Antigenic cell associated dengue 2 virus proteins detected in vitro using dengue fever patients sera

At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients' sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients' sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient's serum, it was further confirmed that all patients' sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.     

Antibody responses of dengue fever patients to dengue 2 (New Guinea C strain) viral proteins

The present study was undertaken to investigate the antibody responses of dengue fever (DF) patients to specific dengue virus proteins. Partially purified dengue 2 New Guinea C (NGC) strain virus was used as antigen. Under the present experimental protocols, it was observed that almost all DF patients' sera had detectable presence of antibodies which recognize the dengue 2 envelope (E) protein. The convalescent-phase sera especially had significant detectable IgG, IgM and IgE against the protein. In addition, IgGs specific against the NS1 dimer and PrM were also detected. Antibody against the core (C) protein, however, was not detectable in any of the DF patients' sera. The substantial presence of IgG against the PrM in the convalescent-phase sera, and the presence of IgE specific for the E, reflect the potential importance of these antibody responses in the pathogenesis of dengue.     

Detection of some dengue-2 virus antigens in infected cells using immuno-microscopy

Summary Immunoelectron microscopy was used to detect the distribution of some dengue-2 virus proteins in infected Vero andAedes albopictus (C 6/36) cells. It was found that the envelope protein (GP 60) was located in clumps on the surface of plasma membrane, and accumulated very little in the infected cytoplasm. However no envelopment of dengue-2 virus nucleocapsids through the plasma membranes was observed. In contrast, the NS 3 (P 67) protein was distributed throughout the whole cytoplasm. No specific association of this protein with the proliferated virus-induced structures was seen. The NS 1 (GP 46) protein showed almost similar distribution as the NS 3 but its quantity appeared to be lower. The NS 3 and NS 1 distribution patterns observed supported the results of immunofluorescent staining but results on E protein were not consistent in both methods.     

2型登革病毒04株基因组结构特点的研究

报告了２型登革病毒０４株结构蛋白Ｃ，ＰｒＭ（Ｍ）、Ｅ和非结构蛋白ＮＳ１基因的核苷酸及氨基酸序列，并与国际参考株新几内亚Ｃ株、牙买加株和候选疫苗Ｓ１株进行了比较。结果表明，从壳体蛋白Ｃ到主要非结构蛋白ＮＳ１，这一开放读码框架核苷酸总数为３３８１，编码１１２７个氨基酸；在Ｅ蛋白区有３个保守的氨基酸序列，分别位于Ｅ区的９８－１１１、３７１－３７８、４１６－４２２；有２个潜在的糖基化位点，分别位于Ｅ区的Ａｓｎ－６７和Ａｓｎ－１５３位；有１２个保守的半胱氨酸残基。非结构蛋白ＮＳ１有两个糖基化位点，位于ＮＳ１区的１３０和２０７位，有１０个保守的半胱氨酸残基。在结构蛋白Ｃ、ＰｒＭ（Ｍ）、Ｅ和非结构蛋白ＮＳ１中，Ｅ蛋白表现最为保守，其次是Ｃ蛋白。Ｄ２－０４株病毒在核苷酸水平与氨基酸水平上与其他２型病毒株比较，我国０４株较类似于牙买加株，其次是ＮＧＣ株，与Ｓ１株类似性最小。说明我国病毒株与牙买加株相关性较大，与Ｓ１株相关性较小。     

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F). In addition, the PDK50 virus contained a C to T change of nucleotide 57 in the 5′ non-coding region and four silent mutations of nucleotides 591, 987, 6471, and 8907. An infectious PDK50 cDNA clone virus was produced and characterized for growth kinetics in monkey (LLC-MK₂, Vero) and mosquito (C6/36) cells. Identification of mutations in the vaccine strain and availability of an infectious clone will permit systematic analysis of the importance of individual or collective mutations on attenuation of dengue virus.     

Detection of flavivirus antigens in purified infected Vero cell plasma membranes.

The types of Kunjin virus-specified proteins present in purified Vero cell plasma membrane were studied. Immunofluorescence of unfixed Kunjin virus-infected whole cell monolayers, indicated that two structural proteins (envelope and prM) and three non-structural proteins (NS1, 3 and 5) were found at the plasma membrane. There was no obvious progressive accumulation of the observed antigens over the time periods between 8 to 24 h p.i. Thus SDS-PAGE analysis was performed using purified radiolabelled Vero cell plasma membranes. From the protein profiles, all five antigens detected by immunofluorescent staining were also present. In addition, two smaller molecular weight non-structural proteins NS4B and NS2B were also observed. Generally, all the non-structural proteins found in the purified plasma membranes were of the same molecular weights as those found in infected whole cell lysate. Interestingly, both the structural proteins, i.e., envelope (E) and prM proteins in the plasma membrane sample were of higher molecular weights as compared to the counterparts in the infected whole cell lysate. The envelope protein of purified extracellular Kunjin virus was also lower in molecular weight compared to the same protein in the plasma membrane.     

