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1.
Arch Virol ; 164(10): 2599-2603, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278422

RESUMO

This work describes the characterization and genome annotation of a new lytic Enterococcus faecalis siphovirus, vB_EfaS_AL3 (referred to as AL3), isolated from wastewater samples collected in Liaoning Province, China. The genome of phage AL3 is composed of linear double-stranded DNA that is 40,789 bp in length with a G + C content of 34.84% and 61 putative protein-coding genes. Phylogenetic and comparative genomic analyses indicate that phage AL3 should be considered a novel phage.


Assuntos
Bacteriófagos/genética , Enterococcus faecalis/virologia , Genoma Viral , Filogenia , Análise de Sequência de DNA , Águas Residuárias/virologia , Bacteriólise , Composição de Bases , China , DNA/química , DNA/genética , DNA Viral/química , DNA Viral/genética , Microscopia Eletrônica de Transmissão , Anotação de Sequência Molecular , Ensaio de Placa Viral , Vírion/ultraestrutura
2.
Prep Biochem Biotechnol ; 49(7): 686-694, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31035907

RESUMO

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness-in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120 min), and temperature (25 °C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.


Assuntos
Técnicas de Cultura de Células/métodos , Herpesvirus Humano 1/fisiologia , Inativação de Vírus , Álcoois/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Cercopithecus aethiops , Desenho de Equipamento , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Projetos de Pesquisa , Temperatura Ambiente , Células Vero , Ensaio de Placa Viral , Inativação de Vírus/efeitos dos fármacos
3.
BMC Vet Res ; 15(1): 178, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142304

RESUMO

BACKGROUND: Avian infectious bronchitis (IB) is a disease that can result in huge economic losses in the poultry industry. The high level of mutations of the IB virus (IBV) leads to the emergence of new serotypes and genotypes, and limits the efficacy of routine prevention. Medicinal plants, or substances derived from them, are being tested as options in the prevention of infectious diseases such as IB in many countries. The objective of this study was to investigate extracts of 15 selected medicinal plants for anti-IBV activity. RESULTS: Extracts of S. montana, O. vulgare, M. piperita, M. officinalis, T. vulgaris, H. officinalis, S. officinalis and D. canadense showed anti-IBV activity prior to and during infection, while S. montana showed activity prior to and after infection. M. piperita, O. vulgare and T. vulgaris extracts had > 60 SI. In further studies no virus plaques (plaque reduction rate 100%) or cytopathogenic effect (decrease of TCID50 from 2.0 to 5.0 log10) were detected after IBV treatment with extracts of M. piperita, D. canadense and T. vulgaris at concentrations of extracts ≥0.25 cytotoxic concentration (CC50) (P < 0.05). Both PFU number and TCID50 increased after the use of M. piperita, D. canadense, T. vulgaris and M. officinalis extracts, the concentrations of which were 0.125 CC50 and 0.25 CC50 (P < 0.05). Real-time PCR detected IBV RNA after treatment with all plant extracts using concentrations of 1:2 CC50, 1:4 CC50 and 1:8 CC50. Delta cycle threshold (Ct) values decreased significantly comparing Ct values of 1:2 CC50 and 1:8 CC50 dilutions (P < 0.05). CONCLUSIONS: Many extracts of plants acted against IBV prior to and during infection, but the most effective were those of M. piperita, T. vulgaris and D. canadense .


Assuntos
Antivirais/farmacologia , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais , Animais , Antivirais/toxicidade , Cercopithecus aethiops , Testes de Sensibilidade Microbiana , Extratos Vegetais/toxicidade , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Ensaio de Placa Viral
4.
PLoS Negl Trop Dis ; 13(4): e0007269, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30986252

