Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 188
Filter
1.
Chinese Journal of Biotechnology ; (12): 3179-3189, 2021.
Article in Chinese | WPRIM | ID: wpr-921415

ABSTRACT

Bluetongue virus (BTV) causes Bluetongue (BT) of ruminants vectored by culicoides midges. It is also a classic model for studying the release mechanism of non-enveloped virus. This review begins with the infection and assembly of BTV, then summarizes the advances of different ways of releasing BTV. This includes BTV-induced autophagy and the release as extracellular vesicles via multivesicular bodies, BTV-induced apoptosis and the lytic release, as well as different pathways of release through budding via plasma membrane. The regulatory mechanisms of NS3 which is a key non-structural protein during the release of BTV are also discussed, providing a basis for further understanding the molecular mechanisms underpinning the infection, proliferation and release of BTV.


Subject(s)
Animals , Bluetongue , Bluetongue virus , Ceratopogonidae , Sheep , Viral Nonstructural Proteins
2.
Chinese Journal of Biotechnology ; (12): 1334-1345, 2021.
Article in Chinese | WPRIM | ID: wpr-878635

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.


Subject(s)
COVID-19 , Codon/genetics , Cysteine Endopeptidases/genetics , Escherichia coli/genetics , Humans , Peptide Hydrolases , SARS-CoV-2 , Viral Nonstructural Proteins/genetics
3.
Braz. J. Vet. Res. Anim. Sci. (Online) ; 57(2): e166086, mai. 2020. ilus, tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1122174

ABSTRACT

Avian coronavirus (AvCoV) infects a range of tissues in chickens and several other avian species. Although the virus can be isolated in chicken embryos, only a few strains of the 6 genotypes/33 lineages can grow in cell lines, with the Beaudette strain (GI-1 lineage) being the most used for in vitro studies. Considering the differences between cell lines and chicken embryos as habitats for AvCoV, this study aimed to assess the diversity of the genes coding for the nonstructural protein 3 (nsp3) and spike envelope protein (S) after serial passages in BHK-21 and Vero cells. After 14 passages of an embryo-adapted Beaudette strain, the virus loads fluctuated in both cell lines, with the highest loads being 8.72 log genome copies/µL for Vero and 6.36 log genome copies/µL for BHK-21 cells. No polymorphisms were found for nsp3; regarding S, not only aa substitutions (Vero: 8th passage A150S, and 14th S150A; BHK-21: 4th S53F, 8th F53Y, and 8th S95R), but also minor variants could be detected on chromatograms with fluctuating intensities. As the regions of these aa substitutions are within the receptor-binding domain of S, it can be speculated that differences in cell receptors between Vero and BHK-21 cells and the speed of cell death led to the selection of different dominant strains, while the stability of nsp3 supports its function as a protease involved in AvCoV replication. In conclusion, AvCoV quasispecies evolution is influenced by the biological model under consideration, and a gradual transition is seen for minor and major variants.(AU)


O Coronavírus aviário AvCoV infecta uma variedade de tecidos de galinhas e de outras espécies aviárias. Apesar de este vírus poder ser isolado em ovos embrionados de galinha, apenas alguns dos 6 genótipos / 33 linhagens podem crescer em cultivo celular, sendo a cepa Beuadette (linhagem GI-11) a mais utilizada para estudos in vitro. Considerando as diferentes linhagens celulares e ovos embrionados como habitats para o AvCoV, este estudo teve por objetivo estudar a diversidade de genes que codificam para a proteína não-estrutural 3 (nsp3) e espícula (S) após passagens seriadas em células BHK-21 e VERO. Após 14 passagens, de uma amostra Beuadette adaptada a ovos embrionados, os títulos virais variaram em ambas as células, com os maiores títulos sendo de 8,72 log cópias genômicas/µL para Vero e 6,36 cópias genômicas/µL para BHK-21. Nenhum polimorfismo foi encontrando para nsp3. Considerando a proteína S, não somente foram encontradas substituições de aminoácidos (Vero: 8a passagem A150S e 14a passagem S150A; BHK-21: 4a passagem S53F, 8a passagem F53Y e S95R), mas também, variantes subconsensuais foram detectadas pelos cromatogramas com intensidades flutuantes. Uma vez que as regiões destes aa se encontram no domínio de ligação de receptor de S, pode-se especular que diferenças em receptores celulares entre Vero e BHK-21, além da velocidade da morte celular, levaram à seleção de diferentes cepas dominantes, enquanto que a estabilidade de nsp3 concorda com sua função como protease com papel na replicação de AvCoV. Como conclusão, a evolução de quase-espécies de AvCoV é influenciada pelo modelo biológico sob consideração e uma transição gradual é vista para variantes dominantes e subdominantes.(AU)


