A multiple-target mRNA-LNP vaccine induces protective immunity against experimental multi-serotype DENV in mice

Dengue virus(DENV)is a mosquito-borne virus with a rapid spread to humans,causing mild to potentially fatal illness in hundreds of millions of people each year.Due to the large number of serotypes of the virus,there remains an unmet need to develop protective vaccines for a broad spectrum of the virus.Here,we constructed a modified mRNA vaccine containing envelope domain III(E-DIII)and non-structural protein 1(NS1)coated with lipid nanoparticles.This multi-target vaccine induced a robust antiviral immune response and increased neutralizing antibody titers that blocked all four types of DENV infection in vitro without significant antibodydependent enhancement(ADE).In addition,there was more bias for Th1 than Th2 in the exact E-DIII and NS1-specific T cell responses after a single injection.Importantly,intramuscular immunization limited DENV transmission in vivo and eliminated vascular leakage.Our findings highlight that chimeric allogeneic structural and non-structural proteins can be effective targets for DENV vaccine and that they can prevent the further development of congenital DENV syndrome.

Transplacental transmission of dengue infection

We specifically addressed the persistent challenge of dengue in endemic regions,highlighting the potential seriousness of dengue infection through vertical trans-mission.Vertical dengue transmission has been well documented,particularly in hyper-endemic regions,including Ecuador.Herein,we present a neonate diagno-sed with congenital dengue and review similar cases from previously published reports.Although congenital dengue is commonly infected with severe serotypes of DENV(DENV-1 and DENV-2)infections,favorable outcomes are generally observed.

Dengue outbreaks in Taiwan, 1998-2017: Importation, serotype and temporal pattern

Objective: To ascertain the role of imported cases and serotypes on dengue outbreaks in Taiwan which have been sporadic yet highly volatile during the past two decades, exhibiting record-breaking magnitude in recent years. Methods: Confirmed case and serotype data from Taiwan Centers for Disease Control during 1998-2017 were fully examined, with fitting of weekly and daily case data of each city/county to a mathematical model to pinpoint the waves of cases and their locations. Moreover, we quantify the timing of turning point and transmission potential of each wave and determine its circulating serotype, to ascertain any pattern or connection between the variations in circulating serotypes and the magnitude/transmissibility of outbreak. Results: While the number of imported case increased steadily during past two decades, the yearly number of indigenous cases fluctuated wildly. Moreover, while yearly percentages of serotypes for imported cases remains steady, that of indigenous cases does not exhibit any clear pattern. There was at least one wave of reported cases somewhere in Taiwan every year from 1998 to 2015, except in 2016-2017. The effective reproduction number R for all waves in all locations ranged from 1.14 to 2.87, with the exception of two Tainan waves, in 2010(3.95) and 2015(6.84). Four major outbreaks of over 2000 cases reveal circulation of one dominant serotype. Conclusions: Correlation between imported cases and indigenous outbreak prove to be difficult to ascertain, even with the availability of serotype data. However, although there had been occasional co-circulation of serotypes in one location, and for some years with different serotypes circulating in different locations, all major outbreaks of over 2 000 cases during the past two decades are due to circulation of mainly a single serotype, perhaps indicating greater transmission potential with one dominating serotype.

Contribution of phylogenetics to understanding the evolution and epidemiology of dengue virus

Dengue virus(DENV)is one of the most important arboviral pathogens in the tropics and subtropics,and nearly one-third of the world's population is at risk of infection.The transmission of DENV involves a sylvatic cycle between nonhuman primates(NHP)and Aedes genus mosquitoes,and an endemic cycle between human hosts and predominantly Aedes aegypti.DENV belongs to the genus Flavivirus of the family Flaviviridae and consists of four antigenically distinct serotypes(DENV-1-4).Phylogenetic analyses of DENV have revealed its origin,epidemiology,and the drivers that determine its molecular evolution in nature.This review discusses how phyloge-netic research has improved our understanding of DENV evolution and how it affects viral ecology and improved our ability to analyze and predict future DENV emergence.

