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1.
J Viral Hepat ; 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32564517

RESUMO

Although a high seroprevalence of antibodies against hepatitis A virus (HAV) has been estimated in Central Africa, the current status of both HAV infections and seroprevalence of anti-HAV antibodies remains unclear due to a paucity of surveillance data available. We conducted a serological survey during 2015-2017 in Gabon, Central Africa, and confirmed a high seroprevalence of anti-HAV antibodies in all age groups. To identify the currently circulating HAV strains and to reveal the epidemiological and genetic characteristics of the virus, we conducted molecular surveillance in a total of 1007 patients presenting febrile illness. Through HAV detection and sequencing, we identified subgenotype IIA (HAV-IIA) infections in the country throughout the year. A significant prevalence trend emerged in the young child population, presenting several infection peaks which appeared to be unrelated to dry or rainy seasons. Whole-genome sequencing and phylogenetic analyses revealed local HAV-IIA evolutionary events in Central Africa, indicating the circulation of HAV-IIA strains of a region-specific lineage. Recombination analysis of complete genome sequences revealed potential recombination events in Gabonese HAV strains. Interestingly, Gabonese HAV-IIA possibly acquired the 5'-untranslated region (5'-UTR) of the rare subgenotype HAV-IIB in recent years, suggesting the present existence of HAV-IIB in Central Africa. These findings indicate a currently stable HAV-IIA circulation in Gabon, with a high risk of infections in children aged under 5 years. Our findings will enhance the understanding of the current status of HAV infections in Central Africa and provide new insight into the molecular epidemiology and evolution of HAV genotype II.

2.
J Gen Virol ; 101(6): 573-586, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32375950

RESUMO

Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2.

3.
Virus Res ; 278: 197868, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31962066

RESUMO

Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.

4.
Int J Infect Dis ; 91: 129-136, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31821892

RESUMO

OBJECTIVES: Dengue outbreaks, mainly caused by dengue virus serotype 2 (DENV-2), occurred in 2007 and in 2010 in Gabon, Central Africa. However, information on DENV infections has been insufficient since 2010. The aim of this study was to investigate the current DENV infection scenario and the risk of repeated infections in Gabon. METHODS: During 2015-2017, serum samples were collected from enrolled febrile participants and were tested for DENV infection using RT-qPCR. DENV-positive samples were analyzed for a history of previous DENV infection(s) using ELISA. The complete DENV genome was sequenced to analyze the phylogeny of Gabonese DENV strains. RESULTS: DENV-3 was exclusively detected, with a high rate of anti-DENV IgG seropositivity among DENV-3-positive participants. DENV-3 showed higher infection rates in adults and the infection was seasonal with peaks in the rainy seasons. Phylogenetic analysis revealed that Gabonese DENV-3 originated from West African strains and has been circulating continuously in Gabon since at least 2010, when the first DENV-3 case was reported. CONCLUSIONS: These findings indicate stable DENV-3 circulation and the risk of repeated DENV infections in Gabon, highlighting the need for continuous monitoring to control DENV infections.


Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Feminino , Gabão/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Estudos Soroepidemiológicos , Sorogrupo , Adulto Jovem
5.
J Med Virol ; 92(2): 251-256, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31538666

RESUMO

Hepatitis B virus (HBV) infection remains to be a major public health issue worldwide, although there is currently a safe vaccine and effective antiviral treatments. In surveillance of infectious diseases in Gabon, HBV viremia was detected in patients with febrile. Whole-genome sequencing was conducted to characterize the HBV strains currently circulating in Gabon and to investigate HBV genome diversity during viremia. Phylogenetic analysis revealed the existence of former subgenotype A5, which exhibits a particular pattern of distribution from several West and Central African countries to Haiti. Furthermore, sequencing analysis identified two similar HBV strains mixed in one sample, and a very rare 1-base pair insertion in the viral precore region. This insertion caused a frameshift mutation, indicating the production of an aberrant fusion protein of the HBV x and e antigens. Our data showed that the detected HBV strain was possibly in an "evolving" state during viremia, a phase of active replication.

