Molecular mimicry between Zika virus and central nervous system inflammatory demyelinating disorders: the role of NS5 Zika virus epitope and PLP autoantigens / Mimetismo molecular entre o vírus Zika e os distúrbios inflamatórios desmielinizantes do sistema nervoso central: o papel do epítopo NS5 do vírus Zika e dos autoantígenos PLP

Abstract Background Evidence indicates a strong link between Zika virus (ZikV) and neurological complications. Acute myelitis, optic neuritis, polyneuropathy, and encephalomyelitis that mimic inflammatory idiopathic demyelination disorders (HDD) after ZikV infection have been reported in Brazil. Objective The present study aims to investigate the possible occurrence of molecular mimicry between ZikV antigens and Multiple Sclerosis (MS) autoantigens, the most frequent HDD of the central nervous system (CNS). Methods A retrospective cohort study with 305 patients admitted due to suspected arbovirus infection in Rio de Janeiro was performed, all subjects were submitted to neurological examination, and a biological sample was collected for serologic and molecular diagnostic. Bioinformatics tools were used to analyze the peptides shared between ZikV antigens and MS autoantigens. Results Of 305 patients, twenty-six were positive for ZikV and 4 presented IDD patterns found in MS cases. Sequence homology comparisons by bioinformatics approach between NS5 ZikV and PLP MS protein revealed a homology of 5/6 consecutive amino acids (CSSVPV/CSAVPV) with 83% identity, deducing a molecular mimicry. Analysis of the 3D structures revealed a similar conformation with alpha helix presentation. Conclusions Molecular mimicry between NS5 Zika virus antigen and PLP MS autoantigens emerge as a possible mechanism for IDD spectrum in genetically susceptible individuals.

Resumo Antecedentes Evidências indicam uma forte ligação entre o vírus Zika (ZikV) e complicações neurológicas. Mielite aguda, neurite óptica, polineuropatia e encefalomielite que mimetizam distúrbios inflamatórios de desmielinização idiopáticos (DDII) após infecção por ZikV têm sido relatadas no Brasil. Obejtivo O presente estudo tem como objetivo investigar a possível ocorrência de mimetismo molecular entre antígenos do ZikV e autoantígenos da Esclerose Múltipla (EM), a DDII mais frequente do sistema nervoso central (SNC). Métodos Foi realizado um estudo de coorte retrospectivo com 305 pacientes internados por suspeita de infecção por arbovirus no Rio de Janeiro, todos os indivíduos foram submetidos a exame neurológico e coleta de amostra biológica para diagnóstico sorológico e molecular. Ferramentas de bioinformática foram usadas para analisar os peptídeos compartilhados entre antígenos do ZikV e autoantígenos da EM. Resultados Dos 305 pacientes, vinte e seis foram positivos para ZikV e 4 apresentaram padrão IDD encontrado em casos de EM. As comparações de homologia de sequência por abordagem de bioinformática entre a proteína NS5 ZikV e PLP EM revelaram uma homologia de 5/6 aminoácidos consecutivos (CSSVPV/CSAVPV) com 83% de identidade, deduzindo um mimetismo molecular. A análise das estruturas 3D revelou uma conformação semelhante com apresentação em alfa-hélice. Conclusões O mimetismo molecular entre o antígeno NS5 do vírus Zika e o autoantígeno PLP da EM surge como um possível mecanismo para o espectro IDD em indivíduos geneticamente suscetíveis.

In Silico Screening of Natural Products as Potential Inhibitors of SARS-CoV-2 Using Molecular Docking Simulation / 中国结合医学杂志