RESUMO

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 has been used in Nepal for catch-up campaigns and is now included in the routine immunisation schedule. Previous studies have shown good vaccine efficacy after one dose in districts with a high incidence of JE. The first well-documented dengue outbreak occurred in Nepal in 2006 with ongoing cases now thought to be secondary to migration from India. Previous infection with dengue virus (DENV) partially protects against JE and might also influence serum neutralising antibody titres against JEV. This study aimed to determine whether serum anti-JEV neutralisation titres are: 1. maintained over time since vaccination, 2. vary with historic local JE incidence, and 3. are associated with DENV neutralising antibody levels. We conducted a cross-sectional study in three districts of Nepal: Banke, Rupandehi and Udayapur. Udayapur district had been vaccinated against JE most recently (2009), but had been the focus of only one campaign, compared with two in Banke and three in Rupandehi. Participants answered a short questionnaire and serum was assayed for anti-JEV and anti-DENV IgM and IgG (by ELISA) and 50% plaque reduction neutralisation titres (PRNT50) against JEV and DENV serotypes 1-4. A titre of ≥1:10 was considered seropositive to the respective virus. JEV neutralising antibody seroprevalence (PRNT50 ≥ 1:10) was 81% in Banke and Rupandehi, but only 41% in Udayapur, despite this district being vaccinated more recently. Sensitivity of ELISA for both anti-JEV and anti-DENV antibodies was low compared with PRNT50. DENV neutralising antibody correlated with the JEV PRNT50 ≥1:10, though the effect was modest. IgM (indicating recent infection) against both viruses was detected in a small number of participants. We also show that DENV IgM is present in Nepali subjects who have not travelled to India, suggesting that DENV may have become established in Nepal. We therefore propose that further JE vaccine campaigns should be considered in Udayapur district, and similar areas that have had fewer vaccination campaigns.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/prevenção & controle , Programas de Imunização , Vacinas contra Encefalite Japonesa/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Nepal/epidemiologia , Testes de Neutralização , Estudos Soroepidemiológicos , Inquéritos e Questionários , Ensaio de Placa Viral , Adulto Jovem
5.
Molecules ; 24(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901934

RESUMO

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.


Assuntos
Antivirais/farmacologia , Desoxiaçúcares/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Uridina/farmacologia , Antivirais/química , Linhagem Celular Tumoral , Células Cultivadas , Desoxiaçúcares/química , Relação Dose-Resposta a Droga , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Uridina/análogos & derivados , Uridina/química , Ensaio de Placa Viral
6.
Vet Res ; 50(1): 18, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823888

RESUMO

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.


Assuntos
Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Animais , Galinhas/imunologia , Galinhas/virologia , Regulação da Expressão Gênica , Genoma Viral/genética , Testes de Inibição da Hemaglutinação/veterinária , Imunidade Inata , Técnicas In Vitro , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/imunologia , Influenza Aviária/patologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA/veterinária , Ensaio de Placa Viral/veterinária , Virulência , Eliminação de Partículas Virais
7.
mSphere ; 4(1)2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787120

RESUMO

Coxsackievirus typically infects humans via the gastrointestinal tract, which has a large number of microorganisms collectively referred to as the microbiota. To study how the intestinal microbiota influences enteric virus infection, several groups have used an antibiotic regimen in mice to deplete bacteria. These studies have shown that bacteria promote infection with several enteric viruses. However, very little is known about whether antibiotics influence viruses in a microbiota-independent manner. In this study, we sought to determine the effects of antibiotics on coxsackievirus B3 (CVB3) using an in vitro cell culture model in the absence of bacteria. We determined that an aminoglycoside antibiotic, neomycin, enhanced the plaque size of CVB3 strain Nancy. Neomycin treatment did not alter viral attachment, translation, or replication. However, we found that the positive charge of neomycin and other positively charged compounds enhanced viral diffusion by overcoming the negative inhibitory effect of sulfated polysaccharides present in agar overlays. Neomycin and the positively charged compound protamine also enhanced plaque formation of reovirus. Overall, these data provide further evidence that antibiotics can play noncanonical roles in viral infections and that this should be considered when studying enteric virus-microbiota interactions.IMPORTANCE Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown the bacteria promote infection with a variety of enteric viruses. However, it is possible that antibiotics have microbiota-independent effects on viruses. Here we show that an aminoglycoside antibiotic, neomycin, can influence quantification of coxsackievirus in cultured cells in the absence of bacteria.


Assuntos
Antibacterianos/farmacologia , Enterovirus/efeitos dos fármacos , Neomicina/farmacologia , Linhagem Celular , Células Cultivadas , Microbioma Gastrointestinal , Células HeLa , Humanos , Orthoreovirus de Mamíferos/efeitos dos fármacos , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
8.
MBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782664

RESUMO

Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.


Assuntos
Antivirais/farmacologia , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antivirais/síntese química , Linhagem Celular , Cercopithecus aethiops , Humanos , Ligação Proteica , Conformação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Ensaio de Placa Viral
9.
Nat Microbiol ; 4(3): 527-538, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30718847

RESUMO

Tapping into the metabolic crosstalk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed 'in plaque-MSI'. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying 'in plaque-MSI' may facilitate the discovery of bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.


