Objective To establish a multiplex real-time fluorescent quantitative reverse transcription polymerase chain reaction (multiplex real-time qRT-PCR) method for rapid detection of nucleic acids of Alongshan virus (ALSV) and Songling virus (SGLV). Methods Specific primers and TaqMan probes were designed for the conserved regions of the NS 3 gene of ALSV and the S gene of SGLV. A multiplex real-time qRT-PCR method was established for the two viruses, and the specificity, sensitivity, and repeatability of the method were evaluated. Tick specimens were used to verify the method. Results The detection method had no cross-reactivity with six other arboviruses, such as tick-borne encephalitis virus, with a sensitivity up to 1×10 ¹ copies/μl, and the coefficient of variation of cycle threshold for repeatability testing was less than 2.00%. Through this method, two groups of ALSV-positive specimens and one group of SGLV-positive specimens were detected from 30 groups of tick specimens collected from Heilongjiang, China in 2019. This method was verified to be 100% consistent with the general PCR method in terms of test results. Conclusion In this study, a highly sensitive and highly specific multiplex real-time qRT-PCR method for rapid detection of ALSV and SGLV has been successfully established.

Objective To investigate the species distribution of hematophagous midges in 31 counties (banners and cities) in 8 provinces (autonomous regions) of China, including Jiangxi and Yunnan. Methods From 2013 to 2018, hematophagous midges were captured with light traps at the residences, cattle pens, sheepfolds, chicken houses, pig pens, duck pens, and vegetable plots in 31 counties (banners and cities) in 8 provinces (autonomous regions) of China, including Jiangxi and Yunnan; specimens were slide-mounted in phenol-balsam. Results A total of 184 314 hematophagous midges from 72 species belonging to 2 genera were collected from 31 counties (banners and cities) in 8 provinces (autonomous regions) of China, of which 63 species belonged to the genus Culicoides and 9 species belonged to the genus Lasiohelea. There were 7 species in 3 counties (banners) in Inner Mongolia, 4 species in a county in Xinjiang, 16 species in a county in Guizhou, 9 species in 2 counties (cities) in Sichuan, 27 species in 10 counties (cities) in Jiangxi, 20 species in 3 counties in Hunan, 36 species in 6 counties (cities) in Yunnan, and 28 species in 5 counties (cities) in Hainan. In terms of distribution in provinces and cities, dominant midges species were distributed as follows: C. punctatus in 19 counties (cities) in 7 provinces (autonomous regions), C. homotomus in 18 counties (cities) in 7 provinces (autonomous regions), C. arakawai in 27 counties (cities) in 6 provinces (autonomous regions), C. oxystoma in 23 counties (cities) in 6 provinces (autonomous regions), C. parahumeralis in 12 counties (cities) in 6 provinces, and C. nipponensis in 12 counties (cities) in 5 provinces (autonomous regions). C. punctatus, C. homotomus, C. arakawai, C. oxystoma, and C. nipponensis each were distributed in cattle pens, sheepfolds, pig pens, duck pens, and residences. Conclusion Hematophagous midges from 72 species belonging to 2 genera are collected in 31 counties (banners and cities) in 8 provinces (autonomous regions) of China; C. punctatus, C. homotomus, C. arakawai, C. oxystoma, C. nipponensis, and C. parahumeralis are widely distributed hematophagous midge species in the 31 counties (cities) in 8 provinces (autonomous regions) under this investigation in China.