Subject(s)
Chick Embryo , Viral Nonstructural Proteins , Coronavirus Infections/veterinary , Spike Glycoprotein, Coronavirus , Gammacoronavirus
4.
Mem. Inst. Oswaldo Cruz ; 115: e200179, 2020. graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135266

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on viral polyprotein processing, catalysed by the main proteinase (Mpro). The solution of the SARS-CoV-2 Mpro structure allowed the investigation of potential inhibitors. This work aims to provide first evidences of the applicability of commercially approved drugs to treat coronavirus disease-19 (COVID-19). We screened 4,334 compounds to found potential inhibitors of SARS-CoV-2 replication using an in silico approach. Our results evidenced the potential use of coagulation modifiers in COVID-19 treatment due to the structural similarity of SARS-CoV-2 Mpro and human coagulation factors thrombin and Factor Xa. Further in vitro and in vivo analysis are needed to corroborate these results.


Subject(s)
Humans , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Betacoronavirus , Structure-Activity Relationship , Computer Simulation , Cysteine Endopeptidases , Coronavirus Infections/drug therapy , Coronavirus 3C Proteases , SARS-CoV-2 , COVID-19/drug therapy
5.
Mem. Inst. Oswaldo Cruz ; 115: e200207, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135237

ABSTRACT

BACKGROUND Since the World Health Organization (WHO) declared Coronavirus disease 2019 (COVID-19) to be a pandemic infection, important severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural proteins (nsp) have been analysed as promising targets in virtual screening approaches. Among these proteins, 3-chymotrypsin-like cysteine protease (3CLpro), also named main protease, and the RNA-dependent RNA polymerase (RdRp), have been identified as fundamental targets due to its importance in the viral replication stages. OBJECTIVES To investigate, in silico, two of the most abundant flavonoid glycosides from Dysphania ambrosioides; a medicinal plant found in many regions of the world, along with some of the putative derivatives of these flavonoid glycosides in the human organism as potential inhibitors of the SARS-CoV-2 3CLpro and RdRp. METHODS Using a molecular docking approach, the interactions and the binding affinity with SARS-CoV-2 3CLpro and RdRp were predicted for quercetin-3-O-rutinoside (rutin), kaempferol-3-O-rutinoside (nicotiflorin) and some of their glucuronide and sulfate derivatives. FINDINGS Docking analysis, based on the crystal structure of 3CLpro and RdRp, indicated rutin, nicotiflorin, and their glucuronide and sulfate derivatives as potential inhibitors for both proteins. Also, the importance of the hydrogen bond and π-based interactions was evidenced for the presumed active sites. MAIN CONCLUSIONS Overall, these results suggest that both flavonoid glycosides and their putative human metabolites can play a key role as inhibitors of the SARS-CoV-2 3CLpro and RdRp. Obviously, further researches, mainly in vitro and in vivo experiments, are necessary to certify the docking results reported here, as well as the adequate application of these substances. Furthermore, it is necessary to investigate the risks of D. ambrosioides as a phytomedicine for use against COVID-19.


