Development and characterization of serotype-specific monoclonal antibodies against Dengue virus NS1 / 生物工程学报

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.

NS1 codon usage adaptation to humans in pandemic Zika virus

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.

Construction and expression of a synthetic gene encoding nonstructural glycoprotein NS1 of dengue 2 virus in Pichia pastoris

Objectives To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris). Methods A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested. Results The recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test. Conclusions The resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.

Construction and expression of a synthetic gene encoding nonstructural glycoprotein NS1 of dengue 2 virus in Pichia pastoris

Objectives: To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris). Methods: A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA af-finity chromatography, and its antigenicity was tested. Results: The recombinant DENV-2 NS1 protein was secreted as a protein with a mo-lecular weight of ～45 kDa, and the optimal expression condition was achieved by in-duction with 2％(v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test. Conclusions: The resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.

Análisis filogenético de secuencias suramericanas del gen ns1 del serotipo dengue-2, relacionadas con sangrado clínico severo / Phylogenetic analysis of South American sequences of the nonstructural protein-1 (ns1) of dengue serotype 2 associated with severe clinical bleeding

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)

Comparison of cellular localization of NS1 from different subtypes of influenza A virus / 生物工程学报

The non-structural (NS1) protein is a multifunctional molecular protein encoded by influenza A virus genome. NS1 plays an important role in inhibition of host immune responses. In order to assess the cellular localization of NS1 in different influenza A virus subtypes, we performed the immunofluorescence assay to observe the cellular location of NS1 after infection with influenza A virus WSN (H1N1), PR8 (H1N1), CA04 (H1N1), SD (H9N2) and AH01 (H7N9) in A549 cells and MDCK cells respectively. According to the results, NS1-WSN and NS1-PR8 accumulated mainly in cytoplasm at 24 h post infection, while the NS1-CA04 and NS1-SD appeared major in the nucleus. We also observed localization of NS1 by transfected 293T cells with plasmids which encoding the full-length NS1 from WSN, SD and AH01. The key sites which might determine the different cellular localization of NS1 were chosen by sequence alignment, and seven residues which were different between WSN, PR8 and CA04, SD and AH01 were finally focused. However, we found that single mutation of these residues could not alter the localization of NS1. The data indicated that the difference of location might not be caused by substitution of a single site, which contributes to our understanding of the diverse regulation of host factors during different subtypes of influenza virus infection.

Homology Modeling and Docking Studies of Japanese Encephalitis Virus Proteins Like Envelope Protein and NS1 Protein.

Japanese Encephalitis (JE) is a vector- borne, viral zoonosis that may affect humans. The disease periodically becomes endemic in areas such as northern India, parts of central and southern India. Japanese Encephalitis virus belongs to the mostly vector-borne flaviviriade, which are single stranded RNA viruses. The envelope glycoprotein of JE Viruses contain specific as well as cross relative, neutralizing epitopes. The objective of this research to find out the best ligand molecule each for the two drug targeting protein present in the JEV. This will be done by studying the complete structure of JEV drug targeting proteins and their interaction with their respective ligand. The envelope protein and NS1 protein have been studied. The minimum energies were recorded after the docking studies for all the inhibitors docked with the protein. After comparison of the minimum energies recorded, the ligand with the least minimum docking energy has been considered as the best ligand. The entire study indicates that the inhibitor Mycophenolate with minimum energy -5.00605kj/mol is the most effective against Envelope protein. However in case of NS1 protein, the inhibitor Deoxynojirimycin with the minimum energy of - 6.75932kj/mol is found to be the most effective.

NS1’ protein expression in the JaOArS982 strain of Japanese encephalitis virus does not enhance virulence in mice

Using a mouse model,we previously demonstrated that subcutaneous infection with the JaTH160 strainof Japaneseencephalitis virus (JEV) causes significantly higher virulence and strongervirus propagation in the brain compared with that of the JaOArS982strain. We also showed that the JaTH160 strain,but not JaOArS982, expresses the NS1’ protein and that NS1’ enhances JEVproduction in avian cells and embryonated chicken eggs. In this study, weexamined whether NS1’ expression affects virulence in mice infected with theJaOArS982 and JaTH160 strains using the corresponding recombinant viruses S982-ICand JaTH-IC.Expression of the NS1’ protein in S982-IC diminishedthe mortality in mice, whereas S982-IC viruses without NS1’ caused 40% mortality.However, the viral loads in the brains of these mice were not significantlydifferent despite the difference in NS1’ expression. JaTH-IC viruses depletedof the NS1’ protein exhibited high mortality levels, similar to those of thevirus expressing NS1’.Previousstudies showed that the NS1’ protein plays a role in the enhanced virulenceof the JEV SA14 strain in mice. However, ourcurrent data suggest that NS1’ protein expression in S982-IC reduces,rather than enhances, the mortality in mice. Thus, the effect of NS1’ on pathogenicity <i>invivo</i> may vary among virus strains. Our data also suggest that the reducedmortality resulting from NS1’ expression in S982-IC is not simply due to viralreplication in the brains. Furtherinvestigation is needed to uncover the mechanism by which NS1’ affectspathogenicity in JEV-infected animals.

