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1.
Diagn Microbiol Infect Dis ; 95(4): 114885, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31607514

RESUMO

Adenoviral epidemic keratoconjunctivitis (EKC) is a major cause of ocular morbidity worldwide and specific antiviral therapies are not available. EKC is primarily caused by Human adenovirus D (HAdV-D) types 8, 37, 53, 54, 56 and 64. Considering the genomic variation in HAdV-D, we hypothesized that clinical signs could be differentiated by virus type. The hypothesis was retrospectively tested with clinical signs recorded from 250 patients with ocular infections visiting an ophthalmological clinic in southern Japan between 2011 and 2014. The results showed that conjunctival opacity, corneal epithelial disorders and pre-auricular lymphadenopathy, were more frequently associated with EKC than other ocular infections. Furthermore, HAdV types 8, 37 and 54, caused corneal complications and longer infections significantly more frequently than infections by types 53 and 56 (P < 0.05). Our descriptive results supported that symptoms severity vary with the infecting type, however, further research is needed to improve diagnosis of EKC.


Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/patologia , Adenovírus Humanos/fisiologia , Ceratoconjuntivite/epidemiologia , Ceratoconjuntivite/patologia , Células A549 , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Efeito Citopatogênico Viral , Humanos , Lactente , Japão/epidemiologia , Ceratoconjuntivite/virologia , Pessoa de Meia-Idade , Tipagem Molecular , Estudos Retrospectivos , Adulto Jovem
2.
Biomed Pharmacother ; 118: 109271, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377467

RESUMO

Enterovirus 71 (EV71) brainstem encephalitis (BE) is divided into-uncomplicated BE, autonomic nervous system (ANS) dysregulation, and pulmonary edema (PE)-based on cytokine-mediated severe systemic and central nervous system (CNS) inflammatory responses. Minocycline has been found to have anti-inflammatory and immunomodulatory properties in infectious and inflammatory neurological disease models. The effects of minocycline on EV71 infection were studied in vitro and in vivo experiments. The minocycline treatment (100-300 µg/mL) on cytokine expressions and viral replications were investigated in rhabdomyosarcoma (RD), U-87MG, and THP-1 cells. The mouse-adapted-EV71 strain (MP4)-infected 7-day-old ICR mice model was used to explore the anti-inflammatory and antiviral effects of minocycline (1 and 5 µg/g) for the treatment of EV71 infection. In in vitro, minocycline reduced cytopathic effects (CPEs), viral protein expressions, viral titers, the levels of interleukin (IL)-6 and IL-8 and relative mRNA expressions of IL-12p40, IL-1ß, and tumor necrosis factor (TNF) after EV71 infection. The levels of TNF, IL-1ß, IL-6, and IL-8 decreased with a single dose of minocycline in EV71-infected THP-1 cells. Double-dose minocycline treatment demonstrated more effective reduction in cytokines. In the MP4-infected animal model, clinical scores, mortality rates and viral titers in various brain tissues were decreased evidently after double-dose minocycline treatment. Minocycline inhibited IL-6 and granulocyte colony-stimulating factor (G-CSF) in plasma and TNF in the cerebellum. Minocycline has properties that enable it to function both as an anti-inflammatory and antiviral agent in EV71 infection. These results evidence its potential usefulness in clinical treatment.


Assuntos
Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Minociclina/farmacologia , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Efeito Citopatogênico Viral/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Carga Viral , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Virol J ; 16(1): 94, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366366