Subject(s)
Humans , Flavonoids/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Betacoronavirus/drug effects , Glycosides/pharmacology , Pneumonia, Viral , Cysteine Endopeptidases , Coronavirus Infections , Pandemics , Molecular Docking Simulation , Coronavirus 3C Proteases , SARS-CoV-2 , COVID-19
6.
Journal of Integrative Medicine ; (12): 229-241, 2020.
Article in English | WPRIM | ID: wpr-829101

ABSTRACT

OBJECTIVE@#Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.@*METHODS@#To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.@*RESULTS@#For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.@*CONCLUSION@#RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.


Subject(s)
Antiviral Agents , Chemistry , Therapeutic Uses , Betacoronavirus , Chemistry , Coronavirus Infections , Drug Therapy , Virology , Cysteine Endopeptidases , Chemistry , Drugs, Chinese Herbal , Chemistry , Therapeutic Uses , Humans , Mass Spectrometry , Medicine, Chinese Traditional , Molecular Docking Simulation , Pandemics , Pneumonia, Viral , Drug Therapy , Virology , Protein Interaction Maps , Viral Nonstructural Proteins , Chemistry
7.
Chinese Journal of Biotechnology ; (12): 2357-2366, 2020.
Article in Chinese | WPRIM | ID: wpr-878492

ABSTRACT

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.


Subject(s)
Animals , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease Virus , Viral Nonstructural Proteins/genetics , Viral Vaccines
8.
Chinese Journal of Biotechnology ; (12): 2206-2215, 2020.
Article in Chinese | WPRIM | ID: wpr-878479

ABSTRACT

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.


Subject(s)
Animals , Antibodies, Monoclonal , Antibodies, Viral/metabolism , Dengue/diagnosis , Dengue Virus/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Mice, Inbred BALB C , Sensitivity and Specificity , Serogroup , Viral Nonstructural Proteins/immunology
9.
Mem. Inst. Oswaldo Cruz ; 115: e200287, 2020. tab, graf
Article in English | LILACS | ID: biblio-1154869

ABSTRACT

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.


Subject(s)
Humans , Immunologic Tests/methods , Enzyme-Linked Immunosorbent Assay/methods , Viral Nonstructural Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction/methods , Dengue/diagnosis , Dengue Virus/isolation & purification , Antigens, Viral/blood , Predictive Value of Tests , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics , Dengue/blood , Dengue/virology , Dengue Virus/genetics , Dengue Virus/immunology , Antibodies, Viral/blood , Antigens, Viral/immunology
10.
Infectio ; 23(3): 259-265, July-Sept. 2019. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1002160

ABSTRACT

Resumen Objetivo: Se considera que el diagnóstico del dengue es fundamentalmente clínico; sin embargo, las pruebas rápidas basadas en la detección de IgM o NS1/IgM están siendo utilizadas en los servicios de salud. Este estudio determinó la contribución de las pruebas rápidas al diagnóstico de dengue en un área endémica antes de la introducción del virus zika. Metodología: Diseño de corte transversal de pruebas diagnósticas realizado a partir del análisis secundario de un estudio previo en 14 instituciones de salud del Valle del Cauca. Se obtuvo información de 632 participantes con resultados de prueba rápida, diagnóstico clínico y pruebas de referencia ELISA NS1, ELISA IgM y RT-PCR. Se compararon la sensibilidad, especificidad, valores predictivos y razones de verosimilitud del uso solo, en serie, y paralelo de los componentes NS1, IgM, NS1/IgM de la prueba rápida y el diagnóstico clínico con las pruebas Q de Cochran y McNemar para datos pareados. Resultados: La sensibilidad del diagnóstico clínico (61,4% IC95% 56%-66,7%) fue superior a la de las pruebas rápidas (37% IC95% 29,6%-44,7%) (P Conclusión: El diagnóstico clínico tiene una mayor sensibilidad que las pruebas rápidas, pero por si solo no es suficiente para confirmar o descartar dengue. Un resultado positivo en pruebas rápidas en pacientes con diagnóstico clínico de dengue es útil para confirmarlo, pero un resultado negativo no lo descarta.