Detection of dengue virus serotype 3 in Cajamarca,Peru:Molecular diagnosis and clinical characteristics

Objective:To describe and molecularly characterize an outbreak of dengue virus(DENV)infection in Cajamarca,an Andean region in Peru.Methods:A total of 359 serum samples from patients with acute febrile illness were assessed for the presence of DENV via RT-PCR,ELISA NS1,Ig M and Ig G in Cajamarca,Peru from January 2017 to June 2017.The evaluation of the different diagnostic tests and their applicability was performed.Results:Dengue virus was detected in 24.7%of samples by RTPCR.Meanwhile,serological analysis detected 30.3%positive cases via ELISA NS1 antigen,16.7%via ELISA Ig G and 9.7%via ELISA Ig M.Most of the cases corresponded to DENV-3(77.5%).The use of RT-PCR performed better in primary infections(P<0.01),while detection of ELISA Ig M performed better in secondary infections(P<0.01).The combination of NS1 and Ig M performed better than the other assays in detecting primary(92.5%)and secondary infections(96.6%).The most frequent symptoms associated with fever were headaches,myalgias,and arthralgias across all groups.Conclusions:We report an important outbreak of dengue infection caused by DENV-3 in Cajamarca,Peru.Our findings encourage the use of NS1 antigen and Ig M co-detection.These findings demonstrate an increasing expansion of DENV-3 in Peru and highlight the importance of molecular diagnosis and serotype characterization among the clinically defined dengue cases to strengthen the Peruvian epidemiological surveillance.

Construction and expression of a synthetic gene encoding nonstructural glycoprotein NS1 of dengue 2 virus in Pichia pastoris

To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.

Emerging peril of post-dengue mucormycosis:A case report

Rationale:Dengue fever is a leading cause of death in tropical and subtropical countries.Although most patients have a self-limited febrile illness,the viral infection can induce virus-mediated host changes,making immunocompetent persons susceptible to deadly fungal infections.However,there are only a few reports of such an association.Here we present a case of this deadly co-infection.Patient’s Concern:A 17-year-old male patient was diagnosed with dengue fever.He presented to us with facial swelling,periorbital edema,and black discoloration over the palate during the second week of his illness.Diagnosis:Diagnostic tests confirmed the presence of fungal hyphae.A diagnosis of post-dengue mucormycosis was made.No other comorbidity or underlying immune deficit was detected.Interventions:The patient underwent surgical debridement and antifungal treatment.Outcomes:The patient recovered and showed signs of palatal healing with an advancing mucosal edge.Lessons:Dengue virus and mucor co-infection has brought to light a new pathogenic paradigm.Clinicians need to be aware of this emerging medical condition and maintain a high index of suspicion for mucor co-infections while treating dengue patients.

利用Aag2细胞过表达体系筛选埃及伊蚊对DENV2易感相关免疫基因

埃及伊蚊Aedes aegypti是登革病毒的主要传播媒介,埃及伊蚊感染登革Ⅱ型病毒(dengue virus typeⅡ,DENV2)后产生先天免疫反应的相关研究,有助于进一步理解埃及伊蚊感染DENV2的过程和机制。本研究通过埃及伊蚊和Aag2细胞感染DENV2进行转录组分析,与未染毒的蚊虫和细胞进行对比,筛选出12个表达变化较大的基因,之后分别构建含有这12个基因的PSL1180polyUBdsRED质粒载体,并使用细胞转染技术在Aag2细胞中建立目标基因的瞬时转染过表达模型,观察了目标基因过表达后细胞上清中DENV2的复制水平。结果显示,12个基因中羟基类固醇脱氢酶样蛋白、蜕皮激素20单加氧酶、3β-羟基类固醇脱氢酶3个基因过表达后细胞上清中病毒拷贝数出现了具有统计学意义的降低,表明这3个基因的表达可能影响DENV2在Aag2细胞中的复制。这3个基因的相关蛋白都涉及到20-羟基脱皮酮的合成,这提示20-羟基脱皮酮可能在蚊虫感染DENV2时介导了重要的免疫调节途径,为进一步研究蚊虫与虫媒病毒的互作机制提供了新的思路。