Assuntos
Eutrofização , Ácidos Graxos/análise , Haptófitas/virologia , Interações entre Hospedeiro e Microrganismos , Espectrometria de Massas , Phycodnaviridae/fisiologia , Metabolômica , Análise Espaço-Temporal , Ensaio de Placa Viral , Viroses/metabolismo
10.
Virology ; 529: 7-15, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30641481

RESUMO

H9N2 avian influenza viruses (AIVs) were prevailing in chickens, causing great economic losses and public health threats. In this study, turkey herpesviruses (HVT) was cloned as an infectious bacterial artificial chromosomes (BAC). Recombinant HVT (rHVT-H9) containing hemagglutinin (HA) gene from H9N2 virus were constructed via galactokinase (galK) selection and clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. The recombinant rHVT-H9 showed no difference with parent HVT in plague morphology and virus replication kinetics. H9 protein expression of rHVT-H9 could be detected by western blot and indirect immunofluorescence assay (IFA) in vitro and in vivo. Immunization with rHVT-H9 could induce robust humoral and cellular immunity in chickens. In the challenge study, no chicken shed H9N2 virus from oropharynx and cloaca, and no H9N2 virus was found in viscera in vaccination groups. The result suggests that rHVT-H9 provides effective protection against H9N2 AIV in chickens.


Assuntos
Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Herpesvirus Meleagrídeo 1/metabolismo , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária/prevenção & controle , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Engenharia Genética , Vacinas contra Influenza , Interferon gama , Ensaio de Placa Viral
11.
J Virol Methods ; 265: 113-116, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30639413

RESUMO

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.


Assuntos
Clonagem Molecular , DNA Complementar/genética , Biologia Molecular/métodos , Recombinação Genética , Vírus da Estomatite Vesicular New Jersey/crescimento & desenvolvimento , Vírus da Estomatite Vesicular New Jersey/genética , Virologia/métodos , Animais , Linhagem Celular , Cricetinae , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Estomatite Vesicular/patologia , Estomatite Vesicular/virologia , Ensaio de Placa Viral
12.
Virology ; 526: 91-98, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30388630

RESUMO

Highly pathogenic alphaviruses display complex glycans on their surface. These glycans play a crucial role in viral pathogenesis by facilitating glycan-host interaction during viral entry which can be targeted. Various studies have reported antiviral activity of lectins that bind to the glycans present on the surface of enveloped viruses. This study evaluates the antiviral potential of a chitinase (chi)-like lectin from Tamarind (TCLL) having specificity for N-acetylglucosamine (NAG). Thus, TCLL might bind to N-glycan rich surface of alphavirus and inhibit the entry of virus into the host cells. The direct treatment of TCLL with virus reduced the virus infection. Remarkably, the addition of NAG to TCLL abolished antiviral activity confirming that NAG binding property of TCLL is accountable for its antiviral activity. Further, an ELISA assay confirmed the binding of TCLL to alphaviruses. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycan.


Assuntos
Acetilglucosamina/metabolismo , Antivirais/farmacologia , Vírus Chikungunya/efeitos dos fármacos , Quitinases/farmacologia , Lectinas de Plantas/farmacologia , Polissacarídeos/metabolismo , Tamarindus/enzimologia , Animais , Antivirais/isolamento & purificação , Antivirais/metabolismo , Antivirais/uso terapêutico , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/metabolismo , Quitinases/isolamento & purificação , Quitinases/metabolismo , Relação Dose-Resposta a Droga , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Sementes/enzimologia , Tamarindus/química , Proteínas do Envelope Viral/metabolismo , Ensaio de Placa Viral , Internalização do Vírus/efeitos dos fármacos
13.
Virus Res ; 261: 21-30, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30543872

RESUMO

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo.