Abstract Objective: Dengue diagnosis is considered to be mainly clinical; however, rapid tests that detect IgM or NS1/IgM are being used in health services. This study assessed the contribution of rapid tests to dengue diagnosis in an endemic area before the emergence of zika virus in Colombia. Methods: Cross-sectional study of diagnostic tests based on a secondary analysis of a previous study in 14 health care institutions in Valle del Cauca department. Results of dengue rapid test, clinical diagnosis, and reference tests ELISA NS1, ELISA IgM, and RT-PCR were obtained for 632 participants. The sensitivity, specificity, predictive values and likelihood ratios of the use alone, serial and parallel combinations of NS1, IgM, NS1/IgM of the rapid test and clinical diagnosis were compared using Cochran´s Q and MacNemar tests for paired data. Results: The sensitivity of clinical diagnosis (61.4% 95%IC 56-66.7) was higher than the sensitivity of rapid tests (37% 95% IC 29.6-44.7) (P<0.001). The serial used of NS1/IgM rapid test when clinical diagnosis was negative increased the sensitivity to 79.5% and, the serial use when clinical diagnosis was positive increased the specificity (from 66.3% to 98.7%). However, the latter decreased the sensitivity to 32.2%. While all negative likelihood ratios (LR-) were close to 1, the serial use of rapid tests when clinical diagnosis was positive had LR+ higher than 10. Conclusion: The clinical diagnosis is more sensitive than rapid tests, but by itself does not confirm or rule out dengue. A positive result in rapid tests is useful to confirm dengue but a negative result does not rule it out.


Subject(s)
Humans , Male , Female , Dengue , Dengue/diagnosis , Zika Virus , Sensitivity and Specificity , Viral Nonstructural Proteins/analysis , Colombia , Clinical Laboratory Techniques , Point-of-Care Testing
11.
Chinese Journal of Biotechnology ; (12): 1126-1134, 2019.
Article in Chinese | WPRIM | ID: wpr-771815

ABSTRACT

Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.


Subject(s)
Human bocavirus , Promoter Regions, Genetic , Transcriptional Activation , Viral Nonstructural Proteins , Virus Replication
12.
Rev. Soc. Bras. Med. Trop ; 51(2): 168-173, Mar.-Apr. 2018. graf
Article in English | LILACS | ID: biblio-897058

ABSTRACT

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.


Subject(s)
Humans , Leukocytes, Mononuclear/virology , Dengue/diagnosis , Dengue Virus/genetics , Dengue Virus/immunology , Antibodies, Viral/blood , Cell Separation , Chromatography, Affinity , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Flow Cytometry , Fluorescence
13.
Mem. Inst. Oswaldo Cruz ; 113(1): 38-44, Jan. 2018. tab, graf
Article in English | LILACS | ID: biblio-894888

ABSTRACT

BACKGROUND A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.


Subject(s)
RNA, Viral/genetics , Genome, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Zika Virus/genetics , Phylogeny , Viral Nonstructural Proteins , High-Throughput Nucleotide Sequencing
14.
Article in English | WPRIM | ID: wpr-758781

ABSTRACT

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.


Subject(s)
Animals , Classification , Clone Cells , Enterotoxins , Epitopes , Genetic Variation , Genotype , Humans , Protein Folding , Protein Processing, Post-Translational , Rotavirus , Sequence Analysis , Viral Nonstructural Proteins , Virulence
15.
Mem. Inst. Oswaldo Cruz ; 113(5): e170385, 2018. tab, graf
Article in English | LILACS | ID: biblio-894923

ABSTRACT

BACKGROUND Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.


Subject(s)
Viral Nonstructural Proteins/genetics , Zika Virus Infection/epidemiology , Zika Virus Infection/virology , Brazil/epidemiology , Codon , Genome, Viral
16.
Mem. Inst. Oswaldo Cruz ; 113(5): e170393, 2018. tab, graf
Article in English | LILACS | ID: biblio-894924

ABSTRACT

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.


