0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.
Animals ; Cell Line ; China ; Culicidae ; virology ; Phylogeny ; Reoviridae ; classification ; genetics ; isolation & purification ; ultrastructure

OBJECTIVETo investigate the species and distribution of mosquitoes and mosquito-borne arboviruses in Yuncheng city of Shanxi province, China.

METHODSMosquito samples were collected in 19 collection sites from Linyi county and Yongji city in Yuncheng city, in August, 2012. After identification and classification, all the specimens were homogenized and centrifuged to acquire supernatant before being inoculated to both C6/36 and BHK21 cells for viral isolation. Positive isolates were identified with arbovirus species-specific primers under RT-PCR, for further sequencing and phylogenetic analysis.

RESULTSA total of 10 455 mosquitoes of 7 species in 4 genuese were collected. The predominant mosquito species in Linyi county was Culex pipens pallens (91.96%, 3 911/4 253), but the one in Yongji city was Culex tritaeniorhynchus (72.85%, 4 518/6 202). A total of 23 strains of viruses were isolated from the mosquito pools. 15 strains from Culex tritaeniorhynchus and Culex pipens pallens were identified as genotype I Japanese encephalitis virus (JEV). Four strains from Culex pipens pallens were identified as Culex flavivirus (CxFV). Three strains from Culex pipens pallens were identified as Culex pipiens pallens densovirus (CppDNV). One strain from Armigeres subalbatus and Aedes albopictus was identified as Getah virus (GETV).

CONCLUSIONFour kinds of arboviruses were isolated from the mosquito pools, including GETV and CxFV, which were isolated and documented in Shanxi province for the first time. In the city of Yuncheng, Culex tritaeniorhynchus had been the predominant species and major vector for transmitting JEV. Genotype I JEV remained the major JEV circulating in the local natural environment.

Animals ; Arboviruses ; genetics ; isolation & purification ; China ; Cities ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; Phylogeny ; Species Specificity

OBJECTIVETo monitor and discover medically important mosquito-borne viruses circulating in Xinjiang, China.

METHODSMosquitoes were collected from Abbey Lake wetland in Bortala, in Northern Xinjiang. Viral isolates were obtained through inoculating and serial passaging into susceptible mammalian host cells (BHK-21), identified by cytopathogenic effect (CPE) observation and plague forming assay. Genetic identification of viral isolates was conducted by RT-PCR, sequencing and phylogenetic analysis.

RESULTSA virus strain which causing CPE on BHK-21 cells, was isolated from the predominant Culex modestus (36.6%) and tentatively designated as Abbey Lake virus. Information on molecular identification revealed that Abbey Lake virus belonged to Orthobunyavirus genus within Bunyaviridae. Partial sequences (651 bp and 980 bp) of viral genomic S and M segment showed that Abbey Lake virus was phylogenetically related to Germiston virus that uniquely found in South Africa with 90.6% nucleotides and 95.0% amino acids similarities in S segment. However, viral M segment displayed much variability with 78.6% nucleotides and 86.1% amino acid similarities, suggesting a new member of Orthobunyavirus genus was discovered in the area.

CONCLUSIONIn this study, Abbey Lake virus was isolated and characterized indicating its potential circulation nature of this newly-emerged mosquito-borne virus.

Animals ; China ; Culicidae ; virology ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Analysis, RNA ; Viral Proteins ; genetics

OBJECTIVETo grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province.

METHODSMosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0).

RESULTSTotally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I.

CONCLUSIONGenotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.

Animals ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; Genotype ; Phylogeny ; Time Factors

OBJECTIVETo investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983.

METHODSA total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software.

RESULTSFrom the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology.

CONCLUSIONThe JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.

Amino Acid Sequence ; Animals ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; isolation & purification ; Encephalitis, Japanese ; virology ; Genotype ; Phylogeny ; Rats

OBJECTIVETo investigate arboviruses in some regions of Shanxi province, isolation and identification for arbovirus activity from mosquitoes was conducted.

METHODSMosquitoes were collected from these area in 2007 and then used for virus isolation by cell culture. The virus isolates were identified by molecular biology and the sequences were analyzed by bioinformatics.

RESULTSTen Banna virus (strains SX0765, SX0766, SX0767, SX0771, SX0789, SX0790, SX0793, SX0794, SX0795, SX0796) were isolated, and two Liaoning virus were also isolated from isolates SX0771, SX0794. Phylogenetic tree of the Banna virus isolates showed that ten strains are located in a distinct branch from all of the other Chinese Banna virus isolates. The homology is between 89.7% and 94.1%.

CONCLUSIONTen Banna virus and two Liaoning virus were isolated during this arbovirus investigation in Shanxi province. New Banna virus isolates showed a distinct phylogenetic relationship with the other Chinese Banna virus strains.

