A non-coated enzyme-linked immunosorbent assay for screening zika virus envelope protein / 南方医科大学学报

OBJECTIVE@#To establish a non-coated enzyme-linked immunosorbent assay (ELISA) based on zika virus envelope (E) protein for detecting the expression of E protein in infected cells.@*METHODS@#Adherent Vero-143 cells infected with zika virus in a 96-well plate were fixed, and the antibodies against zika virus E protein were added at an optimized concentration to establish the non-coated ELISA method for E protein. The antiviral activities of lignans compound C1 was evaluated using this method. The accuracy of this non-coated ELISA was verified by RT-PCR, and the cross reaction with dengue virus was assessed.@*RESULTS@#After optimization, the background absorbance at 450 nm of uninfected cells was reduced to about 0.20. The antiviral activities of lignans compound C1 detected by this method were basically consistent with the results of RT-PCR. No cross reaction with dengue virus was found in this assay.@*CONCLUSIONS@#A non- coated ELISA method based on zika virus E protein was established, which can be used for screening antiviral agents against zika virus.

Physiological Cyclic Stretch Inhibits Vascular Smooth Muscle Cell Apoptosis via Inducing Expression of Nuclear Envelope Protein Emerin / 医用生物力学

Objective To investigate the mechanical response of Emerin, a nuclear envelope protein, and its role in apoptosis of vascular smooth muscle cells (VSMCs) during cyclic stretch, and the potential effect of transcriptional factors in this process. Methods Physiological cyclic stretch with the magnitude of 5% and frequency of 1.25 Hz was subjected to VSMCs in vitro by using FX-5000T cyclic stretch loading system. VSMCs cultured under the same conditions but without applying mechanical stretch were used as the static control. The apoptosis of VSMCs was detected by using Cleaved-caspase3 ELISA kit, and the expression of Emerin was revealed by using Western blotting. The effects of Emerin on activities of 345 kinds of transcriptional factors in VSMCs were demonstrated with Protein/DNA array after Emerin specific RNA interference (RNAi) under static condition, and the potential transcriptional factors involved in VSMC apoptosis were analyzed with Ingenurity Pathway Analysis (IPA) software. Furthermore, the binding abilities of Emerin to the motif of 2 kinds of apoptosis-related transcriptional factors were detected with chromatin immunoprecipitation (CHIP) and qPCR. ResultsCompared with the static control, the apoptosis of VSMCs was significantly decreased by 5% cyclic stretch, which suggested a protective effect of physiological cyclic stretch. The expressions of Emerin in VSMCs was remarkably increased with 5% cyclic stretch applied for 6 h, 12 h and 24 h. Specific RNAi under static condition decreased the expressions of Emerin but increased the apoptosis of VSMCs. Emerin siRNA transfection remarkably increased (more than 2 times) the activities of 10 transcriptional factors that participated in cellular apoptosis, i.e. CREB-BP1, p300, p55, MAX, NRF-1, STAT1, STAT3, TEF1, TR and BZP. CHIP-qPCR result revealed that the binding ability of Emerin to specific mofit of STAT1 or STAT3 was significantly repressed with Emerin RNAi. Conclusions Physiological cyclic stretch could increase the expression of Emerin which might modulate the binding of Emerin to motifs of apoptosis-related transcriptional factors such as STAT1 and STAT3, regulate the activities of these factors, and then subsequently repress the VSMC apoptosis. The investigation on mechanobiological mechanisms of VSMC apoptosis induced by cyclic stretch may contribute to further understanding the physiological and pathological mechanisms of vascular homeostasis and vascular remodeling.

Prokaryotic expression and preparation of the polyclonal antibodies of E protein and domain Ⅲ in Zika virus / 中国人兽共患病学报

To clone,express and purify the E Protein and EDⅢ of Zika virus in E.coli and prepare two kinds of polyclonal antibodies,the virus was amplified by Vero E6 cell culture.Total RNA was extracted by RT-PCR and reverse transcribed into cDNA.The prokaryotic expression vectors pET32a/E and pET28a/EDⅢ were constructed by cDNA sequence of E and EDⅢ gene.Then,recombinant plasmids were transformed into E.coli BL21 and induced by IPTG,and purified by Ni+ column affinity chromatography.BALB/C mice were immunized with purified recombinant proteins.Antiserum was collected and titer was determined by indirect ELISA.Western blot was used to detect the specificity.Results showed that the recombinant proteins were successfully expressed and purified.The titer of the polyclonal antibodies both reached 1:409 600.Western Blot analysis showed that the polyclonal antibodies could specifically recognize the recombinant proteins.Thus,the specific polyclonal antibody were successfully prepared,laying a foundation for further study on the pathogenesis,detection methods and immune strategies of Zika virus.