RESUMO

BACKGROUND: Although enterovirus 71 (EV71) is an important public health threat, especially in the Asia-Pacific region, there are still no effective drugs or vaccines to treat and prevent EV71 infection. Therefore, it is critical to develop prophylactic and therapeutic agents against EV71. Rosmarinic acid (RA), a phytochemical, has been discovered to possess a broad spectrum of biological activities. METHODS: The virucidal effects of RA on EV71 were determined by MTT, western blot, median cell culture infectious dose, apoptosis detection, plaque reduction, semi-quantitative real-time polymerase chain reaction, immunofluorescence detection, molecular docking analysis, and mouse protection assay. RESULTS: RA showed a strong protective effect against EV71 infection in human rhabdomyosarcoma cells when the multiplicity of infection was 1, with a low IC50 value (4.33 ± 0.18 µM) and high therapeutic index (340). RA not only protected cells from EV71-induced cytopathic effects, but also from EV71-induced apoptosis. The results of time-of-addition analysis demonstrated that the inhibitory activity of RA was highest at the early stage of viral infection. Consistent with this, the infectivity of EV71 in the early stage of viral infection also was observed to be limited in neonatal mice treated with RA. Further, molecular docking predicts that RA could replace the natural pocket factor within the VP1 capsid-binding hydrophobic pocket. CONCLUSIONS: This study suggests that RA has the potential to be developed as an antiviral agent against initial EV71 infection to prevent or reduce EV71-induced pathogenesis and complications, since RA can effectively reduce EV71 infection in the early stages of viral infection.


Assuntos
Antivirais/farmacologia , Cinamatos/farmacologia , Depsídeos/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Enterovirus Humano A/fisiologia , Humanos , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Replicação Viral/efeitos dos fármacos
4.
Arch Virol ; 164(10): 2551-2558, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31321588

RESUMO

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.


Assuntos
Infecções por Enterovirus/veterinária , Enterovirus/classificação , Enterovirus/isolamento & purificação , Doenças das Cabras/virologia , Filogenia , Animais , Análise por Conglomerados , Efeito Citopatogênico Viral , Enterovirus/genética , Infecções por Enterovirus/virologia , Genoma Viral , Cabras , Microscopia Eletrônica de Transmissão , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Vírion/ultraestrutura , Cultura de Vírus
5.
Virology ; 535: 279-282, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31357167

RESUMO

Bovine viral diarrhea viruses (BVDV) are a common global viral pathogen of ruminants. Considerable genetic variability is found amongst BVDV1 isolates, with at least 21 subgenotypes being described. In the United States, BVDV1a and 1b are the only subgenotypes described to date. Here, the genomic sequence of CA2005, a cytopathic BVDV1, was determined. This virus, isolated in California, did not segregate into either BVDV1a or 1b subgenotypes. BLAST analysis showed CA2005 was most closely related to BVDV1i isolates. CA2005 was also the first cytopathic BVDV1i and one of few non-1a, non-1b cytopathic viruses reported. The genomic sequence was 15,752 nucleotides in length. Cytopathogenicity was conferred by duplication of the NS3 protein with a small ubiquitin B insertion at the border of the NS2/NS3 proteins. Virus neutralization assays using antisera against BVDV1a vaccine viruses revealed variable neutralization, suggesting modified live vaccines may not be totally protective against CA2005 and similar viruses.


Assuntos
Antígenos Virais/genética , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Testes de Neutralização , Animais , Aspartato Aminotransferases/sangue , California , Bovinos , Doenças dos Bovinos/virologia , Análise por Conglomerados , Efeito Citopatogênico Viral , Diarreia/veterinária , Diarreia/virologia , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Genoma Viral , Genótipo , Filogenia , Sequenciamento Completo do Genoma
6.
Int J Mol Sci ; 20(12)2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234341

RESUMO

Zika virus (ZIKV) transmission can cause serious fetal neurological abnormalities. ZIKV persistence in various human cells and tissues can serve as infectious reservoirs and post serious threats to public health. The human embryonic kidney (HEK293) cell line with known neuronal developmental properties was readily infected by ZIKV in a strain-dependent fashion. Significant cytopathic effect in HEK293 cells infected by the prototype MR 766 strain of ZIKV resulted in complete loss of cells, while small numbers of HEK293 cells infected by contemporary ZIKV isolates (PRV or FLR strain) continued to survive and regrow to confluency in the culture around two months after initial infection. Most, if not all, of the cells in the two resulting persistently ZIKV-infected HEK293 cell lines tested positive for ZIKV antigen. Compared to HEK293 control cells, the persistently ZIKV-infected HEK293 cells had slower growth rates with some cells undergoing apoptosis in culture. The "persistent ZIKVs" produced constitutively by both PRV and FLR strains ZIKV-infected HEK293 cells had significantly attenuated cell infectivity and/or cytopathogenicity. Comparative genome sequence analyses between the persistent ZIKVs and the original inoculum ZIKVs showed no clonal selection with specific gene mutations in the prolonged process of establishing persistently PRV strain ZIKV-infected HEK293 cells; while selection of ZIKV subclones with mutations in the envelope, protein pr and multiple NS genes was evident in developing persistently FLR strain ZIKV-infected HEK293 cell line. Our study provides molecular insights into the complex interplays of ZIKV and human host cells in establishing ZIKV persistence.


