Studies On Genomic Evolution And Molecular Diagnosis Of Mosquito-borne Dengue Viruses

Insect-vectored or arthropod-borne viruses(arboviruses) are a large group of viruses that are spread mainly by blood-sucking insects such as mosquito,sandfly and midge.Those viruses are just carried and transmitted by insects,rather than causing their disease,therefore as natural sources for human disease emergence.Currently,more than 586 specieses of insect have been identified for transmission of 500 viruses,of which 130 viruses could cause serious zoonotic disease.Arboviruses could also be transmitted to other yet-epidemic area by host(human,insect) movement.Despite the serious threat of arbovirus to human health,few specific medicine or vaccine are available for their control,highlighting the necessity of continuing research.Dengue viruses(DENVs),which are transmitted by Aedes albopictus and Ae.Aegypti,belong to the genus of flavivirus with more than one of the four serotypes(DENV-1,-2,and -3) circulating in tropical and subtropical regions. Dengue virus is epidemic in over one hundred countries,threatening 2.5 billion people all over the world.Due to the unavailability of effective vaccine,early diagnosis and timely medical care are of significance for dengue patient.In China,the first epidemic of dengue happens in 1978 in Foshan city,Guangzhou.But now dengue has caused serious public health impact in southern China,and has been documented as an official infectious disease.A systematic understanding of the molecular aspects of dengue epidemiology over thirty-year period is of meaningful for dengue control.As such, this work is focusing on 1) molecular epidemiology of dengue viruses in China from its first emergence in 1978 to the latest outbreak in 2006,and 2) development of real-time PCR TaqMan probe for rapid and early diagnosis of dengue virus.The main results were as follows:1.Genomic evolution of dengue virus1.1 Genomic sequencing and comparisonBased on published dengue sequences from GenBank,different specific primers for DENV-1, -2 and -4 full genome amplification were designed,followed by RT-PCR amplification,cloning into pGEM-T vector,and sequencing.By assembling of sequenced fragments,totally,six E gene and fourteen full-genome sequences were obtained.The full-length of genome of DENV-1,-2 and -4 are 10735bp,10723bp,and 10649bp,encoding 3392,3391,3387 amino acids,respectively.The 5' and 3' non-coding region of DENV-1 were 94 and 462 nts,respectively,while DENV-2 were 94 and 452 nts,and DENV-1 were 101 and 485 nts,respectively.Nucleotide sequence similarity of eight DENV-1(2001-2006) full genomes was ranging from 91.8%to 99.7%,while their corresponding amino acid sequence similarity was ranging from 97.0% to 98.8%.Nucleotide sequence similarity of four DENV-2(1993-2001) full genomes was ranging from 92.3%to 98.8%,while their corresponding amino acid sequence similarity was ranging from 96.5%to 98.8%.Nucleotide sequence similarity of two DENV-4(1978 and 1990) full genomes was 93.6%,while their corresponding amino acid sequence similarity was ranging from 96%.The sequences of 5'UTR and 3'UTR of the same serotype were really conservative,with only several nucleotides difference.According to the amino acid comparison of single protein encoded by sequences determined in the study,the amino acid sequences of NS3,NS4A,NS4B,and NS5 showed superior similarity to that of structural proteins and other nonstructural proteins.We further explored the amino acid change of envelope protein genes collected in the article,and found amino acid position 461 of envelope protein(E461) was Ile(I),Val(V),L(Leu),and A(Ala) at the time point of 2002-2003,2004,2006 and 2007,respectively.And the corresponding hydrophobic parameters for each amino acid were 4.5,4.2,3.8 and 1.8,demonstrating a decreasing trend of hydrophobic parameters.E461 was on the C-terminal of envelope protein,a transmembrane region involving in the host identification and attachment.Whether the trend indicates an adaption of the dengue virus to host remains undetermined.1.2 Genomic evolution of dengue virusA dataset of 14 full-length DENV genome sequences from 1978 to 2006 and six complete E gene sequences from patients' sera in 2006 was obtained.These sequences were combined with 60 DENV full coding region sequences and 125 E gene sequences taken from GenBank representing a wide range in geographic localities.The longitudinal study of dengue(DENV-1,-2 and -4) molecular epidemiology in south China over the thirty-year period revealed,that 1) introduction from neighboring countries contributes most to the dengue outbreak in China,although endemic strain was inferred and China might also be a dengue source,that 2) genetic diversity of Chinese isolates in genotype and clade level was seen,but it was further complicated by diverse potential intra-serotype recombination,that 3) purifying selection was predominating evolutionary drive, while positively selected site on NS1 of DENV-1 was detected that shows to drive specific lineage evolution by changing hydrophilic amino acids to hydrophobic ones.Those highlight the necessity of concern on natural recombination and nonstructural protein gene for dengue evolution.2.Development of real-time PCR diagnosis of dengue viruses2.1 Establishment of real-time PCR diagnosisAll the available sequences from GenBank and the sequences determined in the study were aligned to find their serotype-specific regions for real-time PCR primers and probe exploration by Primer Press 3,followed by evaluation of their specificity and sensitivity by RNA extracted from dengue virus cell culture suspension.Results show that 1) the four sets of primers and TaqMan probes have significant specificity,with no cross reaction between different serotypes,close genus, human genome,and mosquito cell RNA,high sensitivity,with the detection limitation ranging from 15 to 80 copies/reaction.Those indicate the developed real-time PCR methods were reliable for dengue diagnosis.2.2 Preliminary evaluation of real-time PCR for dengue diagnosisRecently,DENV-1 is the main serotype circulating in south China.Sixty-four clinical blood samples with different time after disease onset were collected during dengue epidemic in 2006 in Guangzhou to evaluate the applicability of the established method.Results show that 1) the positive rate of real-time PCR for detecting dengue is 71.9%(46/64),while the immuno-fluorescence assay (IFA) of IgM is 54.7%(35/64),IgG is 34.4%(22/64),showing superior performance of real-time PCR over IFA,that 2) during the first 1-3 days after the symptom onset,the positive rate of real-time PCR is as high as 86.4%(19/22),while in IFA,the IgM and IgG are 18.2%(4/22) and 9.1(2/22), respectively,indicating real-time PCR detecting dengue in the study is superior,especially in early dengue diagnosis.Those above results indicate real-time PCR developed in the study can be used to dengue diagnosis,especially in early diagnosis.