Objective To investigate the species and prevalence of blood-sucking insects and their viruses in Qiongzhong county, Hainan province, China. Methods In August 2019, UV mosquito light-trapas (Gongfu Xiaoshuai) were used to collect specimens in the local environment (sheep pens and pig pens) where blood-sucking insects bred, and dry ice was used to transport the specimens to the laboratory. Virological and molecular biological methods were used to isolate viruses and identify the virus isolates. Results A total of 34 mosquitoes from 3 genera (including 26 from Culex, 1 from Anopheles, and 7 Armigeres subalbatus mosquitoes) and 1 450 midges (to be identified) were collected. A virus isolate (HNQZ1927) was isolated from the collected midge specimens, which could cause cytopathic effect in mosquito cells (C6/36), but did not cause cytopathic effect in BHK-21 cells. The gene amplification and nucleotide sequencing and analysis showed that HNQZ1927 was a 10-gene segment orbivirus. The nucleotides (amino acids) in the coding region of HNQZ1927 had 79.4%-98.7% (94.6%-100%) homology with those of Tibet orbivirus (TIBOV) from the genus Orbivirus, and had 1.2%-78.1% homology with those of other orbiviruses except TIBOV. The phylogenetic analysis of the viral genome showed that the HNQZ1927 virus was in the same evolutionary branch as TIBOV XZ0906, which was previously isolated from An. maculatus collected in Tibet, and the TIBOV isolated from Cx. tritaeniorhynchus, Cx. fatigans, and some species of midges in China. Conclusion The HNQZ1927 virus isolated from midges in Hainan province is TIBOV, which is the first time that TIBOV has been isolated from midge specimens collected from nature in Hainan province, China.

Objective To investigate the species distribution and ecological habit of hematophagous midges in 10 counties or cities in the northeastern and midwestern Jiangxi province, China. Methods Hematophagous midges were captured in 10 counties or cities of Jiangxi (Lichuan, Zixi, Yingtan, Qianshan, Yushan, and Fuliang in the northeast as well as Yongfeng, Xiajiang, Anfu, and Jinggangshan in the midwest) using the light-trap method. The intact individuals of hematophagous midges were selected, and the specimens were slide-mounted in Canada balsam-phenol and subjected to taxonomic identification. Results A total 45 999 hematophagous midges belonging to 27 species in 2 genera were captured in the 10 counties or cities of Jiangxi from June to July, 2018, including 25 species in Culicoides and 2 species in Lasiohelea. Of the 27 species, 11 were recorded for the first time in Jiangxi: C. clavipalpis, C. flaviscutatus, C. innoxius, C. jacobsoni, C. luteolus, C. marginus, C. newsteadi, C. parahumeralis, C. tainanus, C. tenuipalpis, and Lasiohelea diaoluoensis. There were 26 species in the northeastern region and 13 species in the midwestern region. The dominant species were C. arakawai (45.31%) and C. oxystoma (16.71%). The dominant species in the northeastern region were C. arakawai (28.54%) and C. oxystoma (22.05%), and the dominant species in the midwestern region was C. arakawai (91.91%). The species distribution and dominant species varied in different places. The overall blood-sucking rate of 7 dominant species was 16.56%, of which, C. oxystoma had the highest blood-sucking rate (43.90%) and C. arakawai had the lowest blood-sucking rate (6.06%). The total light-trap index of hematophagous midges was 943.54/lamp in six sites, with the highest index in the cattle pen (1 297.78/lamp) and the lowest index in sheepfold (684.25/lamp). The blood-sucking rate and light-trap index varied with different midges species and places. Conclusion The faunal distribution and ecological habit of hematophagous midges in Jiangxi province have been mastered, which provides a reference for investigation as well as prevention and control of midge-borne diseases.

This article reports 23 species of hematophagous midges belonging to 2 genera in Wenchang city, Danzhou city, and Chengmai county of Hainan province, China in 2017, among which there were 22 species of Culicoides and 1 species of Lasiohelea. A new species of C. danzhouensis Liu et Liang, sp. nov. was found, and C. pangkorensis Wirth et Hubert, 1989 was a new record in China. The species of C. calcaratus Wirth et Hubert, 1989 was recorded for the first time in Hainan province. There were 17 species in Wenchang, 18 species in Danzhou, and 19 species in Chengmai. C. danzhouensis Liu et Liang sp. nov. had similar wings as C. recurvus Delfinado, 1961, but the latter had shorter palpus, with a palpus ratio of 2.2; male C. recurvus had narrow parameres, no horn process outside the near middle area, a papillae minute at the middle area of the rear edge of the ninth tergum, and no microtrichia on the ventral membrane of the ninth sternum, which were significantly different from C. danzhouensis Liu et Liang sp. nov. C. danzhouensis Liu et Liang sp. nov. had similar male terminalia as C. lushuiensis Liu et Feng, 2018, but there were significant differences in the numbers and shapes of pale spots on wings between the two species; the latter had no microtrichia on the ventral membrane of the ninth sternum, with aedeagus basal arch extending to nearly one-fifth of total length, which were significantly different from C. danzhouensis Liu et Liang sp. nov. The model specimen of the new species was deposited in the North Command Center for Disease Control and Prevention (Shenyang 110034)

