Molecular evolution and phylogeny of dengue type 4 virus in the Caribbean

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.     

Sustained transmission of dengue virus type 1 in the Pacific due to repeated introductions of different Asian strains

Outbreaks of dengue due to dengue virus type 1 (DENV-1) occurred almost simultaneously in 2001 in Myanmar and at multiple sites almost 10,000 km away in the Pacific. Phylogenetic analyses of the E protein genes of DENV-1 strains recovered from Asia and the Pacific revealed three major viral genotypes (I, II, and III) with distinct clades within each. The majority of strains from the Pacific and Myanmar, and a number of other Asian strains fell into genotype I. Genotype II comprised a smaller set of Asian and Pacific strains, while genotype III contained viruses from diverse geographical localities. These analyses suggested that the continuing outbreak of dengue in the Pacific has been due to multiple, direct, introductions of dengue viruses from a variety of locations in Asia followed by local transmission. There was no evidence that the introduction of these viruses into the Pacific was associated with any adaptive changes in the E protein of the viruses.     

小鼠抗登革病毒2型单克隆抗体的制备及鉴定

目的 制备小鼠抗登革病毒2型(TR1751株)的单克隆抗体(Monoclonal antibody mAb)，并对其性质进行鉴定。方法免疫原为病毒感染的Blabfc乳鼠脑组织，匀浆后离心，去除细胞成分。免疫Blab／c小鼠，将小鼠脾细胞和SP2／0细胞融合后，通过ELISA、间接免疫荧光染色、噬斑减少中和实验(Plaque reduction neutralization test，PRNT)及Western blot进行筛选和鉴定。结果筛选到I1、I9和B152 3株杂交瘤细胞系，其分泌的mAb均为识别登革病毒E蛋白的中和抗体。结论获得了3株分泌小鼠抗登革病毒2型mAb的杂交瘤细胞系，利用所制备的抗体，为进一步研究登革病毒致病机理和开发疫苗提供了免疫学工具。     

Natural attenuation of dengue virus type-2 after a series of island outbreaks: A retrospective phylogenetic study of events in the South Pacific three decades ago

Dengue is an expanding arboviral disease of variable severity characterized by the emergence of virus strains with greater fitness, epidemic potential and possibly virulence. To investigate the role of dengue virus (DENV) strain variation on epidemic activity we studied DENV-2 viruses from a series of South Pacific islands experiencing outbreaks of varying intensity and clinical severity. Initially appearing in 1971 in Tahiti and Fiji, the virus was responsible for subsequent epidemics in American Samoa, New Caledonia and Niue Island in 1972, reaching Tonga in 1973 where there was near-silent transmission for over a year. Based on whole-genome sequencing and phylogenetic analysis on 20 virus isolates, Tonga viruses were genetically unique, clustering in a single clade. Substitutions in the pre-membrane (prM) and nonstructural genes NS2A and NS4A correlated with the attenuation of the Tongan viruses and suggest that genetic change may play a significant role in dengue epidemic severity.     

Comprehensive mapping of immunodominant and conserved serotype- and group-specific B-cell epitopes of nonstructural protein 1 from dengue virus type 1

The dengue virus (DENV) nonstructural protein 1 (NS1) is an immunogenic protein that holds potential for the development of vaccines and diagnostic reagents; however, the epitopes of NS1 have not been comprehensively mapped. We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. This strategy identified three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15, 71-85, and 338-352. We also identified five group-specific B-cell epitopes that were highly conserved among isolates of the four DENV serotypes. These novel immunodominant serotype- and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.     