Assuntos
Infecção por Zika virus/patologia , Zika virus/fisiologia , Animais , Apoptose , Chlorocebus aethiops , Efeito Citopatogênico Viral , Genoma Viral , Genômica , Células HEK293 , Humanos , Mutação , Células Vero , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/virologia
7.
Arch Pharm (Weinheim) ; 352(6): e1800330, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31073993

RESUMO

A series of 1-thia-4-azaspiro[4.5]decan-3-ones bearing an amide group at C-4 and various substitutions at C-2 and C-8 were synthesized and evaluated against human coronavirus and influenza virus. Compounds 7m, 7n, 8k, 8l, 8m, 8n, and 8p were found to inhibit human coronavirus 229E replication. The most active compound was N-(2-methyl-8-tert-butyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-3-phenylpropanamide (8n), with an EC50 value of 5.5 µM, comparable to the known coronavirus inhibitor, (Z)-N-[3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-3-oxo-1-phenylprop-1-en-2-yl]benzamide (K22). Compound 8n and structural analogs were devoid of anti-influenza virus activity, although their scaffold is shared with a previously discovered class of H3 hemagglutinin-specific influenza virus fusion inhibitors. These findings point to the 1-thia-4-azaspiro[4.5]decan-3-one scaffold as a versatile chemical structure with high relevance for antiviral drug development.


Assuntos
Antivirais/síntese química , Compostos Aza/síntese química , Coronavirus/efeitos dos fármacos , Desenho de Fármacos , Compostos de Espiro/síntese química , Animais , Antivirais/química , Antivirais/farmacologia , Compostos Aza/química , Compostos Aza/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Humanos , Células Madin Darby de Rim Canino , Estrutura Molecular , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
8.
Comp Immunol Microbiol Infect Dis ; 63: 97-103, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30961825

RESUMO

Bovine gammaherpesvirus 4 (BoHV4) is a member of the family Herpesviridae. In Argentina, BoHV4 was isolated and characterized in 2007 from samples of aborted cows. Argentinean isolates are highly divergent and are classified as: Genotype 1(Movar-like), Genotype 2 (DN599-like) and Genotype 3 (a novel group). The aim of this study was to comparatively evaluate the biological characteristics of six Argentinean BoHV4 field isolates in cell lines from different origins. All strains induced productive infection in the cell lines used, with different degrees of permissiveness. A direct relationship among the times of appearance of cytopathic effect, the growth kinetics, the size of the lysis plaques and the virulent-like behaviour in vitro could not be established. However, although slight, there are differences in the biological behaviour of the BoHV4 fields isolates analyzed. This variability is independent of their genetic classification but would be conditioned by the nature of the infected cells.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/fisiologia , Infecções Tumorais por Vírus/veterinária , Replicação Viral/fisiologia , Animais , Argentina , Bovinos , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral , Cães , Células HeLa , Células Hep G2 , Herpesvirus Bovino 4/genética , Herpesvirus Bovino 4/isolamento & purificação , Humanos , Células Madin Darby de Rim Canino , Células Vero
9.
Emerg Infect Dis ; 25(5): 1003-1006, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31002049

RESUMO

We retrieved Nipah virus (NiV) sequences from 4 human and 3 fruit bat (Pteropus medius) samples from a 2018 outbreak in Kerala, India. Phylogenetic analysis demonstrated that NiV from humans was 96.15% similar to a Bangladesh strain but 99.7%-100% similar to virus from Pteropus spp. bats, indicating bats were the source of the outbreak.