This paper reports 2 genera, 16 species of hematophagous midges from Lushui, Fugong, Gongshan, 3 counties of Yunnan province in China-Burma border, 2017, among which 15 species in Culicoides and 1 species in Lasiohelea. A new species C. ( Culicoides) lushuiensis Liu et Feng, sp.nov. is described. Three species: C. cylindratus, C. newsteadi, La. hainana are recorded for the first time in Yunnan province. Culicoides ( Culicoides) lushuiensis Liu et Feng, sp.nov. is closely allied to C. dubius Arnaud, 1956, but is distinctly different from distal portion anal cell of wing with a ouvm shap pale spot, PR 3.00 of female; and ninth sternum with wide "V" caudomedion excavationninth, tergum apically with fine break out, Basistyle with rectangulate ventra root, aedeagus distal portion short, stout, aedeagus basal arch extending nearly to half of total length of male of C. dubius Arnaud, 1956. The type specimens are deposited in the Shenyang Command Center for Disease Control and Prevention (Shenyang 110034).

Objective To determine the whole genome nucleotide sequence and carry out molecular genetic analysis of Banna virus (BAV) isolated from Anopheles sinensis. Methods The 12-segment nucleotide sequence of BAV was determined by molecular virological methods. Sequence alignment, homology, and molecular phylogenetic analysis were performed by using bioinformatics software. Results The BAV isolated from An. sinensis (YN12234 strain) caused cytopathic effect (CPE) and stable passage in C6/36 cells. The virus was composed of 12 segments of double-stranded RNA (dsRNA). Phylogenetic analysis of the viral VP1 protein (RdRp) amino acid sequence revealed that the YN12234 virus belongs to BAV of genus Seadornavirus of family Reoviridae. Further analysis of the 12 ^th segment of the gene sequence revealed that the YN12234 virus belongs to the BAV genotype A2. The results of phylogenetic analysis also showed that the BAV isolated from An. sinensis in this study was in a different evolutionary branch from the BAV previously isolated from An. sinensis, and the BAV isolated from different mosquitoes was located in the different evolutionary cluster. Conclusion The YN12234 strain isolated from An. sinensis belongs to the BAV with 12 segments. The results of molecular evolution analysis suggest that vector adaptability between virus and mosquito species does not exist for the BAV isolated from the mosquitoes at home and abroad, including the YN12234 virus isolated in Yunnan province.

Objective To investigate the distribution patterns of mosquitoes and mosquito-borne viruses in northeast Guizhou province. Methods In 2017, host-seeking mosquitoes were collected from livestock sheds using Ultraviolet lights in Dejiang and Jiangkou, Guizhou province. The mosquito specimens were classified according to morphology and stored in liquid nitrogen. All the samples were incubated with BHK-21 and C6/36 cells for virus isolation and detected the virus genes. The virus isolates were identified and analyzed by molecular biological methods. Results In total, there were 7 380 mosquitoes from 3 genera and 3 species collected from Dejiang and Jiangkou, Guizhou province, 61.55% (4 542/7 380) of which were Armigeres subalbatus and 26.80% (1 978/7 380) were Anopheles sinensis. Out of the 3 virus strains isolated from the mosquitoes, One (GZDJ1765) was identified as typeⅠof Japanese encephalitis virus (JEV) isolated from Culex tritaeniorhynchus and two (GZDJ1746-1, GZDJ1752-1) were identified as Getah virus isolated from A. subalbatus. Three mosquito pools (GZDJ1704, GZDJ1743-2, GZDJ1751-2) tested positive for JEV genes y by PCR. Conclusion Armigeres subalbatus was the predominant species in northeast Guizhou. JEV and GETV were isolated.

