Arbovirus Detection And Gene Sequence Analysis Of Viral Encephalitis And Unknown Fever Patients In Yunnan Province

ObjectiveYunnan Province is located in the Southwest, most of the regions are tropical and subtropical climates, complicated natural conditions, suit insects and insect-borne viruses for media distribution and dissemination, research findings had confirmed that at least 10 arboviruses with distribution and prevalence of related disease exist in Yunnan Province, In addition to widespread distribution of Japanese encephalitis (Japanese encephalitis, JE), but also there are other insect-borne virus disease epidemic. there are ofen prevalence of patients with Viral Encephalitis and Unknown Fever in border areas of Yunnan Province. Since many viral encephalitis and fever of unknown Fever diseases and insect-borne virus infection is related, mster the prevalence situation of insect-borne virus disease in the regions, the subject of some areas in Yunnan Province, collect serum and cerebrospinal fluid specimens of viral encephalitis and unknown Fever patients, detect nucleic acid of related arbovirus and virus isolation, use molecular biology technology to arbovirus positive samples, for measurement and analysis of gene sequences and viral genotype. Through investigation and study, we may master the prevalence of relevant insect-borne virus and molecular epidemiological characteristics of epidemic strains, provide a scientific basis for disease control in the regions. MethodsFrom July-September In 2008, we carry out investigation in the western and southern border areas of Yunnan's Baoshan, Dehong, Dali and Xishuangbanna, collect serum and cerebrospinal fluid specimens with viral encephalitis and unknown fever, and Case is investigated and studied, establish specimen collection and test database, using samples collected from patients in RT-PCR and real time-PCR methods to detect nucleic acid of insect-borne virus, using C6/36 cell culture and suckling mouse inoculation methods for virus isolation.carry out nucleotide sequence alignment by ClastalX1.83, using MegAlin software in the DNAstar to nucleotide sequence, amino acid sequence analysis. And using MEG4 software to nucleotide sequence phylogenetic tree analysis of JE-PrM-C section and PrM-C and NS5 section of Dengue virus in turn.ResultsThe 106 samples are collected with serum and cerebrospinal fluid of viral encephalitis and unknown fever patients in Four city,using RT-PCR method to detect nucleic acid of Flavivirus, Alphavirus and Bunya virus, resulting in five positive samples for nucleic acid of Flavivirus. Positive samples number:JB 114, JB135, DLN24, RLB61 and RLC31. After further nucleic acid detection and sequence analysis, five samples, three strains (JB114, JB135, DLN24 strain) for Japanese encephalitis virus, two strains of genotype I (JB114, JB135), one strain of genotypeⅢ(DLN24; two strains of dengue virus (RLB61 and RLC31 strains), and one strain of dengue type 1 (RLB61), one strain of dengue type 3 (RLC31). three Japanese encephalitis virus and two Dengue virus carry out the analysis of molecular epidemiological characteristics.JB 114 and JB 135 strains of Japanese encephalitis virus are cerebrospinal fluid samples with virus encephalitis from Jinghong City, DLN24 strain of Japanese encephalitis virus is cerebrospinal fluid samples with virus encephalitis from DaLi City, three cases of Japanese encephalitis patients that are local infections. Dengue virus (RLB61 and RLC31) are from Dehong Ruili City, unexplained fever patients in acute phase serum, two cases were imported cases of Dengue fever patients, including one case of people in border areas of Myanmar Muse entries for medical treatment of patients, and one case of the border residents in Ruili City for traveling back to Myanmar. It is confirmed that imported cases of dengue fever exist in the Western border regions of Yunnan Province from the molecular level for the first time, and It is confirmed that the types 1 and 3 popular of Dengue virus exist in the border areas of Myanmar in 2008.The 106 samples are not detected to nucleic acid positive samples of Alphavirus and Bunya virus. Using real time-PCR method, nucleic acid of Banna virus are detected in the above samples, nucleic acid weakly positive two samples of Banna virus are detected(MNF40 and MNF41), Ct number are 32. However,Using RT-PCR method, nucleic acid of Banna virus (MNF40 and MNF41) are not negative.ConclutionsYunnan Province has genotypeⅠandⅢprevalence of Japanese encephalitis virus, of which the epidemic strain for genotypeⅠin Xi shuang banna, genotypeⅢin Dali. Japanese encephalitis virus nucleotide and amino acid homology and phylogenetic analysis showed that the natural evolution in the same genotype, no significant variation of Japanese encephalitis virus, has a good genetic stability.The typeⅠandⅢof Dengue virus for imported cases exist in the western Yunnan border regions, indirectly evidence that typeⅠandⅢepidemic strains of Dengue virus exist in the summer and autumn of 2008 year near the Myanmar border areas of Yunnan Province. The two epidemic strains of Dengue virus deteced and Southeast Asia strains, the nucleotide and amino acid homology are very high, phylogenetic analysis showes genetic relationships are close among them. Should strengthen prevention and control of dengue fever in the border areas of Yunnan Province, and Prevent imported cases caused by local popular.Cases of viral encephalitis in Yunnan Province may be the existence of Banna virus infection, should further develop the relationship between Banna virus and disease research.