Assuntos
Quirópteros/virologia , Surtos de Doenças , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/virologia , Vírus Nipah/classificação , Vírus Nipah/genética , Animais , Células Cultivadas , Efeito Citopatogênico Viral , Infecções por Henipavirus/história , Infecções por Henipavirus/transmissão , História do Século XXI , Humanos , Índia/epidemiologia , Mutação , Vigilância em Saúde Pública
10.
PLoS Pathog ; 15(4): e1007640, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30998804

RESUMO

Zika virus (ZIKV) has been known for decades to circulate in Africa and Asia. However, major complications of a ZIKV infection have recently become apparent for reasons that are still not fully elucidated. One of the hypotheses for the seemingly increased pathogenicity of ZIKV is that cross-reactive dengue antibodies can enhance a ZIKV infection through the principle of antibody-dependent enhancement (ADE). Recently, ADE in ZIKV infection has been studied, but conclusive evidence for the clinical importance of this principle in a ZIKV infection is lacking. Conversely, the widespread circulation of ZIKV in dengue virus (DENV)-endemic regions raises new questions about the potential contribution of ZIKV antibodies to DENV ADE. In this review, we summarize the results of the evidence to date and elaborate on other possible detrimental effects of cross-reactive flavivirus antibodies, both for ZIKV infection and the risk of ZIKV-related congenital anomalies, DENV infection, and dengue hemorrhagic fever.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Facilitadores/imunologia , Reações Cruzadas/imunologia , Efeito Citopatogênico Viral/imunologia , Dengue/imunologia , Infecção por Zika virus/imunologia , Zika virus/patogenicidade , Dengue/virologia , Humanos , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/virologia
11.
PLoS One ; 14(4): e0215822, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31013314

RESUMO

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.


Assuntos
Linhagem Celular/virologia , Efeito Citopatogênico Viral/genética , Metapneumovirus/crescimento & desenvolvimento , Infecções Respiratórias/virologia , Células A549 , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Metapneumovirus/genética , Metapneumovirus/patogenicidade , Infecções Respiratórias/genética , Infecções Respiratórias/prevenção & controle
12.
PLoS One ; 14(3): e0214016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30893357

RESUMO

Zika virus (ZIKV) has emerged globally as an important pathogen, since it has been recognized as a cause of microcephaly and other neurologic processes and sequalae in newborns. The virus shares homology with Hepaciviruses and therefore may be a cause of hepatitis. We sought to characterize ZIKV replication in hepatocyte-derived cell lines. Huh7.5 and HepG2 cells were infected with ZIKV and replication potential was evaluated by multiple methods including plaque assay, qRT-PCR, negative-strand ZIKV RNA production, and ZIKV NS1 protein production. Growth curves in cells and supernatant were compared to replicative capacity in Vero cells. Overall, viral replication in both hepatocyte lines approximated that observed in the Vero cells. Cell cytopathology was observed after 3 days of infection and apoptosis markers increased. Transmission electron microscopy revealed evidence of viral capsids in cells and negative staining revealed ZIKV particles in the supernatant. Conclusions: Hepatocyte-derived cell lines are permissive for ZIKV replication and produce an overt cytopathic effect consistent with development of an acute viral hepatitis. Further evaluation of replication and injury is warranted.


Assuntos
Fígado/virologia , Infecção por Zika virus/virologia , Zika virus/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Efeito Citopatogênico Viral/genética , Células Hep G2 , Hepatócitos/virologia , Humanos , Células Vero , Carga Viral/genética , Proteínas não Estruturais Virais/genética , Vírion/genética , Replicação Viral/genética
13.
J Ethnopharmacol ; 236: 124-128, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-30853644