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9

Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV-receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.

Phylogeny of Dengue virus type 2 isolated in the Central Highlands, Vietnam / Filogenia del virus del Dengue tipo2 identificado en el Altiplano Central de Vietnam

AbstractDengue fever is perhaps the most important viral re-emergent disease especially in tropical and sub-tropical countries, affecting about 50 million people around the world every year. In the Central Highlands regions of Vietnam, dengue fever still remains as a major public health issue. Although four viral serotypes have been currently identified, dengue virus type 2 (DENV-2) was involved in the most important outbreaks during 2010-2012, especially, 2010 when the fatality rate highly increased. Detection of genotype of DENV2 provided information on origin, distribution and genotype of the virus. In this study, DEN-2 isolated from dengue patients during the 2010-2012 epidemics was amplified and sequenced with E gene. The consensus sequences were aligned with reference E gene sequences of globally available Genbank. Phylogenetic analysis was performed using Neighbor-joining and Kimura 2-parameter model to construct phylogenetic tree. A total of 15 isolates (seven from 2010; one from 2011 and seven from 2012) were obtained from human serum samples. Phylogenetic analysis revealed that Asian genotype 1 is currently circulating locally in Central Highlands region. Isolates of this genotype were closely related to viruses from Thailand, Laos, and Cambodia. It indicated that these epidemics maybe imported into the Central Highlands region from South-East Asia neighbor countries. The study results would help in planning for prevention and control of dengue virus in Vietnam. Continuous monitoring of DENV genotypes is necessary to confirm the current findings and detect possible genotype shifts within the dengue viruses in the future.

ResumenPosiblemente, la fiebre del dengue es la enfermedad viral recurrente más importante en los países tropicales y subtropicales que afecta cerca de 50 millones de personas cada año en todo el mundo. En las regiones del Altiplano Central de Vietman, la fiebre del dengue aun se considera como una gran preocupación de salud pública. Aunque los cuatro serotipos virales han sido identificados, el virus del dengue tipo 2 (DENV-2) estuvo involucrado en el brote más importante durante el 2010-2012, especialmente en el 2010 cuando los índices de mortalidad aumentaron considerablemente. El descubrimiento del genotipo DENV-2 proporcionó información del origen, distribución y genotipo del virus. En este estudio, el serotipo DENV-2 identificado de pacientes con dengue durante las epidemias de 2010-2012 se amplificaron y secuenciaron con E gene. Las secuencias consenso se alinearon con secuencias de referencia mundiales de E gene disponibles en GenBank. El análisis filogenético se llevó a cabo utilizando el modelo Neighbor-joining y Kimura 2-parámetros para construir el árbol filogenético. Un total de 15 cepas (siete de 2010, una de 2011 y 7 de 2012) se obtuvieron de las muestras de suero humano. El análisis filogenético reveló que el genotipo asiático 1 circula localmente en la región del Altiplano Central. Las cepas de este genotipo estan muy relacionadas con los virus de Tailandia, Laos y Camboya. El análisis también indicó que estas epidemias pudieron migrar a la región del Altiplano Central desde los países vecinos del sureste asiático. Los resultados de este estudio pueden ayudar en la planificación de la prevención y el control del virus del dengue en Vietnam. Un monitoreo contante de los genotipos DENV es necesario para confirmar los hallazgos recientes y detectar los posibles cambios del genotipo de los virus del dengue en el futuro.

Expression of envelope protein of Zika virus in baculovirus expression system / 中华实验和临床病毒学杂志

Objective@#To express envelope protein of ZIKA virus in baculovirus expression system.@*Methods@#Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1. The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells. The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells, and the constructed recombinant baculovirus rBac-E was determined for titer, for insertion of E gene by PCR, and for expression of E protein by IFA and Western blotting.@*Results@#PCR proved that skeleton plasmid rBacmid-E was constructed correctly. The titer of rBac-E of passage 3 was 2.58×105pfu/ml. The genome of infected cells virus was extracted, the gene band at length of 3 830 bp was observed after PCR amplification. Indirect immunofluorescence of the infected cells showed the specific green fluorescence, 55×103specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBac-E.@*Conclusions@#The recombinant baculovirus with E gene of ZIKA virus was successfully constructed, which laid a foundation of further study on the function of E protein and the vaccine of ZIKA virus.

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.

Isolation, Cloning and Co-expression of Hepatitis C Virus Envelope Proteins: As Potential HCV Detecting Antigens.