6.
J Gen Virol ; 100(7): 1099-1111, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31184566

RESUMO

Ebola virus (EBOV) VP40 is a major driving force of nascent virion production and a negative regulator of genome replication/transcription. Here, we showed that the YIGL sequence at the C-terminus of EBOV VP40 is important for virus-like particle (VLP) production and the regulation of genome replication/transcription. Accordingly, a mutation in the YIGL sequence caused defects in VLP production and genome replication/transcription. The residues I293 and L295 in the YIGL sequence were particularly critical for VLP production. Furthermore, an in silico analysis indicated that the amino acids surrounding the YIGL sequence contribute to intramolecular interactions within VP40. Among those surrounding residues, F209 was shown to be critical for VLP production. These results suggested that the VP40 YIGL sequence regulates two different viral replication steps, VLP production and genome replication/transcription, and the nearby residue F209 influences VLP production.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Vírion/fisiologia , Replicação Viral , Motivos de Aminoácidos , Sequência de Aminoácidos , Ebolavirus/química , Ebolavirus/genética , Genoma Viral , Humanos , Alinhamento de Sequência , Proteínas da Matriz Viral/genética , Vírion/química , Vírion/genética , Liberação de Vírus
7.
J Virol Methods ; 269: 30-37, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30974179

RESUMO

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

8.
J Virol ; 93(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30814285

RESUMO

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel emerging virus that has been identified in China, South Korea, and Japan, and it induces thrombocytopenia and leukocytopenia in humans with a high case fatality rate. SFTSV is pathogenic to humans, while immunocompetent adult mice and golden Syrian hamsters infected with SFTSV never show apparent symptoms. However, mice deficient for the gene encoding the α chain of the alpha- and beta-interferon receptor (Ifnar1-/- mice) and golden Syrian hamsters deficient for the gene encoding signal transducer and activator of transcription 2 (Stat2-/- hamsters) are highly susceptible to SFTSV infection, with infection resulting in death. The nonstructural protein (NSs) of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT proteins. Here, we demonstrated that SFTSV induces lethal acute disease in STAT2-deficient mice but not in STAT1-deficient mice. Furthermore, we discovered that NSs cannot inhibit type I IFN signaling in murine cells due to an inability to bind to murine STAT2. Taken together, our results imply that the dysfunction of NSs in antagonizing murine STAT2 can lead to inefficient replication and the loss of pathogenesis of SFTSV in mice.IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTSV, which has been reported in China, South Korea, and Japan. Here, we revealed that mice lacking STAT2, which is an important factor for antiviral innate immunity, are highly susceptible to SFTSV infection. We also show that SFTSV NSs cannot exert its anti-innate immunity activity in mice due to the inability of the protein to bind to murine STAT2. Our findings suggest that the dysfunction of SFTSV NSs as an IFN antagonist in murine cells confers a loss of pathogenicity of SFTSV in mice.


Assuntos
Infecções por Bunyaviridae/metabolismo , Phlebovirus/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Antivirais/metabolismo , Infecções por Bunyaviridae/virologia , Glicoproteínas/metabolismo , Células HEK293 , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Febre por Flebótomos/virologia , Phlebovirus/patogenicidade , Fosforilação , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/fisiologia , Especificidade da Espécie , Trombocitopenia/metabolismo , Proteínas não Estruturais Virais/metabolismo , Virulência
9.
PLoS Negl Trop Dis ; 12(11): e0006971, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30500827

RESUMO

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.