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Enterovirus 71 (EV71) has a propensity to cause hand-foot-and-mouth disease (HFMD) epidemics associated with neurological sequelae. Unfortunately, no drugs are currently available for the clinical treatment of EV71 infections. Sophoridine (SRI) is one of the most abundant alkaloids in Sophora flavescens Aiton (Leguminosae), which has been used to treat fever, throat inflammation, cancer, and other diseases. MATERIALS AND METHODS: In this study, we found that SRI inhibits EV71 infection in Vero cells. To study the antiviral activity of SRI, Vero cells were divided into 3 treatment groups based on the timing of SRI dosing: prior to viral adsorption (Group A), during viral adsorption (Group B), and after viral adsorption (Group C). We further revealed the antiviral activity of SRI with the attachment assay and the penetration assay. For Group A, 50% viability of Vero cells was observed at a SRI concentration of 61.39 µg/mL, whereas for Groups B, 50% viability was observed at SRI concentrations of 196.86 µg/mL. Furthermore, 29.7% cell viability was observed even at a SRI concentration of 1000 µg/mL in Groups C. The results show that SRI was highly effective against EV71 when Vero cells were pretreated with SRI for 2 h (Group A). Further researches indicate SRI was highly effective at inhibiting EV71 attachment when the SRI concentrations over 250 µg/mL (P < 0.001). CONCLUSIONS: We have shown that Vero cell viability increases when SRI is administered prior to viral adsorption. This suggests that SRI has the considerable potential as an antiviral for EV71 disease prevention.


Assuntos
Alcaloides/farmacologia , Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Quinolizinas/farmacologia , Alcaloides/isolamento & purificação , Animais , Antivirais/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinolizinas/isolamento & purificação , Sophora/química , Células Vero
14.
J Gen Virol ; 100(3): 457-470, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30707666

RESUMO

The NS4A protein of dengue virus (DENV) has a cytosolic N terminus and four transmembrane domains. NS4A participates in RNA replication and the host antiviral response. However, the roles of amino acid residues within the N-terminus of NS4A during the life cycle of DENV are not clear. Here we explore the function of DENV NS4A by introducing a series of alanine substitutions into the N-terminus of NS4A in the context of a DENV infectious clone or subgenomic replicon. Nine of 17 NS4A mutants displayed a lethal phenotype due to the impairment of RNA replication. M2 and M14 displayed a more than 10 000-fold reduction in viral yields and moderate defects in viral replication by a replicon assay. Sequencing analyses of pseudorevertant viruses derived from M2 and M14 viruses revealed one consensus reversion mutation, A21V, within NS4A. The A21V mutation apparently rescued viral RNA replication in the M2 and M14 mutants although not to wild-type (WT) levels but resulted in 100- and 1000-fold lower titres than that of the WT, respectively. M2 Rev1 (M2+A21V) and M14 Rev1 (M14+A21V) mutants displayed phenotypes of smaller plaque size and WT-like assembly/secretion by a transpackaging assay. A defect in the virus-induced cytopathic effect (CPE) was observed in HEK-293 cells infected with either M2 Rev1 or M14 Rev1 mutant virus by MitoCapture staining, cell proliferation and lactate dehydrogenase release assays. In conclusion, the results revealed the essential roles of the N-terminal NS4A in both RNA replication and virus-induced CPE. Intramolecular interactions in the N-terminus of NS4A were implicated.


Assuntos
Efeito Citopatogênico Viral , Vírus da Dengue/metabolismo , Dengue/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Células HEK293 , Humanos , Mutagênese , Domínios Proteicos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
15.
Virus Res ; 264: 22-31, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30794895

RESUMO

Emerging viruses are a major threat to human health. Recent outbreaks have emphasized the urgent need for new antiviral treatments. For several pathogenic viruses, considerable efforts have focused on vaccine development. However, during epidemics infected individuals need to be treated urgently. High-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics. Repurposed drugs can bypass part of the early cost and time needed for validation and authorization. In this review we describe recent efforts to find broad spectrum antivirals through drug repurposing. We have chosen several candidates and propose strategies to understand their mechanism of action and to determine how resistance to antivirals develops in infected cells.


Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos , Vírus/efeitos dos fármacos , Animais , Antivirais/química , Efeito Citopatogênico Viral , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Replicação Viral
16.
Circulation ; 139(20): 2326-2338, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30755025

RESUMO

BACKGROUND: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.