Subject(s)
Viral Nonstructural Proteins , Dengue Virus/genetics , Zika Virus , DNA Primers/genetics , Computational Biology/methods , Reverse Transcriptase Polymerase Chain Reaction
17.
Mem. Inst. Oswaldo Cruz ; 112(11): 793-795, Nov. 2017. graf
Article in English | LILACS | ID: biblio-894847

ABSTRACT

A recent study showed that infectivity of Zika virus (ZIKV) Asian genotype was enhanced by an alanine-to-valine amino acid substitution at residue 188 of the NS1 protein, but the precise time and location of origin of this mutation were not formally estimated. Here, we applied a Bayesian coalescent-based framework to estimate the age and location of the ancestral viral strain carrying the A188V substitution. Our results support that the ancestral ZIKV strain carrying the A188V substitution arose in Southeastern Asia at the early 2000s and circulated in that region for some time (5-10 years) before being disseminated to Southern Pacific islands and the Americas.


Subject(s)
Humans , Proteins/genetics , Bayes Theorem , Viral Nonstructural Proteins/genetics , Zika Virus/genetics , Mutation/genetics , Phylogeny , Asia , Genotype
18.
Weekly Epidemiological Monitor. 2017; 10 (40): 1
in English | IMEMR | ID: emr-187578

ABSTRACT

The province of Khyber Pakhtunkhwa of Pakistan has officially notified of an outbreak of dengue fever in its capital city-Peshawar. Since mid July to end of September 2017, a total of 52,926 suspected/clinically diagnosed cases of dengue fever including 38 associated deaths have been reported. Of these, 10,893 cases have been laboratory-confirmed by NS1 antigen test [Non- structural protein]. Samples from the suspected cases were also tested at the National Institute of Health [NIH] in Islamabad and Dengue serotype-2 [DEN-2] was identified as the causative agent for this outbreak in Peshawar


Subject(s)
Humans , Dengue/mortality , Viral Nonstructural Proteins
19.
Medicina (B.Aires) ; 76(6): 390-398, dic. 2016. ilus, graf, tab
Article in Spanish | LILACS | ID: biblio-841617

ABSTRACT

La hepatitis crónica por el virus de la hepatitis C (HCV) es un problema de salud mundial. En el mundo, 170 millones de personas están infectadas. En Latinoamérica la prevalencia se estima entre 1.0 y 2.3% y en Argentina es en promedio 1.0 a 1.5%. La eficacia del tratamiento de esta enfermedad ha mejorado sustancialmente en los últimos 2 a 3 años. Con los nuevos antivirales de acción directa (AAD) disponibles actualmente, pueden lograrse tasas de respuesta viral sostenida (RVS) mayores al 90-95% prácticamente con pocos efectos adversos. Para poder acceder a estos tratamientos con una alta tasa de curación, y así lograr reducir la carga de la enfermedad en la salud pública, es necesario aumentar el número de pacientes diagnosticados y que estos accedan a un cuidado adecuado. El rol de los médicos de atención primaria es fundamental: deben sospechar la infección, diagnosticarla y complementar su atención con la derivación al especialista. El trabajo conjunto de generalistas y especialistas optimizará el manejo de los recursos disponibles, permitiendo que cada vez más personas infectadas con el HCV sean diagnosticadas y tratadas adecuadamente.


Chronic hepatitis C (HCV) is a global health problem. Worldwide, 170 million people are chronically infected. In Latin America its prevalence is estimated between 1.0 and 2.3%, and in Argentina between 1.0 and 1.5%. Treatment efficacy has considerably improved in the last 2 or 3 years. Sustained virological response (SVR) rates around 90-95% can be achieved with the new direct acting antiviral agents (DAAs) currently available, with few side effects. It is necessary to increase the number of diagnosed patients, linking them to adequate management and treatment. Raising treatment rates will increase the percentage of cured patients, reducing the burden of disease. Primary care physicians´ role is essential to achieve this goal. They must identify persons at risk, diagnose them and work with specialists to continue their medical care. Team working of generalists and specialists will permit that more HCV infected people can access to adequate care and treatment.


