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

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 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 Wenshan and Hekou county which are the Sino-Vietnam frontier regions of Wenshan, Yunnan province, China.

METHODSIn September 2007, 6091 Culicines, 1334 Anophelines, 848 Aedes vexans and 53 Armigeres obturbans were collected from 5 field sites. Mosquitoes were collected and stored in liquid nitrogen after classification. The mosquito pools were homogenized, and centrifuged, then the supematant was inoculated onto C6/36 and BHK-21 cells, and the viral isolates were identified by serological and molecular biological methods.Sequence alignment and phylogenetic analysis on the viral isolates were carried out using Clustal X 1.85, GENEDOC and MEGA4 software.

RESULTSA total of 4 pairs of virus isolated with C6/36 cells cytopathic effect were observed, and other mosquito species have not cytopathic effect.Strain WS0704-2 was Banna virus which identified by antibody response and PCR. Strain WS0704-1, WS0708-1, WS0708-2 were Culex pipens pallens densovirus (CppDNV) which identified by PCR. The phylogenetic analysis the 12th segment showed significant difference between the new Banna virus and other strains isolated in China.

CONCLUSIONThere are many mosquito vectors in frontier regions (China and Vietnam) of Wenshan in Yunnan province of China, and mosquito-borne arbovirus, such as BAV were isolated here.

Animals ; Arboviruses ; classification ; genetics ; isolation & purification ; China ; Culicidae ; virology ; Densovirus ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Sequence Analysis, DNA

This study aims to investigate the distribution patterns of mosquito-borne viruses in Menghai County, Xishuangbanna Prefecture, Yunnan Province, China and to provide evidence for the prevention and control of mosquito-borne diseases. Mosquito samples were collected using mosquito lamps. Viruses were isolated from the samples by cell culture, and the isolates were identified by RT-PCR. The genomes of isolates were sequenced for phylogenetic analysis. In July 2012, a total of 1468 mosquitoes were captured in Daluo Town of Menghai County; they were divided into 32 pools, including Culex tritaeniorhynchus (28 pools, 1383 mosquitoes), Culex quinquefasciatus (2 pools, 66 mosquitoes), and Anopheles (2 pools, 19 mosquitoes). Golden hamster kidney cells (BHK-21) and Aedes albopictus cells (C6/36) were used for virus isolation. The results showed that C6/36 cells were susceptible to two isolates recovered from Culex tritaeniorhynchus (BNDL1205 and BNDL1227), with marked cytopathic effect (CPE) of cell fusion. By contrast, the two isolates could not cause CPE in BHK-21 cells. RT-PCR was performed for the two isolates using the flavivirus-specific primers FU2/cFD3, and a 800-bp amplicon was obtained from both of them. Phylogenetic analysis showed that the two isolates shared the same evolutionary branch with the Quang Binh virus (QBV) strain VN180, which had been isolated from Vietnam, with nucleotide sequence homologies of 83.4% and 82.9%, respectively. However, there existed relatively large differences in nucleotide sequence between them and other Culex flavivirus strains previously isolated in China and other regions. In light of the similarity between the two isolates and QBV, BNDL1205 and BNDL122 were referred to as Quang Binh-like virus, which were first reported in China.
Animals ; Cell Line ; China ; Cricetinae ; Culicidae ; virology ; Evolution, Molecular ; Insect Viruses ; isolation & purification ; physiology ; Phylogeny ; Sequence Homology

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

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 detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.

METHODSBy aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.

RESULTSWith the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.

CONCLUSIONA TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Animals ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.
Animals ; Cell Line ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; virology ; Genotype ; Humans ; Mice ; Phenotype ; Viral Plaque Assay ; Virulence

OBJECTIVETo investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province.

METHODSSamples of mosquitoes and sera of swine were collected in three counties (Xianju, Longquan and Cixi) of Zhejiang province from May to October in 2007. 10 662 mosquitoes were collected during 2007, of which, C.pipiens pallens and C.quinquefasciatus were the most dominant species and 204 pig serum samples were detected. Japanese encephalitis virus in mosquitoes were detected by virus isolation and real time RT-PCR. The antibody against Japanese encephalitis virus in swine was detected by ELISA. The isolated strain were identified by real time RT-PCR. The PrM gene of the isolated strain was amplified by RT-PCR. Three strains were typed by the gene of PrM.

RESULTSSeven positive mosquitoe samples were identified by real time RT-PCR. Three strains were isolated and identified by real time RT-PCR. The PrM gene was cloned and sequenced. The phylogenetic analysis showed that three isolates belong to genotype I of Japanese encephalitis virus. Of 204 swine serum samples, 121 positive samples were identified positives. Above 50% sera samples from swine were positive in June.

CONCLUSIONThe vector of Japanese encephalitis virus existed and carried the Japanese encephalitis virus in these areas of Zhejiang province. Three strains of Japanese encephalitis virus were isolated from mosquito pools collected in Zhejiang province. It should be the first isolation of genotype I Japanese encephalitis virus in Zhejiang province in recent years.

Animals ; Antibodies, Viral ; blood ; Arboviruses ; classification ; isolation & purification ; China ; Culicidae ; virology ; Disease Vectors ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Genotype ; Swine ; virology