Early activation of natural killer and B cells in response to primary dengue virus infection in A/J mice

Dengue virus (DEN) causes the most prevalent arthropod-borne viral illness in humans worldwide. Immune mechanisms that are involved in protection and pathogenesis of DEN infection have not been fully elucidated due largely to the lack of an adequate animal model. Therefore, as a first step, we characterized the primary immune response in immunocompetent inbred A/J mice that were infected intravenously with a non-mouse-adapted DEN type 2 (DEN2) strain. A subset (55%) of infected mice developed paralysis by 14 days post-infection (p.i.), harbored infectious DEN in the central nervous system (CNS), and had an elevated hematocrit and a decreased white blood cell (WBC) count. Immunologic studies detected (i). increased numbers of CD69(+) splenic natural killer (NK) and B cells at day 3 p.i., (ii). DEN-specific IgM and IgG responses by days 3 and 7 p.i., respectively, and (iii). splenocyte production of IFNgamma at day 14 p.i. We conclude that the early activities of NK cells, B cells and IgM, and later actions of IFNgamma and IgG likely play a role in the defense against DEN infection.     

Identification of a recombinant dengue virus type 1 with 3 recombination regions in natural populations in Guangdong province,China

Using recombination analysis, we identified a recombinant dengue virus type 1 strain, namely, GD23/95, with three recombination regions, located within the sequences of the prM/E junction, NS1, and NS3, respectively. The recombinant dengue virus was further confirmed by phylogenetic analysis based on its recombination and non-recombination regions. This appears to be the first study to confirm the existence of three recombination regions in a single dengue virus isolate and to report recombination between parent virus strains isolated from the same geographic area (Guangdong province, China). It is also the first to report breakpoints within the NS3 gene of dengue viruses.     

Needle-free Biojector injection of a dengue virus type 1 DNA vaccine with human immunostimulatory sequences and the GM-CSF gene increases immunogenicity and protection from virus challenge in Aotus monkeys

A dengue-1 DNA vaccine containing sequences encoding premembrane and envelope proteins (DIME) was previously shown to elicit virus neutralizing antibodies in rhesus and Aotus monkeys, and the primates were partially protected from viremia upon challenge. To increase the neutralizing antibody levels and subsequent protection from virus challenge, four strategies were evaluated: (a) coimmunization with a plasmid expressing Aotus GM-CSF gene; (b) coimmunization with a plasmid containing human immunostimulatory sequences (ISS); (c) coimmunization with both the GM-CSF gene and ISS; and (d) delivery of vaccine using the needle-free Biojector system. Vaccination with the mixed formulation containing DIME, GM-CSF gene, and ISS, by either needle injection or Biojector, led to neutralizing antibody titers that were stable for up to 6 months after vaccination. Furthermore, 6 of 7 monkeys (85%), and 7 of 8 monkeys (87%) receiving this formulation were completely protected from viremia when challenged 1 and 6 months after vaccination, respectively. This is a significant improvement compared to our previous study in which one of three monkeys (33%) receiving just the DIME vaccine was completely protected from viremia at 6 months after immunization.     

两株登革2型病毒E、NS1蛋白基因序列测定及其系统发生分析

目的　对从登革出血热 (denguehemorrhagicfever,DHF)病人血清分离到的一株登革 2型病毒 (denguevirustype 2 ,DEN 2B株 )和DEN 2NGC株作E、NS1蛋白部分基因序列测定并进行系统发生树分析 ,了解其变异及分子进化特征。方法　利用RT PCR法扩增B株和NGC株E、NS1部分基因片段 ,PCR产物直接测序 ;利用DNAssist软件预测其氨基酸序列 ,以及PHYLIP软件的CLUSTAL方法绘制系统发生树。结果　B株与NGC株E蛋白基因区第 1～ 4 76nt( 4 76bp)碱基序列存在 8个位点的差异 ;编码氨基酸存在 4个位置的不同 ;NS1基因区第 5 89～ 10 0 2nt( 4 13bp)碱基序列存在 7个位点的差异 ,编码氨基酸有 2个位置的改变 ;B株与NGC株和DEN 2 4 4株E区部分片段核苷酸同源性分别为99 .1%和 98.7% ;与NS1区部分片段核苷酸同源性分别为 98.3%和 98.1%。B株E基因、NS1基因序列已登录GenBank ,注册号分别为AY179733和AY2 384 71。结论　B株E、NS1基因序列和氨基酸序列与NGC株存在差异 ;B株与我国海南 1989年流行的DEN 2 4 4株和NGC株系统进化关系较近。     

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.     