A new species and a new record of hematophagous midges, Culicoides enpingensis Wu et Liu, sp. nov., and C. calcaratus Wirth et Hubert, 1989 (new record in China)were described from Enping city of Guangdong province in China. Culicoides enpingensis Wu et Liu, sp. nov. is similar to C. lunchiensis Chen et Tsai, 1962, but distinctly different in the shapes of pale spots in cell R5, and base portion anal cell of wing; distal portion of aedeagus with a pair ear salient, and obtuse round at tip; parameres with hairy tip of male C. lunchiensis Chen et Tsai, 1962. The type specimens were deposited in the Shenyang Command Center for Disease Control and Prevention (Shenyang 110034).

This paper reports 2 genera, 16 species of hematophagous midges in 2016 in Dejiang county, Guizhou province, China, among which 13 species in Culicoides and 3 species in Lasiohelea. A new species C. ( Oecacta) qinglongensis Liu, sp. nov. was described. Five species, C. actoni, C. kongmiaoensis, C. newsteadi, C. parahumeralis, and La. danxinensis were recorded for the first time in Guizhou province. Culicoides ( O.) qinglongensis Liu, sp. nov. wassomewhat allied to C.( O.) okinawensis Arnaud, 1956, but was distinctly different from wing length 1.38 mm, antennasensilla coeloconica present on segments 3-14, antennal ratio (AR)1.28, mandible with 17 teeth of female; and shape of aedeagus, parameres, and basistyle with ventral root of male of C. ( O.) okinawensis Arnaud, 1956. The type specimens are deposited in the PLA Shenyang Command Center for Disease Control and Prevention (Shenyang 110034).

Objective In order to construct fast and efficient genome sequencing platform of Banna virus (BAV), we designed specific-type sequencing primers for BAV. Methods In this study, primer design software Primer Premier v6.0 was used to design the BAV genotype A specific amplification and sequencing primers to analyze the genotype, the sequences of which were based on the information of BAV on GenBank. Meanwhile, the whole-genome sequences of type A1 and A2 of 30 strains of BAV isolated in China, were sequenced by these primers. Results Two sets of specific amplification and sequencing primers for BAV were designed, 26 pairs for gene A1 subtype, and 30 pairs for gene A2 subtype. Moreover, the whole-genome sequences of 30 strains of BAV were amplified. Conclusion Specific amplification primers of BAV with high efficiency and accuracy were designed, laying foundation for further studies on biological feature of BAV.

Objective To study species distribution, community composition and bloodsucking rate of hematophagous midges in 3 cities (countries) of China-Laos border. Methods Hematophagous midges were captured with light trap to generate data on the species distribution in China-Laos border in June to August, 2015. Results A total of 78 336 host-seeking midges belonging to 54 species and 3 genera were collected; genus Leptoconops with 4 species, genus Lasiohelea with 7 species, genus Culicoedes with 43 species. Seven species, C. gentiloides, C. huffi, C. kibabaluensis, C. lansangensis, C. parahumeralis, C. variatus, La. adita are recorded for the first time in Yunnan province. The species distribution of hematophagous midges was recorded 48 species in Mengla, 22 species in Jiangcheng, 16 species in Jinghong. The predominant species included C. jacobsoni, C. parahumeralis and C. palpifer, and their composition were 25.93%, 19.25%, and 17.13% respectively. The community composition and the bloodsucking rate of midges varied with different counties and habitats. The bloodsucking rate was 32.26% in residence district, and 28.24% in cowshed. Conclusion The investigation provides the reference for species distribution, community composition and studies of infectious disease by hematophagous Culicoides.