Assuntos
Febre Lassa/virologia , Vírus Lassa/genética , Vírus Lassa/isolamento & purificação , Evolução Molecular , Variação Genética , Humanos , Febre Lassa/epidemiologia , Vírus Lassa/classificação , Nigéria/epidemiologia , Filogenia , Proteínas Virais/genética
10.
J Am Chem Soc ; 140(48): 16834-16841, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30475615

RESUMO

Immunosensing is a bioanalytical technique capable of selective detections of pathogens by utilizing highly specific and strong intermolecular interactions between recognition probes and antigens. Here, we exploited the molecular mechanism in artificial nanopores for selective single-virus identifications. We designed hemagglutinin antibody mimicking oligopeptides with a weak affinity to influenza A virus. By functionalizing the pore wall surface with the synthetic peptides, we rendered specificity to virion-nanopore interactions. The ligand binding thereof was found to perturb translocation dynamics of specific viruses in the nanochannel, which facilitated digital typing of influenza by the resistive pulse bluntness. As amino acid sequence degrees of freedom can potentially offer variety of recognition ability to the molecular probes, this peptide nanopore approach can be used as a versatile immunosensor with single-particle sensitivity that promises wide applications in bioanalysis including bacterial and viral screening to infectious disease diagnosis.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Nanoporos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Galinhas , Ouro/química , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Compostos de Silício/química , Carga Viral/métodos
11.
PLoS Pathog ; 14(7): e1007172, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30028868

RESUMO

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo.


Assuntos
Antígenos CD/imunologia , Coriomeningite Linfocítica/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Proliferação de Células , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Ativação Linfocitária , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL
12.
Biochem Biophys Res Commun ; 503(2): 631-636, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29906459

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection.


Assuntos
Colesterol/metabolismo , Ácidos Graxos/metabolismo , Febre por Flebótomos/metabolismo , Phlebovirus/fisiologia , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Triglicerídeos/metabolismo , Replicação Viral , Animais , Vias Biossintéticas , Células CHO , Linhagem Celular , Cricetulus , Humanos , Febre por Flebótomos/enzimologia
13.
J Gen Virol ; 99(2): 181-186, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29300152

RESUMO

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann-Pick C1.


Assuntos
Ebolavirus/genética , Glicoproteínas/genética , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno , Modelos Estruturais , Proteína C1 de Niemann-Pick/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Ebolavirus/patogenicidade , Glicoproteínas/metabolismo , Humanos , Mamíferos , Mutação , Proteína C1 de Niemann-Pick/genética , Primatas , Alinhamento de Sequência , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Sci Rep ; 7(1): 13503, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29044149

RESUMO

The recent outbreak of Zika virus (ZIKV) disease caused an enormous number of infections in Central and South America, and the unusual increase in the number of infants born with microcephaly associated with ZIKV infection aroused global concern. Here, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of ZIKV. The assay specifically detected ZIKV strains of both Asian and African genotypes without cross-reactivity with other arboviruses, including Dengue and Chikungunya viruses. The assay detected viral RNA at 14.5 TCID50/mL in virus-spiked serum or urine samples within 15 min, although it was slightly less sensitive than reference real time RT-PCR assay. We then evaluated the utility of this assay as a molecular diagnostic test using 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. The results of this assay were consistent with those of the reference RT-PCR test. This portable RT-LAMP assay was highly specific for ZIKV, and enable rapid diagnosis of the virus infection. Our results provide new insights into ZIKV molecular diagnostics and may improve preparedness for future outbreaks.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecção por Zika virus/sangue , Animais , Chlorocebus aethiops , Sensibilidade e Especificidade , Células Vero , Zika virus/genética , Infecção por Zika virus/urina
15.
Biochem J ; 474(20): 3499-3512, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28899944

RESUMO

Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.