登革1～4型病毒prM-E基因的双价和四价重组质粒DNA在小鼠中的免疫原性观察

目的 在鼠模型中观察登革（DEN）病毒双价和四价重组质粒DNA的免疫原性，为登革多价DNA疫苗的研究奠定基础。方法采用可去除内毒素的试剂盒大量提取质粒DNA。将双价质粒DNA及将它们配伍后再与鼠源GM-CSF进行联合免疫，免疫途径采用肌肉注射，并加以电刺激，以提高质粒DNA的摄入效率。于初次免疫后第2和4周各加强免疫1次。然后在小鼠中分别测定针对登革1-4型病毒的体液和细胞免疫应答水平。采用间接免疫荧光法和中和试验测定血清抗体效价，细胞免疫应答水平通过测定脾淋巴细胞的增殖指数和其分泌的IFN-T浓度进行评价。细胞浸润实验通过对免疫部位的肌肉进行HE染色确定。结果小鼠在初次免疫后第4周即开始产生针对DEN1～4型病毒的抗体，随着时间的延长，抗体效价逐渐上升。第14周中和抗体效价最高达1：32。淋巴细胞的刺激指数及其分泌IFN-T的浓度均显著高于对照组。GM-CSF对体液免疫应答有促进作用，但对细胞免疫应答无显著的促进作用。结论本研究所构建的双价和四价重组质粒DNA在鼠模型中具有较好的免疫原性，这为登革多价DNA疫苗的研究奠定了基础。     

Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1

To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly of a cDNA capable of producing infectious virus was only possible using a bacterial artificial chromosome plasmid, indicating that DV1 sequences were especially difficult to propagate in E. coli. While characterizing our cDNA we discovered a fortuitous temperature-sensitive mutation in the NS1 encoding region. Using our infectious cDNA and a renilla luciferase-expressing replicon we were able to demonstrate that this mutation produced a defect in RNA replication at 37 degrees C, demonstrating that the DV1 NS1 protein plays an essential role in RNA replication.     

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.     

Polyclonal antibodies against properly folded Dengue virus NS1 protein expressed in E. coli enable sensitive and early dengue diagnosis

The non-structural 1 (NS1) protein plays an important role in dengue diagnosis because it has been detected as a soluble serum antigen in both primary and secondary infections. The NS1 protein was expressed in Escherichia coli cells, and the efficiency of four different refolding protocols was tested. All of the protocols generated dimeric NS1 in a conformation similar to that of the protein expressed by eukaryotic cells. A polyclonal antibody produced from the properly folded E. coli recombinant NS1 (rNS1) protein proved to be a useful tool for the diagnosis of Dengue virus because it detected 100% of the Dengue virus 2 (DENV2) in infected patients’ sera and 60% of the DENV IgM-positive sera not detected by commercial NS1-based diagnostic kits. These data suggest a high-efficiency method for correctly folding rNS1 that maintains its structural and immunogenic properties. In addition, a detection method using the polyclonal antibody against correctly folded rNS1 seemed to be more sensitive and efficient for NS1 detection in serum, highlighting its usefulness for developing a high-sensitivity diagnostic kit.     

Clinical isolates of dengue virus with distinctive susceptibility to nitric oxide radical induce differential gene responses in THP-1 cells