Objective To develop a rapid and sensitive detection method for Batai virus (BATV) based on TaqMan Real-time PCR. Methods Based on the BATV NS gene sequences of S segment published in GenBank, BATV specific primers and probe were designed. The specificity and stability of the system were evaluated. Quantitative standard curve of BATV TaqMan Real-time PCR was established. Results The specificity and stability test showed that the system is specific and the coefficient variables were all less than 2.50%. Quantitative standard curve based on the genomic copy was drawn, and the lowest detectable limit (LOD) of system is 10 copies/μl. Conclusion TaqMan Real-time PCR for BATV detection has been developed, which is more sensitive and more efficient than the general PCR.

Objective To continue the investigation on the distribution of mosquito-borne arbovirus in some areas of Hubei province. Methods Mosquitoes were collected from Enshi state, Shennongjia forest region, Jiangling county and Suizhou city in the summer of 2010. Virus was isolated from these mosquitoes by cell culture. The virus isolates were identified by serological and molecular biological methods, and phylogenetic analysis was conducted on virus genome sequences. Results 12 845 mosquitoes were collected. 38 virus strains were isolated from mosquitoes. Through serological and molecular biological identification, 32 strains were identified as Japanese encephalitis virus (JEV), 5 were Getah virus (GETV), 1 was mixed strain of JEV and GETV. Phylogenetic analysis on E sequence of JEV showed all JEV strains belonged to genotype Ⅰ JEV, and analysis on NS2 sequence of GETV showed new strains that were homogenous with strains isolated from Hebei and South Korea, but heterogeneous with strain from Russia. Conclusion GETV was firstly isolated from Hubei province, and genotype Ⅰ JEV was re-confirmed from Hubei province.

Objective To understand the species, distribution and genotype of mosquito-borne arboviruses in some regions of Henan province. Methods Mosquito samples were collected from Xixian county and Xin'an county in Henan province from May to August, 2012. After species identification, mosquitoes were inter-cells cross cultured for viral isolation. RT-PCR using specific primer for common arboviruses was used to identify the positive isolates. Molecular biological analysis were conducted by using software of Clustal X2.1, MegAlign, Genedoc 3.2 and Mega v5.1, and genotype for the virus was identified. Results A total of 7149 mosquitoes which belong to 5 species in 4 genera were collected. The predominant mosquito specie in Xin'an county was Armigeres subalbatus (2055, 51.36%), but the mainly species in Xixian county was Culex pipens pallens (2964, 94.16%). Five strains from Cx. pipens pallens and Cx. tritaeniorhynchus were identified as genotypeⅠ Japanese encephalitis virus (JEV), which caused slightly cytopathic effect (CPE) in C6/36 cells, and no CPE in BHK-21. But severe CPE was observed in BHK-21 when 3 days after inoculating the C6/36 culture of the viruses onto BHK-21 cells. Conclusion In Xixian county and Xin'an county of Henan province, b

Objective To identify the arbovirus strain QH07130 isolated from Culex modestus collected in Qinghai province, China. Methods The virus QH07130 was identified by serological and molecular biological methods, and phylogenetic analysis of this virus was performed. Results QH07130 propagated in C6/36 cells and caused significant cytopathic effects. As shown by indirect immunofluorescence assay (IFA), QH07130 reacted with the monoclonal antibodies against Liaoning virus (LNV). Polyacrylamide gel electrophoresis showed that QH07130 was a virus with 12 double-stranded RNA segments, which was in accordance with the 6-5-1 banding pattern of LNV. Sequence analysis of the 10th segment of QH07130 revealed that the sequence length was 844 bp, and its nucleotide homologies with LNV SX0771 and NE9712 amounted 99.5% and 98.0%, respectively. Phylogenetic analysis demonstrated that QH07130 was located in the same evolutionary branch with LNV and had the closest revolutionary relationship with LNV SX0771 and NE9712. QH07130 was identified as LNV. Conclusion LNV is transmitted by mosquitoes in Qinghai province. And QH07130 is the first strain of LNV isolated in Qinghai province.

