the external wall in winter and summer using ANSYS software showed that the thickness of the insulation layer with the best insulation effect is 40-60 mm. The organic-inorganic composite aerogel provides a simple and environmentally friendly method for the application of external wall insulation systems in low-energy buildings with both mechanical properties and flame retardant properties.


Asunto(s)
Retardadores de Llama , Geles , Mananos , Mananos/química , Geles/química
19.
J Virol ; 86(23): 12861-70, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22993148

RESUMEN

With a new serotype (H17) of hemagglutinin (HA) recently being discovered, there are now 17 serotypes (H1 to H17) of influenza A viruses in total. It is believed that HA is initially expressed as a precursor of HA0 and then cleaved into HA1 and HA2, forming a disulfide bond-linked complex, for its full function. Structural data show that a loop structure exists in the cleavage site between HA1 and HA2, and this flexible loop is crucial for the efficient cleavage of HA0. Here, the crystal structures of H16 (a low-pathogenicity avian influenza virus) in their HA0 form (H16HA0) have been solved at 1.7-Å and 2.0-Å resolutions. To our surprise, an α-helix element in the cleavage site which inserts into the negatively charged cavity with the key residue R329 hidden behind the helix was observed. In vitro trypsin cleavage experiments demonstrated inefficient cleavage of H16HA0 under both neutral and low-pH conditions. The results provide new insights into influenza A virus pathogenicity; both the relatively stable α-helix structure in the flexible cleavage loop and inaccessibility of the cleavage site likely contribute to the low pathogenicity of avian influenza A virus. Furthermore, compared to all of the HAs whose structures have been solved, H16 is a good reference for assigning the HA subtypes into two groups on the basis of the three-dimensional structure, which is consistent with the phylogenetic grouping. We conclude that in light of the current H16HA0 structure, the natural α-helix element might provide a new opportunity for influenza virus inhibitor design.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/patogenicidad , Modelos Moleculares , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Cristalización , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mariposas Nocturnas , Tripsina
20.
Front Public Health ; 11: 1288293, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38026367

RESUMEN

Backgrounds: Frailty is a significant problem for older persons since it is linked to a number of unfavorable consequences. According to observational researches, air pollution may raise the risk of frailty. We investigated the causal association between frailty and air pollution (including PM2.5, PM2.5-10, PM10, nitrogen dioxide, and nitrogen oxides) using Mendelian randomization approach. Methods: We conducted MR analysis using extensive publically accessible GWAS (genome-wide association studies) summary data. The inverse variance weighted (IVW) method was employed as the primary analysis method. The weighted median model, MR-Egger, simple model, and weighted model approaches were chosen for quality control. The Cochran's Q test was utilized to evaluate heterogeneity. Pleiotropy is found using the MR-Egger regression test. The MR-PRESSO method was used to recognize outliers. The leave-one-out strategy was used to conduct the sensitivity analysis. Results: MR results suggested that PM2.5 was statistically significantly associated with frailty [odds ratio (OR) = 1.33; 95%confidence interval (CI) = 1.12-1.58, p = 0.001] in IVW method. We observed no statistical association between PM2.5-10(OR = 1.00, 95% CI = 0.79-1.28, p = 0.979), PM10(OR = 0.91, 95% CI = 0.75-1.11, p = 0.364), nitrogen dioxide (OR = 0.98, 95% CI = 0.85-1.12, p = 0.730), nitrogen oxides (OR = 1.15, 95% CI = 0.98-1.36, p = 0.086) and frailty. There was no pleiotropy in the results. The sensitivity analysis based on the leave-one-out method showed that the individual single nucleotide polymorphisms (SNPs) did not affect the robustness of the results. Conclusion: The current MR investigation shows a causal association between PM2.5 and frailty. Frailty's detrimental progression may be slowed down with the help of air pollution prevention and control.


Asunto(s)
Contaminación del Aire , Fragilidad , Humanos , Anciano , Anciano de 80 o más Años , Dióxido de Nitrógeno/efectos adversos , Estudio de Asociación del Genoma Completo , Análisis de la Aleatorización Mendeliana , Contaminación del Aire/efectos adversos , Material Particulado/efectos adversos
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