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...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.展开更多
Background:Dengue was regarded as a mild epidemic in China's Mainland transmitted by Aedes albopictus.However,the 2014 record-breaking outbreak in Guangzhou could change the situation.In order to provide an early ...Background:Dengue was regarded as a mild epidemic in China's Mainland transmitted by Aedes albopictus.However,the 2014 record-breaking outbreak in Guangzhou could change the situation.In order to provide an early warning of epidemic trends and provide evidence for prevention and control strategies,we seek to characterize the 2014 outbreak through application of detailed cases and entomological data,as well as phylogenetic analysis of viral envelope(E)gene.Methods:We used case survey data identified through the Notifiable Infectious Disease Report System,entomological surveillance and population serosurvey,along with laboratory testing for IgM/IgG,NS1,and isolation of viral samples followed by E gene sequencing and phylogenetic analysis to examine the epidemiological and molecular characteristics of the outbreak.Results:The 2014 dengue outbreak in Guangzhou accounted for nearly 80%of total reported cases that year in China's Mainland;a total of 37,376 cases including 37,340 indigenous cases with incidence rate 2908.3 per million and 36 imported cases were reported in Guangzhou,with 14,055 hospitalized and 5 deaths.The epidemic lasted for 193 days from June 11 to December 21,with the highest incidence observed in domestic workers,the unemployed and retirees.The inapparent infection rate was 18.00%(135/750).In total,96 dengue virus 1(DENV-1)and 11 dengue virus 2(DENV-2)strains were isolated.Phylogenetic analysis indicated that the DENV-1 strains were divided into genotype I and V,similar to the strains isolated in Guangzhou and Dongguan in 2013.The DENV-2 strains isolated were similar to those imported from Thailand on May 11 in 2014 and that imported from Indonesia in 2012.Conclusions:The 2014 dengue epidemic was confirmed to be the first co-circulation of DENV-1 and DENV-2 in Guangzhou.The DENV-1 strain was endemic,while the DENV-2 strain was imported,being efficiently transmitted by the Aedes albopictus vector species at levels as high as Aedes aegypti.展开更多
Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the patho- genesis of severe dengue remains unclear. In this study, we showed that the Racl-microfilam...Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the patho- genesis of severe dengue remains unclear. In this study, we showed that the Racl-microfilament signal pathway was involved in the process of DENV serotype 2 （DENV2） infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Racl activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Racl promoted DENV2 entry but inhibited viral as- sembly, maturation and release. Our findings demonstrated that Racl plays an important role in the DENV2 life cycle by reg- ulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.展开更多
pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two ...pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2—5 days?inoculation. Sequence analysis further confirmed the exis-tence of recovered and recombinant DEN2 viruses, whose 5′ termini had an additional non-virus nucleotides 揋? while the 3′ terminal sequences remained the same as natural. The neuroviru-lence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 105—102 TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.展开更多
基金supported by Guangzhou Science and Technology Program for Public Wellbeing(2014Y2-00185)the Special Program of National Science and Technology of China(2013ZX10004-805)the National Natural Science Foundation of China(81301491 and 31270974)
文摘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.
基金This work was supported by National Natural Science Foundation of China(NSFC)(No.81273139)Guangdong Natural Science Foundation(No.S2013010013637 and 2015A030313784)+3 种基金the Collaborative innovation project of Bureau of Science and technology of Guangzhou Municipality(No.201508020263)the Project for Key Medicine Discipline Construction of Guangzhou Municipality(No.2017-2019-07)the Bureau of Health of Guangzhou Municipality(No.2014A011090004,2015A011056)The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.
文摘Background:Dengue was regarded as a mild epidemic in China's Mainland transmitted by Aedes albopictus.However,the 2014 record-breaking outbreak in Guangzhou could change the situation.In order to provide an early warning of epidemic trends and provide evidence for prevention and control strategies,we seek to characterize the 2014 outbreak through application of detailed cases and entomological data,as well as phylogenetic analysis of viral envelope(E)gene.Methods:We used case survey data identified through the Notifiable Infectious Disease Report System,entomological surveillance and population serosurvey,along with laboratory testing for IgM/IgG,NS1,and isolation of viral samples followed by E gene sequencing and phylogenetic analysis to examine the epidemiological and molecular characteristics of the outbreak.Results:The 2014 dengue outbreak in Guangzhou accounted for nearly 80%of total reported cases that year in China's Mainland;a total of 37,376 cases including 37,340 indigenous cases with incidence rate 2908.3 per million and 36 imported cases were reported in Guangzhou,with 14,055 hospitalized and 5 deaths.The epidemic lasted for 193 days from June 11 to December 21,with the highest incidence observed in domestic workers,the unemployed and retirees.The inapparent infection rate was 18.00%(135/750).In total,96 dengue virus 1(DENV-1)and 11 dengue virus 2(DENV-2)strains were isolated.Phylogenetic analysis indicated that the DENV-1 strains were divided into genotype I and V,similar to the strains isolated in Guangzhou and Dongguan in 2013.The DENV-2 strains isolated were similar to those imported from Thailand on May 11 in 2014 and that imported from Indonesia in 2012.Conclusions:The 2014 dengue epidemic was confirmed to be the first co-circulation of DENV-1 and DENV-2 in Guangzhou.The DENV-1 strain was endemic,while the DENV-2 strain was imported,being efficiently transmitted by the Aedes albopictus vector species at levels as high as Aedes aegypti.
基金supported by the National Key Programs on Basic Research of China (2011CB504703)the National Natural Science Foundations of China (81301435, 81471957, 81271839, 81401676)Beijing Natural Science Foundation (7144194)
文摘Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the patho- genesis of severe dengue remains unclear. In this study, we showed that the Racl-microfilament signal pathway was involved in the process of DENV serotype 2 （DENV2） infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Racl activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Racl promoted DENV2 entry but inhibited viral as- sembly, maturation and release. Our findings demonstrated that Racl plays an important role in the DENV2 life cycle by reg- ulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.
基金The National Natural Science Foundation of China (Grant No. 30000144).
文摘pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2—5 days?inoculation. Sequence analysis further confirmed the exis-tence of recovered and recombinant DEN2 viruses, whose 5′ termini had an additional non-virus nucleotides 揋? while the 3′ terminal sequences remained the same as natural. The neuroviru-lence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 105—102 TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.
文摘将编码登革病毒2型(DV2)氨基末端80％的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E。间接免疫荧光显示其可在COS-7细胞中表达。ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1:640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4^+、CD8^+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4^+淋巴细胞水平略有上升。CD8^+细胞水平有较大升高(p<0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60％。以上结果表明:pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-I限制性杀伤性CD8^+T淋巴细胞水平的提高对清除病毒是十分有利的。因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索。