Objective To investigate the infection with Barmah Forest virus (BFV) and Ross River virus (RRV) among arboviruses in Guizhou province, China, and to provide scientific basis for prevention and control of viral encephalitis of unknown origin. Methods BFV and RRV IgM antibodies were detected in patients with unexplained fever and viral encephalitis from 2005 to 2008 in Guizhou Province, and BFV and RRV IgG antibodies were detected in healthy persons in 2007. Results Out of the 204 cases with unexplained fever examined, 4 (1.96%) had BFV IgM antibody, and 6 (2.94%) had RRV IgM antibody. Out of the 426 cases with unexplained viral encephalitis examined, 12 (2.82%) had BFV IgM antibody, and 15 (3.52%) had RRV IgM antibody. Out of the 870 healthy persons examined, 1.49% had BFV IgG antibody, and 1.15% had RRV IgG antibody. The number of BFV/RRV IgM-positive cases and the number of counties with positive cases increased year by year from 2005 to 2008. Conclusion There might exist some diseases caused by BFV and RRV infection in Guizhou province, China, and we should strengthen monitoring and research work for the diseases caused by the two viruses.

Objective To investigate the species of mosquitoes and the distribution of arboviruses carried by the mosquitoes in Hexi Corridor of Gansu province, China in 2011. Methods Mosquito samples were collected from 6 p.m. to 6 a.m. in the next day with UV lamps. After being identified, the samples were preserved in liquid nitrogen and then transported to a laboratory for examination. Viruses were isolated from the samples by tissue culture, and the virus isolates were identified by serological and molecular biological methods. Homology and phylogenetic analysis was performed on the nucleotide sequences of isolated viruses by bioinformatics. Results A total of 24 028 mosquitoes, belonging to 6 species and 3 genera, were collected in seven counties or cities along Hexi Corridor in August 2011. Among the collected mosquitoes, Aedes vexans was the most abundant species, accounting for 32.80% (7880/24 028), and Culex pipiens pallens accounted for 30.26% (7272/24 028). One virus strain (GS11-155) that could induce pathological changes of two types of cells (BHK-21 and C6/36 cells) was isolated by cell culture. Genes of Culex flavivirus (CxFV), Liaoning virus (LNV), and Getah virus (GETV) were detected in 32 batches of mosquito samples, and in 11 of the 32 batches, 1 had CxFV, 3 had GETV, and 7 had LNV, according to nucleotide sequencing. Conclusion Ae. vexans was the dominant species of mosquitoes in Hexi Corridor of Gansu province, China. The mosquitoes in Hexi Corridor carry several types of arboviruses, including the CxFV, GETV, and LNV

Objective To investigate the species and distribution patterns of mosquito-borne arboviruses in some regions of Hubei province, China. Methods Mosquito samples were collected in Wuxue county of Huanggang city and Tongcheng county of Xianning city in the summer of 2009. Viruses were isolated from the samples by tissue culture, and the obtained virus isolates were identified by serological and molecular biological methods. Sequence homology and phylogenetic analysis was performed on the isolated viruses using bioinformatics software. Results A total of 9424 mosquitoes, belonging to 5 species and 3 genera, were collected. Four virus strains (HBTC0913, HBTC0917, HBTC0919, and HBTC0921) were isolated and identified as Banna virus (BAV). According to the phylogenetic analysis of the 12th segment of BAV, the four strains were in the same subgroup as the isolates from Beijing, Yunnan, and Inner Mongolia, China as well as Vietnam, and but in a different subgroup from the isolates from Indonesia. Compared with previous isolates, the BAV strains showed nucleotide and amino acid homologies of 87.2%-89.8% and 86.1%-90.9%, respectively, according to the homology analysis of the coding region of the 12th segment. Conclusion BAV strains were first isolated in Hubei province, China. They have a close phylogenetic association with YN6 strain isolated in Yunnan, China.