OBJECTIVE@#To explore potential natural products against severe acute respiratory syndrome coronavirus (SARS-CoV-2) via the study of structural and non-structural proteins of human coronaviruses.@*METHODS@#In this study, we performed an in-silico survey of 25 potential natural compounds acting against SARS-CoV-2. Molecular docking studies were carried out using compounds against 3-chymotrypsin-like protease (3CLPRO), papain-like protease (PLPRO), RNA-dependent RNA polymerase (RdRp), non-structural protein (nsp), human angiotensin converting enzyme 2 receptor (hACE2R), spike glycoprotein (S protein), abelson murine leukemia viral oncogene homolog 1 (ABL1), calcineurin-nuclear factor of activated T-cells (NFAT) and transmembrane protease serine 2.@*RESULTS@#Among the screened compounds, amentoflavone showed the best binding affinity with the 3CLPRO, RdRp, nsp13, nsp15, hACE2R. ABL1 and calcineurin-NFAT; berbamine with hACE2R and ABL1; cepharanthine with nsp10, nsp14, nsp16, S protein and ABL1; glucogallin with nsp15; and papyriflavonol A with PLPRO protein. Other good interacting compounds were juglanin, betulinic acid, betulonic acid, broussooflavan A, tomentin A, B and E, 7-methoxycryptopleurine, aloe emodin, quercetin, tanshinone I, tylophorine and furruginol, which also showed excellent binding affinity towards a number of target proteins. Most of these compounds showed better binding affinities towards the target proteins than the standard drugs used in this study.@*CONCLUSION@#Natural products or their derivatives may be one of the potential targets to fight against SARS-CoV-2.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene

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.

Comparison and evaluation of three assays in preliminary screening of dengue infections / 中华检验医学杂志

Objective To compare and evaluate the performance and applications of three assays in preliminary screening of dengue infections.Methods This study was designed as a retrospective study.Clinical data were obtained from 2 137 borderline cases from September to October in 2014 and 2015.Markers of dengue infections of serum samples were detected by NS1 antigen captured ELISA, dengue NS1 detect rapid test and dengue IgM detect rapid test, respectively.Chi square test was used to compare the preliminary screening value of these 3 methods.Forty-eight diagnosed patients were also examined in the 1st-3rd day, 4th-5th day, 6th-7th days and 14th day after infection to access the sensitivities and specificities of three assays.Results Nine hundred and fifty-seven ( 57.2%) cases in 2014 and 48 ( 10.4%) cases in 2015 were diagnosed to be Dengue fever of 2 137 borderline cases.The overall sensitivity of NS1-ELISA was superior to NS1 and IgM rapid detect test (χ2 =40.865,P<0.001;χ2 =151.383,P<0.001).No significant differences were found in specificity between three assays(χ2 =0.661,P=0.416;χ2 =0.548,P=0.459; χ2 =2.397,P=0.122).The NS1 detecting assays were sensitive in 7 days after infection, but the sensitivity of IgM detecting assay increased over time.Conclusions NS1 detecting assays had good sensitivities and specificities, which can be used as an important method in preliminary screening of dengue infection.ELISA or rapid test can be selected according to epidemic situation.

Suppression effect of expressing parvovirus H-1 nonstructural protein 1 gene on human gastric cancer cells / 中华消化杂志

Objective To investigate the suppression effect of expressing parvovirus H-1 nonstructural protein 1 (NS1) gene on human gastric cancer cells and the possible mechanisms.Methods A recombinant enhanced green fluorescent protein (eGFP) labeled NS1 of parvovirus H-1 plasmid was constructed.Human gastric cancer cell line SGC7901 was transfected with recombinant plasmid (experiment group) or blank vector (negative control group) and blank control group was treated with equal amount of phosphate buffered saline (blank control group).After transfection,the distribution of fluorescent signal was observed under fluorescent microscope.The expression of NS1 at gene and protein level was measured.Cell growth curve of each group was drawn.The expression of cell senescence-associated β-galactosidase (SA-β-Gal) was tested.The changes of cell cycle were investigated by flowcytometry.Two groups' comparision was performed by t-test.Results After transfection,NS1 was expressed in SGC7901 cells at gene and protein level.Compared with negative control group,the fluorescent signal accumulated in cell nucleus in experiment group.The percentage of SA-β-Gal positive cell in experiment group ((30.5 ± 1.4) ％) was higher than that of negative control group ((4.4± 1.1) ％) and the difference was statistically significant (t =-12.931,P ＜ 0.01).The growth inhibition rate of SGC7901 cells from the first day to the fourth day was 45％,62％,73％ and 77％,respectively.The cell cycle of eGFP-NS1 expressed SGC7901 cells was arrested at G0/G1 phase.Conclusion Parvovirus H-1 NS1 play the role in cell nucleus of gastric cancer cell line SGC7901 and could make cell cycle arrested at G0/G1 phase,which effectively inhibited the proliferation SGC7901 cell.

Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus / 中国病毒学·英文版

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

Hepatitis C virus: Molecular biology & current therapeutic options.

Hepatitis C virus (HCV) is a small (~55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent development of a robust cell culture system for HCV propagation. HCV proteins assemble and form replication complexes on modified host membranes, called as membranous webs. Even though HCV is detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments; and the viral population is known to rapidly change during the course of a natural infection thereby escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission. The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins during HCV lifecycle is vital for developing a successful cure. This review deals with current status of efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and rapidly changing hepatitis C virus.

Inhibition of IFN-γ receptor signaling by Hepatitis C Virus non-structural protein NS4B / 中国医师杂志

Objective To study the influence of NS4B on host defense system. Methods After cell line stably expressed NS4B was established, cell expression profiling caused by NS4B was studied using DNA microarray, and the results of microarray were verified via IFNGR1 fluorescence intensity analysis. Results The data showed that HCV-NS4B could suppress host defense system-associated gene ex-pression, in particular, IFN-γsignal transduction-related genes. Conclusion NS4B could play a role in persistence and resistance to IFN therapy in HCV infection.

Expression of hepatitis C virus nonstructural 4B in transgenic mice

Hepatitis C virus (HCV) is a pathogen that is of great medical significance in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Although the HCV proteins have been intensively investigated over the past decade, the biochemical functions of the NS4B protein are still largely unknown. To investigate NS4B as a potential causative agent of liver disease, transgenic mice expressing the NS4B protein in liver tissue were produced. The transgenic animals were phenotypically similar to their normal littermates for up to 18 months of age. Our results suggest that the HCV NS4B protein is not directly cytopathic or oncogenic in our transgenic mice model.

SCREENING AND CLONING OF THE GENES OF PROTEIN INTERACTING WITH THE NONSTRUCTURAL PROTEIN 5B OF HEPATITIS C VIRUS / 解放军医学杂志

Protein-protein binding is the basis of virus and host cell interactions. With the application of technology of studying protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) was acquired. Non-structure protein 5B(NS5B) of HCV is a kind of viral protein, which plays an important role in replication of HCV. However, the effect of NS5B is not clear. To investigate the biological function of NS5B, we performed yeast two hybrid to look for proteins in hepatocytes interacting with NS5B. We constructed NS5B bait plasmid by cloning the gene of NS5B into pGBKT7, then transformed it into yeast AH109(a type). The transformed yeast was mated with yeast Y187(? type)containing liver cDNA library plasmid in 2?YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-?-gal for screening. Thirty-three colonies were selected and sequenced. Among them, two colonies were new genes with unknown function. The preliminary successful cloning of gene of protein interacting with NS5B paved the way for the study of the physiological function of NS5B and its associated protein.

Caracterización Molecular de la Secuencia parcialdel Gen de la Glicoproteína NS1 del Virus Dengue 1 proveniente de Máncora, Perú

Se caracterizó una región genética que codifica la glicoproteína NS1 del virus dengue 1 proveniente de Máncora, Piura. Comparaciones de secuencias de nucleótidos revelaron un 93,32% de identidad entre el aislamiento peruano y una cepa de Hawai. A nivel de aminoácidos, se observaron cambios de tipo no conservativos en dominios epitópicos de reconocimiento humoral. De otro lado, el perfil hidropático de la región estudiada fue similar al de otros aislamientos referenciales. Los resultados sugieren realizar mayores análisis de identidad genética y mutaciones en dominios epitópicos en el virus dengue 1 peruano.

A 419bp-NS1 genetic region corresponding to Dengue virus 1 was characterised from an outbreak in Máncora Piura. The comparison of nucleotide sequences revealed that Peruvian isolates showed high correlation (93.32%) with a Hawaii strain. Amino acids comparisons revealed non-conservative changes into humoral response epitope domains. On the other hand, the hydropathy profile of NS1 was similar to other referential strains. The results suggest that more comparisons are needed regarding genetic identity and mutations into the epitope domain of Peruvian dengue 1 virus.