Assuntos
Doenças dos Peixes/prevenção & controle , Deleção de Genes , Glicoproteínas/metabolismo , Infecções por Herpesviridae/veterinária , Herpesviridae/fisiologia , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Carpas , Núcleo Celular/virologia , Células Cultivadas , Citoplasma/virologia , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Herpesviridae/genética , Herpesviridae/imunologia , Herpesviridae/ultraestrutura , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Microscopia Eletrônica , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral , Ensaio de Placa Viral , Vírion/ultraestrutura , Virulência
14.
J Virol Methods ; 263: 88-95, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30381239

RESUMO

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent "objects". The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Imagem Óptica , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Ensaio de Placa Viral/métodos , Algoritmos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação de Medicamentos , Feminino , Humanos , Testes de Neutralização , Reprodutibilidade dos Testes , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Sigmodontinae/imunologia , Sigmodontinae/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
15.
Viruses ; 10(9)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231560

RESUMO

Porcine epidemic diarrhea (PED) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. Therefore, it is urgent for us to find out efficient ways to prevent and control this disease. In this study, the antiviral activity of a cationic amphibian antimicrobial peptide Caerin1.1 against porcine epidemic diarrhea virus (PEDV) was evaluated by an in vitro system using Vero cells. We found that even at a very low concentration, Caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in PEDV infections. In addition, Caerin1.1 showed powerful antiviral activity without interfering with the binding progress between PEDV and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of PEDV.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Vírus da Diarreia Epidêmica Suína/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antivirais/metabolismo , Sobrevivência Celular , Células Cultivadas , Cercopithecus aethiops , Infecções por Coronavirus/veterinária , Efeito Citopatogênico Viral , Relação Dose-Resposta a Droga , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Suínos , Doenças dos Suínos/virologia , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
16.
J Water Health ; 16(4): 600-613, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30067241

RESUMO

Research on microorganism reduction by physicochemical water treatment is often carried out under the assumption that the microbiological enumeration techniques are not affected by the presence of coagulants. Data presented here indicate that bacteriophage enumeration by plaque assay and RT-qPCR (reverse transcription quantitative polymerase chain reaction) can be affected by these water treatment chemicals. Treatment of water samples with an alkaline protein-rich solution prior to plaque assay and optimization of RNA extraction for RT-qPCR were implemented to minimize the interference. The improved procedures were used in order to investigate reduction of three viral pathogens and the MS2 model virus in the presence of three coagulants. A conventional aluminium coagulant was compared to alternative agents (zirconium and chitosan) in a coagulation-filtration system. The highest virus reduction, i.e., 99.9-99.99%, was provided by chitosan, while aluminium and zirconium reduced virus by 99.9% in colour-rich water and by 90% in water with less colour, implying an effect of coagulant type and raw water quality on virus reduction. Although charge characteristics of viruses were associated with virus reduction, the results reveal that the MS2 phage is a suitable model for aggregation and retention of the selected pathogens.


Assuntos
Alumínio/química , Quitosana/química , Vírus , Microbiologia da Água , Purificação da Água/métodos , Zircônio/química , Água Potável/virologia , Ensaio de Placa Viral , Água
17.
Artigo em Inglês | MEDLINE | ID: mdl-30098552

RESUMO

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage ϕ6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of ϕ6. In addition, binding of ϕ6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 1013 PFU/mg of protein and ~65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.


Assuntos
Bacteriófago phi 6/isolamento & purificação , Fracionamento por Campo e Fluxo/métodos , Vírion/isolamento & purificação , Pseudomonas syringae/virologia , Ultracentrifugação , Ensaio de Placa Viral , Cultura de Vírus
18.
Virology ; 523: 110-120, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30119012

RESUMO

Knowledge about the function of varicella-zoster virus glycoprotein M is limited; the requirement of gM for skin and neural tropism are unknown. VZV gM contains two predicted YXXΦ trafficking motifs and a dileucine motif in the carboxyl-terminus. We constructed a recombinant VZV with gM truncated from the first YXXΦ and five additional viruses with YXXΦ tyrosine substitutions, alone and in combination with dileucine substitution. All recombinant viruses grew to high titer but mutation of the membrane-proximal YXXΦ motif reduced plaque size in cultured cells and altered gM localization. C-terminus truncation had a pronounced effect on virion morphogenesis and plaque size, but not on overall replication kinetics in vitro. Mutation of gM trafficking motifs and truncation attenuated replication in human skin xenografts in vivo; gM truncation did not alter neurotropism. Our results demonstrate that the gM C-terminus is dispensable for virus replication in cultured cells but is important for skin pathogenesis.