Objective To investigate the distribution profiles of arboviruses in different regions of Guizhou province, China. Methods Mosquito samples were collected using mosquito lamps in Qianxi county, Dejiang county, Rongjiang county, and Congjiang county, Guizhou province in July 2008 and then used for virus isolation by tissue culture. The obtained virus isolates were identified by serological and molecular biological methods. Homology and phylogenetic analysis were performed on viral sequences by bioinformatics. Results A total of 9160 mosquitoes (4 species, 3 genera) were collected. Out of the 9 virus strains isolated from the mosquitoes, 8 were identified as Getah virus (GETV), and 1 was identified as Japanese encephalitis virus (JEV). Compared with the prototype strain, the GETV strains had a nucleotide homology of 94.5%-94.9% and an amino acid homology of 97.4%-97.6% . The newly isolated JEV belonged to genotype Ⅰ, according to the homology and phylogenetic analysis. Conclusion GETV and genotypeⅠ JEV were first isolated from mosquitoes collected in Guizhou province, China. The GETV strains in Guizhou province have a close phylogenetic association with the strains isolated from other provinces in China, and the JEV strain in Guizhou province has a close phylogenetic association with the strains isolated from Sichuan province, China.

Objective To investigate the distribution profiles of mosquitoes and arboviruses in Xishuangbanna,Yunnan province, China, and to provide evidence for prevention and control of arbovirus diseases. Methods Mosquito samples were collected in Xishuangbanna and then used for virus isolation by cell culture. RT-PCR was used to identify arbovirus. Serum and cerebrospinal fluid samples were collected from fever patients, and the common encephalitis virus IgM antibodies were detected by ELISA. Results A total of 13 337 mosquitoes, belonging to 29 species and 5 genera, were collected. The main mosquitoes were Culex tritaeniorhynchus (79.98%, 10 667/13 337), followed by Anopheles sinensis (7.95%, 1060/13 337) and An. peditaeniatus (7.38%, 984/13 337). Several specific primers for Japanese encephalitis virus (JEV), Banna virus, alphavirus, and circovirus were used for PCR detection in 214 batches of mosquitoes, and no virus was isolated from the mosquitoes. Also, no virus was found in isolation using several types of cells. A total of 52 serum samples and 54 cerebrospinal fluid samples were collected from patients in acute stage by kits for encephalitis viruses and then subject to ELISA. There were 16 cases with JEV IgM, 4 cases with Herpes simplex virus IgM, 13 cases with mumps virus IgM, 3 cases with ECHO virus IgM, and 1 case with dengue virus IgM. Conclusion No JEV, BAV, and circovirus were detected from mosquitoes collected in Xishuangbanna,Yunnan province, China in 2011. However, serological results indicated that local fever patients were infected with several viruses such as JEV.

Objective To investigate the types and distribution profiles of arboviruses at the riverside of Yalu River in Dandong, Liaoning province, China. Methods Mosquito samples were collected with mosquito lamps and then used for virus isolation by tissue culture. The obtained virus isolates were identified by serological and molecular biological methods. Results A total of 3359 mosquitoes (3 species, 3 genera) were collected from 6 sites at the riverside of Yalu River in July 2007. Eleven virus strains were isolated from the mosquito samples. Of the 11 strains, 5 were identified as Banna Virus (BAV), 3 were identified as Orbivirus, and 3 needed further identification. Based on the results of sequence analysis, the 5 BAV strains shared high homology in terms of the 12th segment, with nucleotide and amino acid homologies of 92.1%-99.8% and 92.8%-99.5%, respectively, and were highly homologous with those from Beijing and Yunnan province, China, exhibiting nucleotide and amino acid homologies of 91.3%-98.7% and 90.8%-100%, respectively. Phylogenetic analysis showed that the 5 BAV strains and the BAV strains isolated from Beijing and Yunnan province were located in the same branch. Conclusion BAV and Orbivirus were first isolated at the riverside of Yalu River in Dandong, Liaoning province, China, and the newly isolated 5 BAV strains have a close phylogenetic association with BAV strains isolated from Beijing and Yunnan province, China.