Assuntos
Regiões 5' não Traduzidas/genética , Cardiomiopatia Dilatada/virologia , Cisteína Endopeptidases/genética , Enterovirus Humano B/isolamento & purificação , Miócitos Cardíacos/virologia , RNA Viral/genética , Proteínas Virais/genética , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/patologia , Células Cultivadas , Cisteína Endopeptidases/biossíntese , Efeito Citopatogênico Viral , DNA Complementar/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/complicações , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Miocardite/complicações , Miocardite/virologia , Deleção de Sequência , Transfecção , Proteínas Virais/biossíntese , Latência Viral , Replicação Viral
17.
Virus Res ; 259: 46-53, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385363

RESUMO

Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.


Assuntos
Doenças dos Animais/virologia , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/veterinária , Ursidae/virologia , Doenças dos Animais/patologia , Animais , Linhagem Celular , Efeito Citopatogênico Viral , DNA Viral , Feminino , Gammaherpesvirinae/classificação , Gammaherpesvirinae/isolamento & purificação , Gammaherpesvirinae/ultraestrutura , Masculino , Filogenia , Análise de Sequência de DNA
18.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487275

RESUMO

Alphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative, and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the protein's transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, by using a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from interferon (IFN)-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins.IMPORTANCE Chikungunya virus is an important human pathogen which now circulates in both the Old and New Worlds. As in the case of other Old World alphaviruses, CHIKV nsP2 not only has enzymatic functions in viral RNA replication but also is a critical inhibitor of the antiviral response and one of the determinants of CHIKV pathogenesis. In this study, we have applied a new strategy to select a variety of CHIKV nsP2 mutants that no longer exhibited transcription-inhibitory functions. The designed CHIKV variants became potent type I interferon inducers and acquired a less cytopathic phenotype. Importantly, they demonstrated the same replication rates as the parental CHIKV. Mutations in the same identified peptide of nsP2 proteins derived from other Old World alphaviruses also abolished their nuclear functions. Such mutations can be further exploited for development of new attenuated alphaviruses.


Assuntos
Vírus Chikungunya/metabolismo , Proteínas não Estruturais Virais/genética , Animais , Antivirais , Linhagem Celular , Febre de Chikungunya/genética , Febre de Chikungunya/metabolismo , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Efeito Citopatogênico Viral/genética , Vírus de DNA/genética , Humanos , Interferon Tipo I/genética , Camundongos , Mutação , Células NIH 3T3 , RNA Viral/metabolismo , Replicon , Transdução de Sinais , Vírus Sindbis/genética , Vírus Sindbis/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética
19.
Microb Pathog ; 127: 79-84, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30500407

RESUMO

Recently, a novel type I interferon alphaomega (IFN-αω), also known as IFN-µ, was identified. However, the biological activity of IFN-αω remain poorly understood. In this study, the porcine IFN-αω (PoIFN-αω) was expressed, purified, and its antiviral activities assessed by its ability to inhibit the cytopathic effect caused by FMDV on IBRS-2 cells. In addition, q-PCR was used to evaluate the expression of IFN-stimulated genes induced by PoIFN-αω. It was found that PoIFN-αω exerted effective antiviral activity against FMDV pre- and post-infection. Additionally, PoIFN-αω induced the transcription of IFN-stimulated genes, including Mx1, ISG15, OAS1, and PKR genes. Our study reported a new indication of PoIFN-αω as an effective anti-FMDV agent for the first time.


Assuntos
Antivirais/farmacologia , Vírus da Febre Aftosa/efeitos dos fármacos , Interferon Tipo I/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Antivirais/isolamento & purificação , Antivirais/metabolismo , Linhagem Celular , Efeito Citopatogênico Viral , Perfilação da Expressão Gênica , Fatores Imunológicos/biossíntese , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Interferon Tipo I/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Suínos
20.
Biologicals ; 57: 61-66, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30477957

RESUMO

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.


Assuntos
Antivirais/farmacologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/tratamento farmacológico , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas Imunoenzimáticas/métodos , Cloreto de Amônio/farmacologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos dos fármacos , Vírus da Diarreia Viral Bovina/fisiologia , Estrenos/farmacologia , Técnicas Imunoenzimáticas/normas , Pirrolidinonas/farmacologia , Ribavirina/farmacologia , Replicação Viral/efeitos dos fármacos
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