Molecular surveillance of dengue virus in Bahia State, Brazil

Dengue is an important emerging viruses, posing a threat to one-third of the global human population. In 2002, the introduction of DENV-3 in the state of Bahia produced massive epidemic (about 35,000 cases detected) and the first cases of dengue hemorrhagic fever. To understand the nature of the virus circulating at Bahia, E/NS1 sequence was determined for 31 DENV viruses isolated in Bahia during the 2006 and 2007 transmission season, from patients presenting with different degrees of severity. The carboxi-terminal region of the E gene (220 nt) of 31 viruses, isolated from dengue patients with clinical diagnosis of dengue infection were used to determine the genetic variability of dengue 2 (DENV-2) and dengue 3 (DENV-3). Sequence data were used in phylogenetic comparisons with global samples of DENV-2 and DENV-3. DENV-2 sample grouped in the South East Indian genotype, while DENV-3 samples were grouped within Indian genotype. This study is the first report on Bahia isolates during two transmission seasons. Our data confirms reports from other parts of Brazil and different countries showing the DENV-3 (geno-type III) strains circulating in the Americas are closely related, and cluster within the genotype that has been associated with DHF epidemics in different conti-nents.

登革2型病毒在RAW264.7细胞和THP-1细胞中复制能力及免疫激活能力的比较

目的:比较登革2型病毒(DENV2)在RAW264.7细胞和THP-1细胞中的复制能力及DENV2对这两种靶细胞的免疫激活作用。方法:将DENV2病毒液与靶细胞(RAW264.7细胞和THP-1细胞)分别孵育6h、12h、24h,孵育结束后收集细胞,Trizol法提取不同时间的细胞总RNA,检测RNA纯度和浓度后逆转录为cDNA,使用qRT-PCR检测样本中病毒目的基因DENV2 E及细胞因子IL-1β、IL-6、TNF-α、IFN-β、ISG15、CCL2的mRNA相对表达量。结果:DENV2感染RAW264.7细胞和THP-1细胞后,DENV2 E基因的表达量均显著上调(P<0.05),且呈现逐渐增加的趋势。6h、12h时,DENV2 E基因在两个靶细胞中的相对表达量一致;24h时,DENV2 E基因在RAW264.7细胞中的相对表达量显著高于THP-1细胞。与未感染DENV2组相比,DENV2感染RAW264.7细胞和THP-1细胞6h、12h、24h后,IL-1β、IL-6、TNF-α、ISG15、IFN-β、CCL2基因的相对表达量均显著升高(P<0.05),且在RAW264.7细胞中的相对表达量显著高于THP-1细胞。结论:DENV2在RAW264.7细胞中的复制能力强于THP-1细胞的复制能力,且DENV2在RAW264.7细胞中具有更强的免疫激活能力。

登革病毒感染的检测技术研究进展

登革病毒(dengue virus,DENV)是引起登革热(dengue fever,DF)的病原体,经蚊叮咬而传播疾病.DF主要流行于热带和亚热带地区.目前,DF已经发展成一个日益严重的全球性公共卫生问题.2012年,DF被列为全球最重要的蚊虫传播性疾病.快速而准确的针对DENV感染的检测技术能够有效地控制DF的爆发及减少死亡病例.目前DENV感染的检测技术主要有病毒分离培养、分子生物学诊断及血清学诊断三类,三类检测技术各有优缺点,应该根据具体情况选用适当的检测手段.