Assuntos
Gânglios Espinais/virologia , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidade , Pele/virologia , Proteínas da Matriz Viral/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Gânglios Espinais/patologia , Herpes Zoster/patologia , Herpesvirus Humano 3/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Domínios Proteicos , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/patologia , Carga Viral , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Ensaio de Placa Viral , Tropismo Viral , Virulência , Replicação Viral
19.
Vet Microbiol ; 221: 81-89, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29981713

RESUMO

Porcine epidemic diarrhea (PEDV) has raised growing concerns in the pig-breeding industry because it has caused significant economic losses. To better understand the molecular epidemiology and genetic diversity of PEDV field isolates, in this study, the complete spike (S) and ORF3 genes of 17 PEDV variants in Zhejiang, China during 2014 to 2017, were characterized and analyzed. Phylogenetic analysis based on the S gene and ORF3 gene of these 17 novel PEDV strains and PEDV reference strains indicated that all the PEDV strains fell into two groups designated G1 and G2. Notably, the strains identified in 2014-2015 were in G2, while the other five strains identified from 2016 to 2017 were in G1. Sequencing and phylogenetic analyses showed that recently prevalent Chinese PEDV field strains shared higher identities with United States strains than with South Korean strains. Compared with classical vaccine strains, a series of deletions and frequently occurring mutations were observed in the receptor binding domains of our PEDV strains. Besides, we successfully isolated and reported the genetic characterization two novel PEDV strains, PEDV-LA1 and PEDV-LY4-98, found on the Chinese mainland, which had significant variations in the S gene. Meanwhile, the virulence of the new mutants may be changed, the PEDV-LY4-98 strain, which has multiple mutations in the signal peptide-encoding fragment of the S gene showed delayed cytopathic effects and smaller plaque size compared with strain PEDV-LA1, which lacks these mutations. Three unique amino acid substitutions (L7, G8, and V9) were identified in the SP-encoding fragment of the S1 N-terminal domain of the PEDV-LY4-98 S protein compared with the S proteins of all the previous PEDV strains. The animal experiment revealed that these two novel strains were high pathogenic to neonatal pigs. Whether these amino acids substitutions and the N-glycosylation site substitutions influence the antigenicity and pathogenicity of PEDV remains to be investigated. Meanwhile, amino acid substitutions in the neutralizing epitopes may have conferred the capacity for immune evasion in these PEDV field strains. This study improves our understanding of ongoing PEDV outbreaks in China, and it will guide further efforts to develop effective measures to control this virus.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/genética , Doenças dos Suínos/virologia , Substituição de Aminoácidos , Animais , Cercopithecus aethiops , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Epitopos , Variação Genética , Genótipo , Mutação , Filogenia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Doenças dos Suínos/epidemiologia , Células Vero , Ensaio de Placa Viral , Replicação Viral/fisiologia
20.
Viruses ; 10(7)2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29997331

RESUMO

The baculovirus-insect cell expression system has been widely used for heterologous protein expression and virus-like particles (VLPs) expression. In this study, we established a new method for antiviral screening targeting to glycoprotein E of flaviviruses based on the baculovirus expression system. ZIKV is a mosquito-borne flavivirus and has posed great threat to the public health. It has been reported that ZIKV infection was associated with microcephaly and serious neurological complications. Our study showed that either ZIKV E or prME protein expressed in insect cells can form VLPs and induce membrane fusion between insect cells. Therefore, the E protein, which is responsible for receptor binding, attachment, and virus fusion during viral entry, achieved proper folding and retained its fusogenic ability in VLPs when expressed in this system. The syncytia in insect cells were significantly reduced by the anti-ZIKV-E specific polyclonal antibody in a dose-dependent manner. AMS, a thiol-conjugating reagent, was also shown to have an inhibitory effect on the E protein induced syncytia and inhibited ZIKV infection by blocking viral entry. Indeed the phenomenon of syncytial formation induced by E protein expressed VLPs in insect cells is common among flaviviruses, including Japanese encephalitis virus (JEV), Dengue virus type 2 (DENV-2), and tick-borne encephalitis virus (TBEV). This inhibition effect on syncytial formation can be developed as a novel, safe, and simple antiviral screening approach for inhibitory antibodies, peptides, or small molecules targeting to E protein of ZIKV and other flaviviruses.


Assuntos
Baculoviridae/genética , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Expressão Gênica , Vetores Genéticos/genética , Células Gigantes/virologia , Animais , Antivirais/farmacologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Flavivirus/efeitos dos fármacos , Flavivirus/ultraestrutura , Engenharia Genética , Humanos , Transporte Proteico , Ensaio de Placa Viral , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos
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