Animals ; Arboviruses ; classification ; genetics ; isolation & purification ; Cell Line ; China ; Culicidae ; virology ; Insect Vectors ; virology ; Molecular Sequence Data ; Phylogeny

BACKGROUNDMosquitoes were collected in Heilongjiang province in 2002, four virus strains were isolated by inoculation of homogenates onto BHK cell lines. The viruses were identified. Multiple alignment and phylogenetic analysis were carried out by Clustal X (1.8) program.Amino acid (AA) analysis was carried out by GENEDOS (3.2).

RESULTSBiological characters of four newly isolated strains were examined and it was found that all of them could produce cytopathogenic effect (CPE) in BHK cells, killing sucking mice. Serological tests showed that all of these stains reacted positively to JEV antibodies. PrM and E gene regions were amplified and sequenced. Phylogenic analysis showed that all the newly isolated JEV strains belong to genotype III. Using the vaccine strains (SA14-14-2) as control, analysis of the E gene of the new strains and two JEV strains (47, Ha-3) isolated previously from Heilongjiang province showed that these new strains' nucleotide sequence had a homology of up to 99.9% and the amino acid sequence homology up to 99.8%, respectively. Compared with the standard JE vaccine strain SA-14-14-2 and the four new strains, the nucleotide sequence homology was 97.3% and amino acid sequence homology was between 96.8% and 97.0%, respectively. Compared with vaccine strain, there were seven common variations in all the four newly isolated strains.

CONCLUSIONFour JE virus strains were isolated in Heilongjiang province. As compared to the vaccine strain, six variations were found in the newly isolated strains at the eight sites relevant to the virulence of the virus.

Animals ; Arbovirus Infections ; virology ; Arboviruses ; classification ; genetics ; isolation & purification ; Cell Line ; Culicidae ; virology ; Genotype ; Mice ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.

METHODSA strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar).

RESULTSThe new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%.

CONCLUSIONThe virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.

Animals ; Cell Line ; China ; Coltivirus ; classification ; genetics ; immunology ; isolation & purification ; Culicidae ; virology ; Insect Vectors ; virology ; Mice ; Molecular Sequence Data ; Phylogeny ; Reoviridae Infections ; immunology ; virology

An artificial intron consisting of the 5'-donor site (from the first intron of the human beta-globin gene) and the 3'-acceptor site (from the intron of an immunoglobulin gene heavy chain variable region) was obtained with a splice overlap extension PCR and was then inserted in frame into the coding sequence of nostructural protein NS1 gene fused to GFP gene in a recombinant mosquito densovirus plasmid p7NS1-GFP. The constructed plasmid was named as p7NS1-Intron-GFP. The plasmid p7NS1-Intron-GFP was co transfected with the helper plasmid pUCA into C6/36 cells, then the packaged recombinant and wild type viruses were purified and recovered. The second-instars of Aedes albopictus larvae were exposed to recombinant and wild type virus mixed stock. The high level GFP expression in C6/36 cells and larvae was observed under fluorescence microscope, indicating that the inserted artificial intron exerted its normal function in self-splicing both in vitro and in vivo. This study laid a foundation for application of an artificial intron in insect cells and development of new strategy for genetic engineering technology of mosqtuito and its pathogens.
Animals ; Cell Line ; Culicidae ; genetics ; virology ; Densovirus ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Introns ; genetics ; Polymerase Chain Reaction

OBJECTIVETo investigate the distribution patterns of mosquito and mosquito-borne viruses in Dehong prefecture, Yunnan province, China.

METHODSMosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July, 2007 and 2010. Mosquito were cell cultured for viral isolation, and positive isolates were identified using RT-PCR and sequence analysis.

RESULTSA total of 43 634 mosquito comprised of 29 species representing six genera were collected. Culex tritaeniorhynchus and Anopheles sinensis comprised 78.69% and 14.77% of the total. Six strains of viruses were isolated from the mosquito pools. RT-PCR and phylogenetic analysis revealed three strains from Cx. tritaeniorhynchus, identified as genotype I Japanese encephalitis virus (JEV). One strain was identified from Cx. tritaeniorhynchus, as Getah virus (GETV). Two strains isolated from Cx. tritaeniorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus (CppDNV).

CONCLUSIONCx. tritaeniorhynchus had been the major species of mosquito and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. Genotype I JEV, GETV and CppDNV were the vectors causing transmission of mosquito-borne diseases in this area. Data from phylogenetic analysis showed that these newly discovered isolates seemed to have had close relationship with those viruses previously circulating in Yunnan and other provinces of China.

Alphavirus ; isolation & purification ; Animals ; Arboviruses ; classification ; isolation & purification ; China ; Culicidae ; virology ; Disease Vectors ; classification ; Encephalitis Virus, Japanese ; isolation & purification