应用通用引物聚合酶链反应技术检测不同型别的登革病毒

利用特异抗体吸附的登革病毒，经含ＴｒｉｔｏｎＸ－１００的裂解缓冲液处理，释放出基因组ＲＮＡ，在同一管中进行逆转录聚合酶链扩增反应。采用这种方法利用保守性引物可检测出登革１型、２型和４型病毒参考株及我国海南岛登革２型病毒分离株的基因组序列，扩增产物约为５５０ｂｐ。用＾３２Ｐ－标记的分子克隆的登革２型病毒基因探针进行Ｓｏｕｔｈｅｒｎ印迹杂交分析证实了护增产物的特异性。利用这种技术还可从感染后２４ｈ的Ｃ     

Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris

The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.     

Flaviviruses can mediate fusion from without in Aedes albopictus mosquito cell cultures

Flavivirus-induced polykaryocytes were detected in monolayers of Aedes albopictus (clone C6/36) mosquito cells as early as 20 min after adsorbing virus to these cells. A high multiplicity of infection with dengue (DEN)-1, 2, 3, 4, Japanese encephalitis, and yellow fever viruses was required to demonstrate fusion from without (FFWO) with these flaviviruses. Optimal conditions for FFWO included exposure of adsorbed virus to pH 6.0 and an incubation temperature of 39 degrees C. DEN-2 monoclonal antibodies to the envelope E glycoprotein inhibited cell fusion, whereas monoclonal antibodies to the prM and NS1 proteins did not inhibit cell fusion. These results indicate that flaviviruses cause FFWO soon after adsorption to C6/36 mosquito cells and the process is most likely mediated by the virion envelope E glycoprotein.     

Molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes

The prevalence of four serotypes of dengue virus (DENV) has risen dramatically in recent years accompanied by an increase in viral genetic diversity. The evolution of DENV has had a major impact on their virulence for humans and on the epidemiology of dengue disease around the world. In order to perform disease surveillance and understand DENV evolution and its effects on virus transmission and disease, an efficient and accurate method for genotype identification is required. Phylogenetic analysis of viral gene sequences is the method used most commonly, with envelope (E) gene the most frequently selected target. To determine which gene might be suitable targets for genotyping DENV, phylogenetic analysis was performed on 10 individual coding genes plus the 3'-non-translated region (3'NTR) for 56 geographically divergent DENV strains representing all identified genotypes. These were reflected in eleven maximum likelihood phylogenetic trees. Based on the bootstrap values (over 90%) supporting the major nodes, the best target genes were identified for each serotype: for DENV-1, the sequences of all coding genes except non-structural gene 4A (NS4A), for DENV-2, PrM/M, E, NS1, NS3, NS4A and NS5, for DENV-3, all coding genes and the 3'NTR, and for DENV-4, C, PrM/M, E, NS1, NS2A, NS2B, NS4A and NS5.     

Cellular proteins bind to the 3' and 5' untranslated regions of dengue 2 virus genome

In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.     

Detection of dengue virus RNA using nucleic acid hybridization

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.     

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.     

Detection and sequencing of defective viral genomes in C6/36 cells persistently infected with dengue virus 2

Dengue virus is the most important arbovirus that affects humans, and it can establish persistent infections, especially in insect-derived cell cultures. Defective viral genomes have been implicated in the establishment and maintenance of persistent infections with several flaviviruses; however, there exists almost no information concerning defective dengue virus genomes. Here, we report the detection of defective dengue 2 virus genomes in persistently infected mosquito C6/36 cells. The defective viral genomes were detected at a low ratio compared with the wild-type genome. Deletions of approximately 147 residues (222-368) were found in the E protein, and these mainly affected domain III (73 %) of the protein; deletions of approximately 153 residues (4-156) and 228 residues (597-825) were found in the methyltransferase and polymerase domains, respectively, of the NS5 protein. The truncated versions of NS5 could be detected by western blot only in the protein extracts derived from persistently infected cells.     

Differential dengue cross-reactive and neutralizing antibody responses in BALB/c and Swiss albino mice induced by immunization with flaviviral vaccines and by infection with homotypic dengue-2 virus strains