Ⅲ型登革病毒在THP-1细胞上的ADE模型建立

登革热(dengue fever,DF)是由Ⅰ、Ⅱ、Ⅲ和Ⅳ型登革病毒(dengue virus,DENV)引起的急性传染病,抗体依赖增强感染(antibody-dependent enhancement,ADE)是自限性的登革热以及危及生命的登革出血热(dengue hemorrhagic fever,DHF)或登革休克综合症(dengue shock syndrome,DSS)等重症的主要原因。采用不同稀释度的Ⅱ型登革病毒prM前膜抗体与分离自云南西双版纳重症病人的Ⅲ型登革病毒复合感染THP-1细胞,通过实时荧光定量PCR发现亚中和浓度prM前膜抗体诱发THP-1细胞液中更高浓度的病毒载量。在THP-1细胞系上的研究可为后续研究登革病毒ADE打下坚实的基础。

四种血清型登革病毒包膜蛋白结构域Ⅲ(EDⅢ)中和抗体的制备及鉴定

目的制备四种血清型交叉反应性的登革病毒包膜蛋白结构域Ⅲ(DENV-EDⅢ)特异性单克隆抗体(mAb),并鉴定其特异性及体外中和活性。方法通过毕赤酵母表达具有良好抗原性的1~4型DENV-EDⅢ蛋白,并以四种DENV血清型EDⅢ蛋白混合免疫BALB/c小鼠,采用聚乙二醇融合技术制备杂交瘤细胞,通过间接ELISA筛选出稳定分泌抗DENV-EDⅢ抗体的杂交瘤细胞,进一步通过间接免疫荧光法对所获得mAb的特异性进行鉴定,并通过改良的酶联免疫斑点微中和实验对所获得的mAb进行体外中和活性测定。结果获得6株特异性针对1~4型DENV-EDⅢ蛋白的mAb和11株特异性针对某一血清型DENV的EDⅢmAb,其中有1株mAb同时对四种血清型DENV有均衡的体外强中和活性,其对四种血清型DENV的半数抑制剂量(IC)分别为(0.05、1.89、0.02、3.91)μg/mL。结论成功筛选获得了一株可同时中和四种血清型DENV的EDⅢ特异性mAb。

登革病毒包膜E蛋白III区

登革病毒包膜E蛋白III区(DENV ED3)是登革病毒与敏感细胞受体结合的功能区域,对其结构和功能的研究,将有助于理解登革病毒与其敏感细胞间的相互作用和致病机理,为登革病毒受体研究和研发特异性阻断剂控制DENV感染奠定基础。现就DENV ED3的研究进展作一综述。

半巢式荧光定量PCR快速检测登革Ⅰ型病毒成熟microRNA方法的建立

目的建立外周血中登革病毒微小RNA检测方法,探讨其作为登革热病毒感染生物标记物的可行性。方法设计并筛选半巢式实时荧光定量PCR检测登革病毒miRNA引物,验证方法的灵敏度、特异性和重复性。结果设计的登革病毒茎环引物和扩增引物可区分健康人和登革热Ⅰ型感染者;重复试验确定Ct≤35为登革病毒感染阳性,Ct〉35为阴性,同一样品内相对标准偏差（RSD）为0.05%,样品间的相对标准偏差（RSD）为0.15%,总体样品的相对标准偏差（RSD）为5%;该方法检测miRNA的灵敏度为1×10^（-7）μmol/L,检测1～10^（-4）μmol/L内的miRNA呈良好的线性关系。该方法也显示了较好的特异性。结论成功建立了半巢式SYBR GreenⅠ荧光定量qRT-PCR检测Ⅰ型DENV的miRNA方法。外周血中成熟的miRNA可以作为一种潜在的DENV感染生物标志物。