NS1’ Protein Expression in the JaOArS982 Strain of Japanese Encephalitis Virus Does Not Enhance Virulence in Mice

Using a mouse model, we previously demonstrated that subcutaneous infection with the JaTH160 strain of Japanese encephalitis virus (JEV) causes significantly higher virulence and stronger virus propagation in the brain compared with that of the JaOArS982 strain. We also showed that the JaTH160 strain, but not JaOArS982, expresses the NS1’ protein and that NS1’ enhances JEV production in avian cells and embryonated chicken eggs. In this study, we examined whether NS1’ expression affects virulence in mice infected with the JaOArS982 and JaTH160 strains using the corresponding recombinant viruses S982-IC and JaTH-IC. Expression of the NS1’ protein in S982-IC diminished the mortality in mice, whereas S982-IC viruses without NS1’ caused 40–60% mortality. However, the viral loads in the brains of these mice were not significantly different despite the dvariation in NS1’ expression. JaTH-IC viruses depleted of the NS1’ protein exhibited high mortality levels, similar to those of the virus expressing NS1’. Previous studies showed that the NS1’ protein plays a role in the enhanced virulence of the JEV SA14 strain in mice. However, our current data suggest that NS1’ protein expression in S982-IC reduces, rather than enhances, the mortality in mice. Thus, the effect of NS1’ on pathogenicity <i>in vivo</i> may vary among virus strains. Our data also suggest that the reduced mortality resulting from NS1’ expression in S982-IC is not simply due to viral replication in the brains. Further investigation is needed to uncover the mechanism by which NS1’ affects pathogenicity in JEV-infected animals.

Evaluación del valor diagnóstico de la detección de NS1 en pacientes con dengue agudo / Diagnostic value of NS1 detection in serum from acute dengue patients

El dengue es endemo-epidémico en Colombia y gran parte de nuestra población está en riesgo de padecer esta enfermedad. Uno de los principales problemas en el manejo del dengue es la dificultad para diagnosticar tempranamente esta arbovirosis, ya que la enfermedad presenta un cuadro clínico de evolución inespecífica, por lo cual es indispensable contar con herramientas diagnósticas rápidas y efectivas. El presente trabajo tiene como objetivo evaluar el valor diagnóstico de la detección de la proteína viral NS1 en pacientes con sospecha de dengue agudo, provenientes del Departamento de Cundinamarca, a quienes previamente se les había realizado detección de IgM en el Laboratorio de Salud Pública de Cundinamarca; de éstos, 44 muestras correspondían a pacientes con una prueba IgM positiva y las otras 44 a pacientes con una prueba de IgM negativa para dengue. A todas las muestras de suero se les practicó la prueba mediante el sistema Pan-E Dengue Early ELISA™ (Panbio) para la detección de NS1. Al hacer el análisis estadístico, se obtuvo una concordancia moderada entre las dos pruebas diagnósticas. Además, se encontró que 40 % de las muestras positivas para NS1, habían sido previamente reportadas como negativas de acuerdo con el resultado de IgM. De igual forma, se encontró enfermedad grave a menor edad y diferencias significativas en la presencia de erupción cutánea, ausencia de diarrea, leucocitos de menos de 4.000 células/μl y menos de 180.000 plaquetas/μl, al comparar los resultados positivos y negativos de cada prueba. Se puede concluir que la determinación de NS1 es necesaria en los casos sospechosos de dengue agudo y, junto con la determinación de IgM, representa un complemento eficiente para el diagnóstico, agilizando la instauración de un tratamiento oportuno.

Dengue is an endemic disease in Colombia and a large part of our population is at risk of suffering from this illness. One of the main problems in the management of dengue is the difficulty to do an appropriate and early diagnosis, because the disease presents an inespecific evolution, thus, it is essential to account on fast and effective diagnostic tools. The present work aimed to evaluate the diagnostic value of detection of the viral protein NS1 in patients with suspected acute dengue, from the Department de Cundinamarca, who had previously been submitted to IgM detection in the Cundinamarca Public Health Laboratory; 44 samples came from patients with a positive IgM test and the other 44 to patients with negative IgM for dengue virus. All serum samples were submitted to Pan Dengue Early ELISA (Panbio) system for NS1 detection. The statistical analysis showed a moderate concordance between the two diagnostic tests. We also found that 40% of positive samples for NS1 had previously been reported as negative according to the results of IgM. Simi1arly, it was found that as younger age, severe disease was present, there were found significant differences in the presence of rash, absence of diarrhea, leukocytes <4000 cel/μl and platelets <180,000/μl when comparing positive and negative results of each test. We can conclude that NS1 determination is needed in suspected cases of acute dengue, and in conjunction with the determination of IgM represents an efficient complement to diagnosis, thus expediting the implementation of early treatment.