We investigated whether cross-reactive and/or cross-protective antibodies against dengue virus could be generated in 6-week-old BALB/c mice by immunization with currently approved flaviviral vaccines, i.e., Japanese encephalitis (JE) BIKEN and yellow fever (YF) 17D. Cross-reactivity with dengue antigens was apparent in at least one-third each of JE-vaccinated mouse sera and of JE/YF-vaccinated mouse sera by dengue enzyme immunoassay, but was not detected in sera of mice immunized with YF vaccine alone. All the immunized BALB/c mice failed to generate neutralizing antibodies against the New Guinea C laboratory (NGC-lab) strain of dengue virus type 2. In addition, we determined the specificity of neutralizing antibodies elicited in 3-week-old Swiss albino mice against two homotypic dengue-2 strains, i.e., NGC-lab and Singapore 1999 (SING/99). Although sera from virus-inoculated mice displayed better neutralization against the corresponding strain, antibodies elicited by NGC-lab exhibited a significantly poorer neutralizing response against the SING/99 strain compared to antibodies elicited by SING/99 against NGC-lab. The differences may be related to sequence variations of approximately 3% between the envelope proteins of both strains. Amino acid disparities at positions 71 (Glu --> Ala), 112 (Ser --> Gly) and 124 (Ile --> Asn), which are found in dengue-2 neutralization escape mutants, were also found in the SING/99 strain. The envelope sequence differences may explain diminished binding of NGC-lab-induced neutralizing antibodies to neutralizing epitopes within the envelope of the SING/99 strain, resulting in a lower titer of neutralizing antibodies against another strain of the same serotype.     

Detection and identification of dengue-1 virus in clinical samples by a nested-PCR followed by restriction enzyme digestion of amplicons

Dengue viruses cause a disease with clinical findings ranging from asymptomatic infections to severe manifestations, characterised by haemorrhage and shock and known as dengue haemorrhagic fever/dengue shock syndrome. Since this fever and syndrome usually results from sequential infections by distinct dengue serotypes, rapid detection and identification of dengue viruses circulating in endemic areas are important to implement control measures, and ultimately to avoid secondary infections that could result in dengue haemorrhagic fever/dengue shock syndrome. A nested-PCR was developed followed by restriction enzyme (Kpn I) digestion of the amplicons to differentiate dengue-1 from dengue-2. Seventy-five IgM-containing samples collected from 2 to 17 days after the beginning of the symptoms were examined. These samples were submitted to nested-PCR amplification, the amplicons were digested with Kpn I, and the results compared to virus isolation in C6/36 cells and to results obtained by the standard PCR. Out of 75 tested samples, virus was isolated from 2 (2.6%), 17 (22.7%) were positive by the regular PCR protocol, and 58 (77.3%) were positive by nested-PCR. All of the amplicons digested by Kpn I identified dengue-1 virus as the infecting strain. These results indicate that the nested-PCR provided a high yield of dengue genome amplification even in the presence of IgM antibodies, and restriction enzyme digestion defined rapidly the circulating serotype. Therefore, the combination of these techniques may be useful to rapidly identify dengue viruses in countries where dengue-1 and dengue-2 circulates, and this approach can also be applied to the other two serotypes.     

Nucleotide and deduced amino acid sequences of genes encoding the structural and nonstructural NS1 proteins of a dengue-2 virus isolated in China

We have determined the nucleotide and encoded amino acid sequences of the capsid, membrane precursor, membrane, envelope, and nonstructural NS1 protein genes of a dengue-2 virus (D2-04) isolated from a patient in Hainan, China. The sequenced region contains a gene organization similar to that of other flaviviruses. The overall amino acid sequence similarity between D2-04 and other dengue-2 viruses is greater than 92%, whereas that between D2-04 and members of the other dengue serotypes is about 65%.The nucleotide sequence data reported in this paper have been deposited in the EMBL database under the following accession numbers: X 65239 for the capsid (C), X 65240 for the envelope (E), X 65241 for the nonstructural (NS1), and X 65242 for the premembrane/membrane (prM/M) protein genes.     

深圳市首例本地登革热3型病例的病原学检测与分析

目的对2016年深圳市报告的首例本地疑似登革热病例查明病因.分离鉴定病原体,从分子水平分析分离株的生物学特征.方法对疑似患者血清标本采用ELISA、胶体金免疫层析法和荧光RT-PCR方法分别检测登革病毒抗体、NS1抗原和病毒核酸,并用C6/36细胞分离登革病毒.采用RT-PCR方法扩增病毒PrM/M-E基因后进行序列测定,并与不同国家和地区的登革毒株进行同源性比较和进化树分析.结果从患者血清标本中检测到登革病毒IgM抗体、NS1抗原和登革3型病毒核酸,并成功分离到登革3型病毒,将其命名为DENV3-SZ1648.深圳市登革3型病毒分离株SZ1648与登革3型国际标准株H87株、国内外流行株80-2、GWL-25株在PrM/M-E基因上核苷酸同源性分别为92.0％、91.8％和90.3％,而与登革1、2、4型国际标准株HAWAII、NGC、H241同源性分别为68.7％、64.2％和63.2％.进化树显示SZ1648株与2007年印度尼西亚分离株MKS-0098亲缘关系最近,在进化树的同一分支上,和85-159株(Indonedia 1985)、2167株(Tahiti 1989)、29472株(Fiji1992)等同属基因Ⅰ亚型.患者发病前1个月在深圳居住,无输血史、无外出史.结论从病原学、血清学和分子生物学特征上均证实该本地病例是由登革3型病毒引起,这也是深圳市首次报道存在本地登革3型病毒的疫情,该毒株最有可能来源于印度尼西亚.     

Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.