In the present study, 10 clinical isolates of dengue virus were selected according to their susceptibility to the inhibitory effect of nitric oxide radical, NO. Five of them are nitric oxide-susceptible viruses while the other five are nitric oxide-resistant viruses. These isolates were investigated to identify genetic factors that are responsible for the different phenotypes. Due to the evidence showing that NO suppresses DENV RNA polymerase activity, we, therefore, hypothesized that the RdRp domain of NS5 may responsible for NO inhibition. To answer this question, sequences of NS5 gene of NO-susceptible viruses and NO-resistant viruses were compared. We found that these two groups of viruses contain different amino acid sequence at position 621 to 646 in the active site of NS5. These data suggested that response to the inhibitory effect of nitric oxide radical may, at least in part, be regulated by NS5. The effect of these two different phenotypes of viruses on host cells was studied using cDNA array screening. The cDNA array analysis demonstrated that the nitric oxide-resistant group had a stronger influence on host cells since it induced changes in the expression of a greater number of genes than did the nitric oxide-susceptible group, 97 genes versus 71 genes, respectively. The NO-resistant virus also stimulated cytokines known to be virulent factors, such as IL 6, IL 8, RANTES, and the inflammatory factors. In conclusion, our data demonstrated that dengue viruses isolated from patients show genotypic and phenotypic differences which may correlate with virulence.     

Genetic diversity and purifying selection in West Nile virus populations are maintained during host switching

To investigate differential evolutionary rates and selective forces of WNV in hosts and vectors, we measured the genetic diversity that arose during alternating passage in mosquitoes and birds. Within-host genetic diversity was monitored in each of three experimentally passed replicates, and the complete genome sequence of each WNV strain was determined after passage. The intrahost genetic diversity that arose during alternating passage was significantly greater than the diversity generated during chicken-only passage and similar to mosquito-only passage. dN/dS ratios suggested purifying selection similar to chick-passed virus, but not to mosquito-passed virus. Thus, the abundant genetic variation contributed to WNV populations through infection of mosquitoes and the strong purifying selection contributed by infection of birds may be maintained despite frequent host switching.     

A study on viral haemorrhagic fever due to dengue,chikungunya and Crimean Congo haemorrhagic fever virus among patients attending tertiary care hospital in North East India

PurposeThe present study was undertaken with the objective to study the common etiology of Viral Haemorrhagic Fever (VHF) among patients attending tertiary health care centre in NE India and also to study the clinico-demographic profile of such patients. The agents of VHF included in the study were dengue, chikungunya and Crimean Congo haemorrhagic fever (CCHF) virus. The inclusion of CCHF was based on evidence of seroprevalence in livestock (bovine, sheep and goat) in various North Eastern states.Materials and methodsSerum samples were collected from 51 suspected VHF patients. MAC-ELISA was done to detect dengue and chikungunya specific IgM antibody. The samples were also tested by real-time RT-PCR for detection of dengue, chikungunya and CCHF specific nucleic acid. The laboratory and clinico-demographic profile of these patients were noted in detail.ResultsSerum samples of 16 of 51 suspected cases were confirmed to be suffering from VHF. Among these confirmed cases, 12 were diagnosed with dengue haemorrhagic fever, one was diagnosed with chikungunya and three were diagnosed with dengue-chikungunya co-infection. Based on severity, DHF was further classified into- DHF I- (4,26.6%), DHF II (6,40%), DHF III (3,20%) and DHF IV (2,13.3%). There was no CCHFV infection detected in our study. Retro-orbital pain (P ?= ?0.02) and haematocrit level (P ?= ?0.03) were found to be statistically significant.ConclusionsThis study reiterates the fact that CCHF virus infection is still probably absent in human population of NE India and haemorrhagic symptoms, though rare maybe one of the atypical manifestations of chikungunya infection.     

Antibodies to Epstein-Barr virus and cytomegalovirus in relation to CD4 cell number in human immunodeficiency virus 1 infection.

Interaction between herpesviruses and human immunodeficiency virus (HIV)1 is postulated in the progression of HIV disease. In order to evaluate the specific antibody responses directed to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, we studied IgA and IgG titres to EBV and CMV in the serum of HIV positive patients in relation to the CD4 cell number. The titres of IgG antibodies to EBV and the prevalence of IgG to CMV were significantly higher in HIV positive patients compared to control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation to the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with low titres. IgA antibodies to EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe in HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.     

Genome analysis of dengue type-1 virus isolated between 1990 and 2001 in Brazil reveals a remarkable conservation of the structural proteins but amino acid differences in the non-structural proteins

We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.