Aim: Approximately 3% of the world population is infected with Hepatitis C virus (HCV) which is the main cause of chronic liver disease. Blood transfusion is thought to be the leading cause of global epidemic of HCV. The envelope proteins E1 and E2 are involved in the early stages of the virus life cycle. These proteins have a major role in binding to receptors on the cell surface, fusion and integration of the virus into the host cell. Considering the potency of E1 and E2 in the development of diagnostic methods, the aim of our present study was co-expression of recombinant envelope proteins in eukaryotic HEK293 (human embryonic kidney) cells. Methods: The viral genomic RNA was used for cDNA (complementary DNA) synthesis. Isolation of HCV envelope proteins coding fragment was performed using cDNA and specific primers. The target gene was cloned into pcDNA3.1 expression vector, and transfected into HEK293 cells, an expression host. Accuracy of the cloning and expression was confirmed using PCR and Western blot analysis. Results: The isolation and cloning of the gene fragment encoding the E1 and E2 proteins was successful. Co-expression of these proteins was confirmed using monoclonal antibodies specific for each protein. Conclusion: This study showed that HEK293 host cell is suitable for the expression of hepatitis C virus E1 and E2 coding gene. These proteins can be used in numerous virological studies and detection of HCV infection.

Expression and identification of the functional domains of dengue virus type 1 envelope protein in 293T cells / 中华微生物学和免疫学杂志

Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

Clinical value of combined detection of anti-nuclear envelope protein antibody, anti-soluble acid resistant nucleoprotein and anti-mitochondrial antibody M2 subtype in diagnosis sjogren syndrome and primary biliary cirrhosis / 中国综合临床

Objective To investigate the clinical significance of anti-nuclear envelope protein antibody (gp210),anti-soluble acid resistant nucleoprotein (sp100) and anti-mitochondrial antibody M2 subtype (AMA-M2) in sjogren syndrome (SS) and primary biliary cirrhosis (PBC).Methods A total of 241 hospitalized patients diagnosed with connective tissue disease (CTD) were recruited.Anti-gp210,anti-sp100 and AMA-M2 were detected by indirect immunofluorescence.Results (1) Positive rate of AMA-M2,anti-sp100 and antigp210 in 241 cases CTD patient were 10.4％ (25/241),3.3％ (8/241) and 2.9％ (7/241) respectively.(2) There were 16 cases with SS,5 cases with SS-PBC overlap syndrome and 17 cases with PBC in 241 patients with CTD.Distinction among groups of PBC,SS,SS overlapping PBC of positive incidence of AMA-M2 antibody (x2 =6.584,P =0.03) and anti-gp210 (x2 =8.735,P ＜ 0.01) were significantly different,while there was no apparent difference about positive rate of anti-sp100 among the three groups (x2 =3.343,P =0.18).(3) Positive expression of either antibody of anti-gp210 or anti-sp100 in the three groups of SS,SS overlapping PBC,PBC were 3 cases,4 cases,4 cases respectively.The positive rates of any of three autoantibodies in three groups of were 8 cases,5 cases,13 cases respectively.(4) There were significant difference in terms of serum ALB(t =3.858,P＜0.000 1),TSB(t =5.473,P＜0.000 1),ALT(t =2.235,P=0.026),AKP(t =3.141,P =0.002) and γ-GT (t =2.317,P =0.021) in liver damaged patients of all CTD between AMA-M2 positive and negative patients (P ＜ 0.05).However,serum TSB in anti-sp100 positive and negative patients were differed (t =7.892,P ＜ 0.000 1).Serum AKP was different between anti-gp210 positive and negative patients (t =2.451,P =0.015).Conclusion Positive rate of anti-gp210,anti-sp100 and AMA-M2 are the highest in patients with SS overlap of the PBC among CTD patients.Combined detection can improve the sensitivity of diagnosis.Antisp100 and anti-gp210 are valuable for the diagnosis of SS-PBC overlaps syndrome with negative AMA-M2.

Prediction and identification of B-cell epitopes on monkey B virus envelope protein gD / 中国比较医学杂志

Objective To Identify B-cell epitopes on monkey B virus envelope protein gD.Methods Base on bioinformatics software, secondary structure, hydrophilicity, surface accessibility, antigenic index and flexibility of monkey B virus envelope protein gD was analysed and some potential peptide epitopes were forecasted.Then, the interactions between synthetic peptides and BV positive sera were detected by Enzyme-linked immunosorbent assay ( ELISA) .At last, The sensitivity and specificity of synthetic peptides was evaluated used 20 samples of Standard sera by ELISA.Result Seven epitopes were forecasted by Bioinformatics analysis. Four synthetic peptides, sequence as 46 LPPLEQKTD54 , 106 RGAPEATRSDA116 , 291 PELAPEERGTSRTPGD306 and 361 AVYLVRRRGR370 could be reacted with positive sera pool.The sensitivity of 4 synthetic peptides changed form 40% to 70% and the specificity were 100% for 4 synthetic peptides. Conclusion There are at least four linear epitope on B virus gD protein.