Study on the interaction between hepatitis virus C nonstructural protein 4A and calcium modulating cyclophipin ligand by in vivo coimmunoprecipitation / 中华传染病杂志

Objective To prove the interaction between hepatitis virus C(HCV)nonstruetural protein 4A(HCV NS4A)and calcium modulating cyclophilin tigand(CAML)with yeast-two hybrid- ization and coimmunoprecipitation.Methods The gene encoding CAML was cloned,and subcloned into the yeast expression vector pGADT7 and eucell expression vector pcDNA3.1/His-A.The back- cross test between HCV NS4A and CAML was performed in yeast cells.After that,the pCMV-Myc/ NS4A plasmid and pcDNA3.1/His-A-CAML plasmid were co transfected into 293 cells and,then, coimmunoprecipitation and Western blot were performed.Results The gene encoding CAML was cloned sucessfully,and then the gene was subcloned into yeast expression vectors,pGADT7.After the interaction between NS4A and CAML was ensured in yeast cells,the eukaryotic expression vec- tors of NS4A and CAML were constructed and their interaction was ensured again by Co-immunopre- cipitation.Conclusions The interaction between HCV NS4A and CAML is proved.CAML is one of the proteins involved in Ca~(2+)signaling,which suggests that the interaction of HCV NS4A and CAML may be a new clue of the chronic mechanism of HCV infection.Future studies will be required to de- fine the physiologic significance of this interaction.

Mutations of hepatitis C virus 1b NS5A 2209-2248 amino acid sequence is not a edictive factor for response to interferon-alpha therapy and development of patocellular carcinoma

Genetic changes between codons 2209 and 2248 of NS5A of genotype 1b hepatitis C virus (HCV-1b) have been reported to be associated with the sensitivity to interferon-alpha (IFN-alpha). The present study was performed to analyze such relationship in Korean patients with chronic hepatitis C and HCV-1b (n=19), including 12 chronic hepatitis C patients treated with IFN-alpha, 3 chronic hepatitis C patients without treatment as controls, and 4 patients with hepatocellular carcinoma (HCC). Two serum samples, before and after the treatment, were analyzed for the mutations by reverse transcription-polymerase chain reaction, cloning and sequencing. The mutations were identified in 32% (6/19), including five intermediate type (1-3 mutations) and one mutant type (4 or more). In 12 patients treated with IFN-alpha, the number of amino acid substitutions in NS5A2209-2248 was not associated with outcome of the treatment.

Cloning and expression of NS3 gene of hepatitis C virus in yeast / 中华传染病杂志

Objective HCV NS3 protein plays an important role in disease caused by HCV. We investigate the gene expression of HCV NS3 in yeast for future study of the function of the protein. Methods PCR was performed to amplify the gene of HCV NS3 from the plasmid pBRTM/HCV containing the whole fragment of HCV and the gene was cloned into pGEM T vector. Thereafter, HCV NS3 gene was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, and recombinant pGBKT7∶NS3 was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Results HCV NS3 gene was successfully cloned into pGBKT7. The results of SDS PAGE and Western blotting assay showed that the molecular weight of the expressed product was about 22000 Da and HCV NS3 protein was existed within yeast cells.Conclusions HCV NS3 was successfully expressed in yeast expression system.

The Effects of Parvoviral Nonstructural Proteins Gene Transfection on the Expression of Cytokines in Gastric Cancer Cell / 中国肿瘤生物治疗杂志

Human gastric cancer cells (MKN-45) were transfected with pULB3238 (a plasmid carrying MVMp NS gene). After the NS gene expressed, some of the NS-transfectants were died. The other cells were survived. In comparison with those of control MKN-45, the survived NS- transfected MKN-45 cells had the following characterizations: (a). nucleus/cytoplasm ratio reduced; (b). generation time prolonged;(c). adhesion ability between cells increased;(d). cloning efficiency decreased;(e). formation of tumors in nude mice suppressed. To investigate the possible mechanisms of above mentioned change of NS-transfectants, the effect of NS on the expression of some cytokine genes were explored. RNA dot blot indicated that the NS might enhance the expression of IL-l?(2.7 times), IL-1/?(4.6 times) IL- 6 nuclear factor (2.5 times) and had no effect on the expression of IL-6. The results suggested that the antineoplastic activity of parvoviruses may be medicated by NS interfere with the expression of cytokines in the host cells.

Screening of binding protein of hepatitis C virus NS4A by phage display from human liver cDNA library / 解放军医学杂志

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.