Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong,China in 2014

Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred in Guangdong province,China,resulting in more than 40000 patients including six deaths.To clarify and characterize the causative agent of this outbreak,the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing(HTS).Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2(DENV-2).A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells.The complete genome of GZ05/2014 was determined and deposited in Gen Bank under the access number KP012546.Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade.No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014.The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong,China.These results help clarify the origin of this epidemic and predict the future status of dengue in China.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

乙脑/登革4型嵌合病毒的构建及其小鼠神经毒力测定

目的构建以乙型脑炎病毒(Japanese encephalitis virus,JEV)疫苗株SA14-14-2为基因骨架的乙脑/登革4型嵌合病毒,并分析该嵌合病毒对小鼠的神经毒力。方法通过重叠PCR方法扩增含有登革病毒4型(DENV-4)H241株pr ME基因序列和乙型脑炎病毒疫苗株SA14-14-2的NS1蛋白前177个核苷酸的融合片段,用Nar I和Bgl II双酶切后替换乙型脑炎病毒疫苗株SA14-14-2全长克隆中的相应区域,构建成乙脑/登革4型嵌合全长克隆,通过体外转录和转染BHK21细胞获得嵌合病毒(JEV/DENV-4 chimeric virus,JD4)。通过测定嵌合病毒JD4和2个母本株JEV SA14-14-2株及DENV-4 H241株蚀斑大小、小鼠脑内神经毒力和皮下感染入脑能力、乳鼠脑内神经毒力,比较JD4和母本株之间的差异。通过将JD4在原代地鼠肾(primary hamster kidney,PHK)细胞传代30次,分析传代后嵌合病毒的神经毒力是否减弱及减弱的程度。结果测序结果表明,构建的嵌合病毒JD4基因组序列和预期一致,没有产生新的位点突变。JD4蚀斑较SA14-14-2明显偏小,但和DENV-4 H241株没有明显区别。JD4对3周龄小鼠具有较强的脑内神经毒力,和母本株DENV-4 H241没有差异,对小鼠没有神经侵袭力。乳鼠实验结果表明,嵌合病毒JD4脑内神经毒力虽然略低于母本株DENV-4 H241,但两者之间没有明显差异,都明显强于乙脑疫苗株SA14-14-2。在PHK细胞传代30次后,小鼠神经毒力虽然有所减低,但并不明显。结论成功构建了嵌合病毒JD4,通过测定并比较JD4与母本株的蚀斑特征、小鼠及乳鼠神经毒力等试验,为分析登革疫苗候选株安全性研究奠定了基础。     

Exploring dengue proteome to design an effective epitope-based vaccine against dengue virus

Dengue, a mosquito-borne disease, is caused by four known dengue serotypes. This infection causes a range of symptoms from a mild fever to a sever homorganic fever and death. It is a serious public health problem in subtropical and tropical countries. There is no specific vaccine currently available for clinical use and study on this issue is ongoing. In this study, bioinformatics approaches were used to predict antigenic, immunogenic, non-allergenic, and conserved B and T-cell epitopes as promising targets to design an effective peptide-based vaccine against dengue virus. Molecular docking analysis indicated the deep binding of the identified epitopes in the binding groove of the most popular human MHC I allele (human leukocyte antigens [HLA] A*0201). The final vaccine construct was created by conjugating the B and T-cell identified epitopes using proper linkers and adding an appropriate adjuvant at the N-terminal. The characteristics of the new subunit vaccine demonstrated that the epitope-based vaccine was antigenic, non-toxic, stable, and soluble. Other physicochemical properties of the new designed construct including isoelectric point value, aliphatic index, and grand average of hydropathicity were biologically considerable. Molecular docking of the engineered vaccine with Toll-like receptor 2 (TLR2) model revealed the hydrophobic interaction between the adjuvant and the ligand binding regions in the hydrophobic channel of TLR2. The study results indicated the high potential capability of the new multi-epitope vaccine to induce cellular and humoral immune responses against the dengue virus. Further experimental tests are required to investigate the immune protection capacity of the new vaccine construct in animal models.