Expression and purification of the soluble envelope protein of Japanese encephalitis virus / 中华微生物学和免疫学杂志

Objective To express and purify the envelope ( E) protein of Japanese encephalitis virus (JEV) in soluble form.Methods Various prokaryotic expression vectors , host strains and induction conditions including time and temperatures were screened to obtain an optimum prokaryotic expression system for JEV E protein in soluble form .The expressed protein was purified by using nickel column chromatography and gel filtration chromatography .Results The soluble JEV E protein , accounting for 23% of the totally bacteria soluble protein was effectively expressed by using the recombinant plasmid PBCX -E406 at low tem-perature.The purity of the expressed protein reached up to 85%after the purification by using nickel column and gel filtration chromatography .Conclusion Soluble JEV E protein was successfully expressed and puri-fied in a simple and efficient way .It would provide a useful tool for further investigation on JEV infection , attenuation mechanism of JE live vaccine strain SA 14-14-2 and the quality control of JE vaccine .It can also be used for the development of diagnosis assay for JEV .

Sequence analysis of envelope genes in dengue viruses from Fujian Province, 2004-2010 / 中国人兽共患病学报

Since several dengue viruses (DENV) have been isolated in Fujian Province during the past decade, sequencing and evolution analyses of viral envelope genes are helpful in determining their possible transmission origins. In this study, viral RNA was extracted from 12 DENV strains from Fujian between 20042010. Viral envelope genes were amplified, cloned into TA vectors and sequenced, and the sequence data were subsequently analyzed by bioinformatics software. Full-length E genes of DENV-1 or DENV-2 of 1 485 bp, and DENV- 3 of 1 479 bp were obtained. It was indicated, from BLAST analysis and phylogenetic trees, that DENV strains in Fujian Province during 20042010 shared the highest similarity with Southeast Asian strains, suggesting that DENV circulating in Fujian Province between 20042010 were probably imported from Southeast Asia. Hence, extensive monitoring on passengers from this region at the entry-ports should be strengthened.

The Comparison of Genetic Variation in the Envelope Protein Between Various Immunodeficiency Viruses and Equine Infectious Anemia Virus / 中国病毒学·英文版

The envelope protein(Env) of lentiviruses such as HIV,SIV,FIV and EIAV is larger than that of other retroviruses.The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus,demonstrating that envelope is crucial for vaccine.We compared Env variation of the four kinds of lentiviruses.Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor,and the relationship of HIV and EIAV was the furthest.EIAV had the shortest Env length and the least number of potential N-linked glycosylation sites(PNGS) as well as glycosylation density compared to various immunodeficiency viruses.However,HIV had the longest Env length and the most PNGS.Moreover,the alignment of HIV and SIV showed that PNGS were primarily distributed within extracellular membrane protein gp120 rather than transmembrane gp41.It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env.There are low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

Progress in Research of the Role of Hepatitis C Virus Protein in Cell Signal Transduction Pathway / 中国实验动物学报

Abnormal cell signal transduction is associated with the occurrence and development of human diseases. Some virus pathogenicity and infection mechanism are due to virus antigen protein acting on the host cell signal transduction pathway, leading to host cell signal transduction disorder. Hepatitis C virus (HCV) is a major pathogen of chronic hepatitis C, which causes cirrhosis and hepatocellular carcinoma. But the pathogenesis of HCV and persistent infection mechanism remain far from clear. HCV pathogenesis may be related to the HCV protein expression interfering host cell signal transduction pathways. The studies of hepatitis C virus proteins acting on host cell signal transduction pathways, not only help to clarify the impact of its pathogenic mechanisms, but also benefit to new drug design and development for new treatment methods. This article summarizes the recent progress in research on the effect of hepatitis C virus protein in cell signal transduction pathways in the past few years.