白纹伊蚊rAlb-34k2蛋白对C6/36细胞内的DENV2早期抑制作用

目的探讨白纹伊蚊唾液rAlb-34k2蛋白在DENV2感染C6/36细胞中的作用。方法采用RT-qPCR检测不同浓度rAlb-34k2蛋白作用6 h、60 h后C6/36细胞内DENV2 NS1核酸的变化;采用Dot-ELISA检测rAlb-34k2蛋白与DENV2颗粒结合情况;采用ELISA及Western blot分别检测rAlb-34k2蛋白与C6/36细胞膜蛋白结合的差异。结果rAlb-34k2蛋白作用6 h后,C6/36细胞内DENV2 NS1 RNA的相对表达被抑制,其中10、25、100μg/ml浓度组NS1 RNA的相对表达(2^(-ΔΔCT))值分别为(0.6778±0.04827)、(0.5972±0.06369)和(0.4704±0.05297),差异有统计学意义(P<0.05);rAlb-34k2蛋白作用60 h后,各蛋白组间差异无统计学意义。Dot-ELISA检测rAlb-34k2蛋白(71、118.3、177.5μg/ml)可与DENV2病毒颗粒结合。间接ELISA检测rAlb-34k2蛋白与C6/36细胞疏水性膜蛋白和亲水性膜蛋白结合的A值分别为(1.1±0.26)和(3.1±0.047),差异有统计学意义(P=0.0016),Western blot检测亲水性膜蛋白的阳性结合条带数显著多于疏水性膜蛋白。结论白纹伊蚊雌蚊唾液特异性rAlb-34k2蛋白体外能与DENV2病毒颗粒及C6/36细胞膜蛋白结合,并抑制DENV2病毒早期在C6/36细胞中增殖。

Progress towards understanding the pathogenesis of dengue hemorrhagic fever

Dengue virus(DENV) is a mosquito-borne virus belonging to the Flaviviridae family. There are 4 serotypes of DENV that cause human disease through transmission by mosquito vectors. DENV infection results in a broad spectrum of clinical symptoms, ranging from mild fever to dengue hemorrhagic fever(DHF), the latter of which can progress to dengue shock syndrome(DSS) and death. Researchers have made unremitting efforts over the last half-century to understand DHF pathogenesis. DHF is probably caused by multiple factors, such as virus-specific antibodies, viral antigens and host immune responses. This review summarizes the current progress of studies on DHF pathogenesis, which may provide important information for achieving effective control of dengue in the future.

Recent advances in the identification of the host factors involved in dengue virus replication

Dengue virus(DENV) belongs to the genus Flavivirus of the family Flaviviridae and it is primarily transmitted via Aedes aegypti and Aedes albopictus mosquitoes. The life cycle of DENV includes attachment, endocytosis, protein translation, RNA synthesis, assembly, egress, and maturation.Recent researches have indicated that a variety of host factors, including cellular proteins and micro RNAs, positively or negatively regulate the DENV replication process. This review summarizes the latest findings(from 2014 to 2016) in the identification of the host factors involved in the DENV life cycle and Dengue infection.

Molecular epidemiology demonstrates that imported and local strains circulated during the 2014 dengue outbreak in Guangzhou, China

The dengue virus （DENV） is a vital global public health issue. The 2014 dengue epidemic in Guangzhou, China, caused approximately 40,000 cases of infection and five deaths. We carried out a comprehensive investigation aimed at identifying the transmission sources in this dengue epidemic. To analyze the phylogenetics of the 2014 dengue strains, the envelope （E） gene sequences from 17 viral strains isolated from 168 dengue patient serum samples were sequenced and a phylogenetic tree was reconstructed. All 17 strains were serotype I strains, including 8 genotype I and 9 genotype V strains. Additionally, 6 genotype I strains that were probably introduced to China from Thailand before 2009 were widely transmitted in the 2013 and 2014 epidemics, and they continued to circulate until 2015, with one affinis strain being found in Singapore. The other 2 genotype I strains were introduced from the Malaya Peninsula in 2014. The transmission source of the 9 genotype V strains was from Malaysia in 2014. DENVs of different serotypes and genotypes co-circulated in the 2014 dengue outbreak in Guangzhou. Moreover, not only had DENV been imported to Guangzhou, but it had also been gradually exported, as the viruses exhibited an enzootic transmission cycle in Guangzhou.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection

Mosquito microRNAs （miRNAs） are involved in host-virus interaction, and have been reported to be altered by dengue virus （DENV） infection in Aedes albopictus （Diptera： Culicidae）. However, little is known about the molecular mechanisms of Aedes albopictus midgut--the first organ to interact with DENV--involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA （mRNA） expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.