Communicated by Ramaswamy H. Sarma     

Investigations of dengue-2 susceptibility and body size among Aedes aegypti populations

The mosquito Aedes aegypti (L.) (Diptera: Culicidae) is the primary global vector for dengue virus (DENV), yet considerable genetic variation exists among populations in terms of its competence to vector DENV. Variability in adult body size has also been observed among various mosquito populations and several studies have reported a relationship between body size and arbovirus dissemination, although most of these relied on artificially derived variation in body size. Here we examine the relationship between body size and disseminated DENV infection among 10 Ae. aegypti populations reared under optimum laboratory conditions. Body size variability was inferred from wing length measurements and DENV competence was evaluated as the proportion of individuals with disseminated infections following exposure to the dengue-2 JAM1409 strain. There were significant differences in mean wing lengths among populations (anova, F(9,22)= 7.10, P < 0.0001), ranging from 2.16 mm (Bangkok population) to 2.79 mm (MOYO-S [susceptible] population). We also observed significant differences among some populations in mean DENV infection rates (Waller-Duncan K-ratio t-test), ranging from 19.54% (MOYO-R [refractory] population) to 56.60% (MOYO-S population). However, we did not observe evidence for significant interactions between body size and DENV dissemination. We suggest that either the two traits are genetically independent or that our ability to detect interactions between them was limited by their respective inheritances as quantitative traits.     

Dengue and dengue haemorrhagic fever: Indian perspective

The relationship of this country with dengue has been long and intense. The first recorded epidemic of clinically dengue-like illness occurred at Madras in 1780 and the dengue virus was isolated for the first time almost simultaneously in Japan and Calcutta in 1943–1944. After the first virologically proved epidemic of dengue fever along the East Coast of India in 1963–1964, it spread to allover the country. The first full-blown epidemic of the severe form of the illness, the dengue haemorrhagic fever/dengue shock syndrome occurred in North India in 1996. Aedes aegypti is the vector for transmission of the disease. Vaccines or antiviral drugs are not available for dengue viruses; the only effective way to prevent epidemic degure fever/dengue haemorrhagic fever (DF/DHF) is to control the mosquito vector, Aedes aegypti and prevent its bite. This country has few virus laboratories and some of them have done excellent work in the area of molecular epidemiology, immunopathology and vaccine development. Selected work done in this country on the problems of dengue is presented here.     

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein–membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.     

Worldwide invasion of vector mosquitoes: present European distribution and challenges for Spain

An Asiatic mosquito species, Aedes albopictus, began to spread worldwide in the 1970s thanks to marine transport of tires and other goods, leading to colonization of many areas of the world. This species is a vector of major human diseases such as Dengue, Yellow Fever and the West Nile virus. In Europe, it was established in Albania and Italy and has been detected in other countries such as France; no records exist for Spain as yet. Colonization by Aedes albopictus is a major public health concern considering that the West Nile virus and several other viruses are known to circulate sporadically in the Mediterranean. Additionally, the parent species Aedes aegypti was the vector causing severe outbreaks of Dengue and Yellow Fever two centuries ago. Although Ae. aegypti was also introduced, it was eradicated from Spain. Both mosquitoes shared habitat types, diseases transmitted and many bionomic data. This article contains a review of the present Ae. albopictusdistribution range worldwide and discusses the likelihood of an establishment in Spain in view of climatological and geographical data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

登革热媒介昆虫抗药性的研究进展

近年来,登革热在全球的流行迅速增长,埃及伊蚊Aedes aegypti(L.)和白纹伊蚊Aedes albopictus(Skuse)是登革热传播的主要媒介。目前,化学防治仍然是防治蚊虫的主要手段,每年化学杀虫剂的大量使用直接或间接地导致其产生了抗药性。关于埃及伊蚊和白纹伊蚊的抗药性研究,主要集中于DDT、有机磷和拟除虫菊酯类杀虫剂导致的抗药性,并已深入到抗药性分子机理的研究。文章就埃及伊蚊和白纹伊蚊对杀虫剂诱导的抗药性现状及抗性机理的研究进展进行综述。