Construction of the Porcine Endogenous Retrovirus Envelope Glycoprotein A and B Specific Antibody

Xenotransplantation using porcine organs could potentially associate with the risk of pathogenic infections, because human tropic porcine endogenous retrovirus (PERV) particles could be released from pig cells or organs. While there is no evidence of PERV transmission to human, safety issues become a paramount concern. For the prevention of this transmission, specific immunological tools must be provided for PERV transmission detection. In this study we described the expression of PERV envelope proteins and the production of a specific antibody against PERV envelope (Env) glycoprotein. The nucleotide sequence harboring the partial region of glycoprotein 70 was cloned into the pET vector and envelope protein was expressed in E. coli. Approximately 42 kDa recombinant Env protein (PERV Env-aa357) was purified by the Ni-affinity column. For antibody production, mice were immunized with the recombinant PERV Env-aa357. The generated anti-serum was tested using Western blot and immunocytochemical assay. We found that anti-PERV Env serum displayed the specificity against the PERV Envs (PERV-A and PERV-B) expressed not only in E. coli but also in mammalian cells, and PERV particles within the porcine cell lines (PK 15 and PK-1). Taken together, PERV antibody could be useful for detecting PERV infection or xenotransplantation transmission.

Expression and Evaluation of Chikungunya Virus E1 and E2 Envelope Proteins for Serodiagnosis of Chikungunya Virus Infection

PURPOSE: Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens. MATERIALS AND METHODS: CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA. RESULTS: The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%. CONCLUSION: The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide

The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.

Phylogenetic analysis of the envelope protein (domain lll) of dengue 4 viruses

Objective. To evaluate the genetic variability of domain III of envelope (E) protein and to estimate phylogenetic relationships of dengue 4 (Den-4) viruses isolated in Mexico and from other endemic areas of the world. Material and Methods. A phylogenetic study of domain III of envelope (E) protein of Den-4 viruses was conducted in 1998 using virus strains from Mexico and other parts of the world, isolated in different years. Specific primers were used to amplify by RT-PCR the domain III and to obtain nucleotide sequence. Based on nucleotide and deduced aminoacid sequence, genetic variability was estimated and a phylogenetic tree was generated. To make an easy genetic analysis of domain III region, a Restriction Fragment Length Polymorphism (RFLP) assay was performed, using six restriction enzymes. Results. Study results demonstrate that nucleotide and aminoacid sequence analysis of domain III are similar to those reported from the complete E protein gene. Based on the RFLP analysis of domain III using the restriction enzymes Nla III, Dde I and Cfo I, Den-4 viruses included in this study were clustered into genotypes 1 and 2 previously reported. Conclusions. Study results suggest that domain III may be used as a genetic marker for phylogenetic and molecular epidemiology studies of dengue viruses.

Objetivo. Evaluar la variabilidad genética del dominio III de la proteína de envoltura (E) y estimar la relación filogenética de los virus dengue 4 (Den-4) aislados en México y en otras regiones endémicas del mundo. Material y métodos. En el presente trabajo reportamos un estudio filogenético del dominio III de la proteína de envoltura (E) que se realizó en 1998 con virus Den-4 aislados en distintos años en México y en otras partes del mundo. Se usaron oligonucleótidos específicos para amplificar por RT-PCR la región del dominio III y para obtener la secuencia de nucleótidos. Mediante el análisis de la secuencia de nucleótidos y de la secuencia deducida de aminoácidos se estimó la variabilidad genética y se generó un árbol filogenético. Para facilitar el análisis genético del dominio III se usó la técnica basada en el polimorfismo de fragmentos generados con enzimas de restricción (PFER) utilizando seis enzimas de restricción. Resultados. Los datos demuestran que la información del análisis de la secuencia de nucleótidos y de aminoácidos de la región del dominio III es similar a la del gene completo de la proteína E. El análisis de PFER con las enzimas de restricción Nla III, Dde I y Cfo I, mostró que los virus Den-4 incluidos en este estudio se agruparon en los genotipos 1 y 2 reportados previamente. Conclusiones. Los resultados sugieren que el dominio III se puede utilizar como un marcador para estudios filogenéticos y de epidemiología molecular del virus Den-4.

Isolation and Partial Characterization of a 50 kDa Hemin-regulated Cell Envelope Protein from Prevotella nigrescens / 대한치주과학회지

In the study presented here, identification, purification, and partial characterization of a hemin-regulated protein in Prevotella nigrescens were carried out. The results of this study confirm that the availability of hemin influences the expression of a selected membrane protein as well as the growth rate of P. nigrescens ATCC 33563. The 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin-binding protein from P. nigrescens. Disulfide bonds were not present in this protein, and N'-terminal amino acid sequence analysis revealed that this protein belongs to a new, so far undescribed protein. The 50 kDa protein was found to be rich in hydrophilic amino acids, with glycine comprising approximately 60% of the total amino acids. The study described here is the first to identify, purify, and biochemically characterize a putative hemin-binding protein from P. nigrescens. Work is in progress to further characterize the molecular structure of this protein.