Assuntos
Retrovirus Endógenos/genética , Regulação da Expressão Gênica no Desenvolvimento , Placenta/fisiologia , Transcrição Genética/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Bovinos , Feminino , Gravidez
16.
Sci Rep ; 7(1): 9500, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28842649

RESUMO

Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the currently-approved anti-influenza viral drugs. We performed in silico screening using a structure-based drug discovery algorithm called Nagasaki University Docking Engine (NUDE), which is optimised for a GPU-based supercomputer (DEstination for Gpu Intensive MAchine; DEGIMA), by targeting influenza viral PA protein. The compounds selected by NUDE were tested for anti-influenza virus activity using a cell-based assay. The most potent compound, designated as PA-49, is a medium-sized quinolinone derivative bearing a tetrazole moiety, and it inhibited the replication of influenza virus A/WSN/33 at a half maximal inhibitory concentration of 0.47 µM. PA-49 has the ability to bind PA and its anti-influenza activity was promising against various influenza strains, including a clinical isolate of A(H1N1)pdm09 and type B viruses. The docking simulation suggested that PA-49 interrupts the PA-PB1 interface where important amino acids are mostly conserved in the virus strains tested, suggesting the strain independent utility. Because our NUDE/DEGIMA system is rapid and efficient, it may help effective drug discovery against the influenza virus and other emerging viruses.


Assuntos
Antivirais/química , Antivirais/farmacologia , Descoberta de Drogas , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/metabolismo , RNA Replicase/metabolismo , Proteínas Virais/metabolismo , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Replicação Viral
17.
J Virol Methods ; 249: 102-110, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28837842

RESUMO

This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86-98% and 67-77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.


Assuntos
Doenças dos Bovinos/diagnóstico , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real , Animais , Bovinos , Doenças dos Bovinos/virologia , Primers do DNA/genética , Vírus da Febre Aftosa/genética , RNA Viral/genética , Sensibilidade e Especificidade , Temperatura
18.
J Virol Methods ; 246: 8-14, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28356221

RESUMO

Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.


Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Primers do DNA , Vírus da Dengue/genética , Ebolavirus/classificação , Ebolavirus/genética , Humanos , Vírus Lassa/genética , Limite de Detecção , Marburgvirus/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Transcrição Reversa , Sensibilidade e Especificidade , Temperatura
19.
PLoS One ; 12(2): e0171858, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28199372

RESUMO

In ruminants, Interferon tau (IFNT) is the pregnancy recognition protein produced by the mononuclear trophectoderm of the conceptus, and is secreted into the uterine lumen during the peri-attachment period. In our previous study, the high-throughput RNA sequencing (RNA-seq) data obtained from bovine conceptuses during the peri-attachment period identified two IFNT mRNAs, IFNT2 and IFNTc1. However, how each of these IFNT variants regulates endometrial gene expression has not been characterized. Using RNA-seq analysis, we evaluated how IFNT2 and IFNTc1 affected transcript expression in primary bovine endometrial epithelial cells (EECs). IFNT treatment induced 348 differentially expressed genes (DEGs); however, there are few DEGs in IFNT2 or IFNTc1 treated EECs, indicating that IFNT2-induced DEGs were similar to those induced by IFNTc1 treatment. In in silico analysis, we identified four IFNT2- and IFNTc1-induced pathways: 1) type II interferon signaling, 2) proteasome degradation, 3) type III interferon signaling, and 4) DNA damage response. We further demonstrated that IFNT2 and IFNTc1 up-regulated several transcription factors, among which forkhead box S1 (FOXS1) was identified as the most highly expressed gene. Furthermore, the knockdown of FOXS1 in IFNT2- or IFNTc1-treated EECs similarly down-regulated 9 genes including IRF3 and IRF9, and up-regulated 9 genes including STAT1, STAT2, and IRF8. These represent the first demonstration that effects of each IFNT on EECs were studied, and suggest that endometrial response as well as signaling mechanisms were similar between two IFNT variants existed in utero.


Assuntos
Células Epiteliais/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Interferon Tipo I/farmacologia , Proteínas da Gravidez/farmacologia , Animais , Bovinos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Endométrio/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/genética , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
20.
Genes Cells ; 22(2): 148-159, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28084671

RESUMO

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence.


Assuntos
Ebolavirus/genética , Mutação , Proteínas do Envelope Viral/genética , Células A549 , Ebolavirus/metabolismo , Evolução Molecular , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Análise de Sequência de DNA/métodos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
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