Objective To grasp the genotypic characteristics of Japanese encephalitis virus (JEV) in Shanghai, China. Methods Mosquito samples were collected from two pig farms in Fengxian county in Shanghai from 2002 to 2005. Viruses were isolated from ground samples and then identified by cytological, zoological, serological, and molecular biological methods. The whole genomes of viruses were sequenced. Sequence splicing, sequence alignment, nucleotide and amino acid homology analysis, and phylogenetic analysis were performed with biological softwares, such as ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2, and Mega 5.0. Results A total of 38 347 mosquitoes were collected and the main species was Culex tritaeniorhynchus. Thirteen JEV strains were isolated and identified. The whole genomes of the 13 strains were sequenced. Phylogenetic analysis based on the whole genome sequences of other 5 genotypes of JEV and those JEV strains found in other years revealed that the JEV strains isolated in 2001, 2003, 2005, and 2007 belonged to genotypeⅠ and those isolated in 2004 and 2006 belonged to genotypeⅢ.Conclusion Two genotypes of JEV (Ⅰ and Ⅲ) were co-prevalent in Shanghai, China.

Objective To carry out the etiological investigation of Japanese encephalitis virus (JEV) in Anhui province, China, and to determine the genotype and molecular characteristics of the JEV. Methods Mosquito samples were collected using mosquito lamps in Fuyang, Huainan, and Anqing, Anhui province in August 2010 and then used for virus isolation by tissue culture. The obtained virus isolates were identified by serological and molecular biological methods. Homology and phylogenetic analysis were performed on the viral sequences by bioinformatics. Results A total of 7651 mosquitoes (3 species, 3 genera) were collected, and 11 virus strains were isolated from the mosquitoes. The newly isolated JEV strains had the highest homology with genotype Ⅰ JEV (96.8%-99.5% nucleotide homology and 97.8%-100% amino acid homology) and thus were identified as genotypeⅠ JEV. Conclusion GenotypeⅠ JEV strains were first isolated in Anhui province, China, and they have a close phylogenetic association with JEV strains isolated from Shanghai and Zhejiang province, China.

Objective To investigate the distribution profiles of arboviruses in Jiangxi province, China. Methods Mosquito samples were collected using mosquito lamps at 8 sites in Jiangxi in the summers of 2008 and 2009 and then used for virus isolation by tissue culture. The obtained virus isolates were identified by serological and molecular biological methods. Homology and phylogenetic analysis was performed on the sequences of the isolates by bioinformatics. Results A total of 11 916 mosquitoes (3 species, 3 genera) were collected in 2008, and 5905 mosquitoes (5 species, 4 genera) were collected in 2009. Four virus strains were isolated from the mosquitoes and were all identified as Japanese encephalitis virus (JEV). All the JEV strains belong to genotypeⅠ, according to the homology and phylogenetic analysis. Conclusion GenotypeⅠ JEV strains were isolated from mosquitoes collected in Jiangxi province, China. And they have a close phylogenetic association with JEV strains from Shanghai and Zhejiang province, China.

Objective To investigate mosquito-borne Liaoning virus (LNV) in Xinjiang Uygur Autonomous Region, China, and to provide evidence for prevention and control of arbovirus diseases. Methods Mosquitoes and ticks were collected from Xinjiang in 2011 and then used for virus isolation by cell culture. The isolated viruses were further analyzed by molecular biology techniques. Results The viruses in the mosquitoes collected from Kashi, Xinjiang and ticks collected from Yili, Xinjiang were subject to PCR using the specific primer for LNV, and the results showed that the positive rate of LNV in mosquitoes was 23.0% (87/379) and that no LNV was found in ticks. Nineteen virus strains were isolated from C6/36 cell cultures of mosquitoes and were identified as LNV. Conclusion Several LNV strains were isolated from mosquitoes collected in Kashi, Xinjiang in 2011, while no LNV was found in ticks collected in Yili, Xinjiang in 2011. Further research on LNV pathogenicity for humans and animals is recommended in Xinjiang, China.