Subject(s)
Humans , Male , Female , Adult , Hepatitis C, Chronic/diagnosis , Hepatitis C, Chronic/drug therapy , Antiviral Agents/therapeutic use , Argentina/epidemiology , Protease Inhibitors/therapeutic use , Time Factors , Prevalence , Risk Factors , Treatment Outcome , Viral Nonstructural Proteins/antagonists & inhibitors , Hepatitis C, Chronic/epidemiology
20.
Rev. salud pública ; 18(3): 1-1, mayo-jun. 2016. ilus, tab
Article in Spanish | LILACS | ID: lil-784965

ABSTRACT

Objetivo Comparar secuencias de nucleótidos y de aminoácidos de la proteína no estructural 1-NS1 de cepas DENV-2, aisladas de pacientes febriles de diferentes países suramericanos, que cursaron cuadros clínicos con severidad o sin ella. Materiales y Métodos El análisis filogenético fue realizado a partir de 28 secuencias moleculares completas (1 056 pb) del gen NS1 del serotipo DENV-2. Se realizó un análisis filogenético bayesiano utilizando el software MrBayes v.3.2.0, con el modelo SYM+G y un análisis filogenético con el método Neighbor-Joining con el modelo Jukes-Cantor. Además, las secuencias de aminoácidos fueron alineadas y comparadas entre sí, mediante el programa Clustal W incluido en el software MEGA v. 5.2. Resultados En las secuencias de aminoácidos asociadas a sangrado, la sustitución más frecuente fue isoleucina→ treonina, en la posición 93. Estas secuencias presentaron un mayor porcentaje (94,6 %) de homología de aminoácidos de la proteína NS1 en comparación con el porcentaje de homología (74 %) de los aislamientos DENV-2 no asociados a sangrado. Se identificaron cinco clados que agrupan la mayoría de las secuencias analizadas (19/24; 79,2 %) con valores de probabilidad posterior mayores o iguales al 58 %. Siete (87,5 %) secuencias asociadas a sangrado se relacionan filogenéticamente dentro de los clados 4 y 5, con valores de probabilidad posterior del 58 % y 97 %, respectivamente. Conclusión No se encontraron características filogenéticas ni tampoco diferencias entre las secuencias de aminoácidos de la proteína NS1-DENV-2 estudiadas, que pudieran ser relacionadas, de manera directa, con la severidad de la enfermedad.(AU)


Objective The objective of this in silico study was to compare nucleotide and amino acids DENV-2-NS1 sequences isolated from febrile patients, with and without disease severity, from different South American countries. Matherials and Methods A bayesian MCMC phylogenetic analysis was carried out using 28 complete sequences of the gene NS1 of the DENV-2 serotype (1 056 bp), using MrBayes v.3.2.0 software, with the model SYM+G (2.5 million generations). We also carried out a phylogenetic analysis with Neighbor-Joining method (Jukes-Cantor model). In addition, the amino acids sequences were aligned and compared with each other, using Clustal W included in MEGA v.5.2 software. Results In the amino acids sequences associated with bleeding, the most frequent substitution was isoleucine → threonine at posicion 93. These sequences showed a high percentage (94.6 %) of amino acid homology in comparison with the percentage of amino acids homology (74 %) of DENV-2 isolates not associated with bleeding. Five clades were identified that group the vast majority of the DENV-2-NS1 sequences analyzed (19/24; 79.2 %) with posterior probability values greater than or equal to 58 %. Seven sequences (87.5 %) associated with bleeding were phylogenetically related within clades 4 and 5, the posterior probability values were 58 % and 97 %, respectively. Conclusion Neither phylogenetic characteristics nor differences between amino acids of the DENV-2-NS1 sequences studied were found that could be associated directly with severity of the disease.(AU)


Subject(s)
Humans , Phylogeny , Viral Nonstructural Proteins/analysis , Severe Dengue/diagnosis , Dengue Virus/isolation & purification , South America , Computer Simulation
SELECTION OF CITATIONS
SEARCH DETAIL