During 2009-2012, the Nam Dinh virus (NDiv) was detected from the samples of Culex pipiens quinquefasciatus in Shenzhen China. In this study, cell culture,SYBR Green I based real time RT-PCR and RT-PCR were performed to analyze the cell susceptibility and other biological characteristics of the NDiV isolates. The results showed that C6/36 cell line was susceptible to four isolates of Culex pipiens quinquefasciatus. The "S" type amplification curve and specific melting curve were obtained in the realtime fluorescence quantitative RT-PCR based on SYBR Green I for the detection of the NDiV from the mosquito. The target bands from the RdRp gene and partial fragment of ZmHel1 gene were observed using agarose gel electrophoresis. Both the nucleotide and amino acid sequences of four Shenzhen isolates showed more than 99.00% homology with the Vietnam representative NDiV strain (02VN178). Phylogenetic analysis showed that four Shenzhen isolates shared the same evolution branch as the Vietnam representative NDiV strain. This is the first report of NDiV in China.
Animals ; China ; Culex ; virology ; Nidovirales ; classification ; genetics ; isolation & purification ; Phylogeny

OBJECTIVETo evaluate the susceptibility of Aedes albopictus and Culex pipiens quinquefasciatus to oral infection with bat Japanese encephalitis virus isolates (GD1 and HN2 strains).

METHODSAedes albopictus and Culex pipiens quinquefasciatus were infected orally by GD1 and HN2 strains of bat Japanese encephalitis virus. TaqMan real-time PCR was used to detect the virus and monitor the changes in the viral loads in Aedes albopictus and Culex pipiens quinquefasciatus at a 2-day interval, starting from 4 days till 20 days after the infection.

RESULTSThe infected Aedes albopictus and Culex pipiens quinquefasciatus were found positive for the Japanese encephalitis virus from day 4 to day 20. Both Aedes albopictus and Culex pipiens quinquefasciatus were susceptible to infection by GD1 and HN2 strains, but the latter showed a greater susceptibility. The HN2 strain virus appeared to have a greater virulence than the GD1 strain.

CONCLUSIONAedes albopictus and Culex pipiens quinquefasciatus can carry GD1 and HN2 strains of bat Japanese encephalitis virus isolates.

Aedes ; virology ; Animals ; Chiroptera ; virology ; Culex ; virology ; Disease Susceptibility ; Encephalitis Virus, Japanese ; isolation & purification

Animals ; Culex ; virology ; Flavivirus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction

OBJECTIVETo probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy.

METHODS0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis.

RESULTS0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus.

CONCLUSION0507JS11 virus is a new member in Brevidensovirus.

Animals ; Culex ; virology ; DNA, Viral ; genetics ; Densovirinae ; classification ; genetics ; isolation & purification ; Genome, Viral ; Sequence Analysis, DNA

0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.
Animals ; Cell Line ; China ; Culex ; virology ; Molecular Sequence Data ; Phylogeny ; Reoviridae ; classification ; genetics ; isolation & purification ; ultrastructure

OBJECTIVETo screen DENV-2 binding proteins from Aedes albopictus and Culex. quinquefasciatus.

METHODSThe total proteins of Aedes albopictus and Culex. quinquefasciatus in different developmental stages were prepared and analyzed with SDS-12% polyacrylamide gel. After electrophoresis the proteins were transferred using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad ) to a nitrocellulose membrane. Virus overlay protein-binding assay (VOPBA) was carried out using anti-dengue virus 1-4 monoclonal antibody.

RESULTSIn Aedes albopictus, VOPBA detected DEN-2 binding molecules of 25 000, 35 000, and 50 000 in larvae samples, molecules of 35 000 and 50 000 in pupae samples, a 50 000 molecule in male mosquito samples, and molecules of 35 000 and 50 000 in female mosquito samples. DENV-2 binding protein of 35 000 was found in the larvae, pupae, and female mosquitoes, but not in male mosquitoes. In Culex. Quinquefasciatus, VOPBA detected a molecule of 100 000 in larvae samples, molecules of 40 000, 100 000, and around 50 000 (48 000 and 60 000) in pupae samples, and molecules of 40 000 and 100 000 in male mosquitoes and female mosquito samples.

CONCLUSIONSeveral proteins capable of binding DENV are found in Aedes albopictus and Culex. quinquefasciatus in different development stages. The 35 000 molecule expressed in Aedes albopictus as a putative receptor protein may be related to virus tropism in mosquito tissues.

Aedes ; virology ; Animals ; Culex ; virology ; Dengue Virus ; Female ; Insect Proteins ; isolation & purification ; Larva ; Male ; Pupa ; Receptors, Virus ; isolation & purification

A flavivirus, Culex flavivirus, was first isolated from Chinese mosquitoes with high sequences similarities to those of flaviviruses found in America and Japan. In this study, a total of 48 pools of field-collected mosquitoes were sampled from Dandong of Liaoning Province, China during July to September of 2011. Six isolated viruses showing cytopathic effect (CPE) in C6/C36 cells were tested by reverse transcription polymerase chain reaction(RT-PCR)using Flavivirus genus--specific primers and Culex flavivirus-specific primers and the positive PCR-product was sequenced and compared with the sequences of 10 isolates from GenBank. Phylogenetic tree of NS5 and enevelop genes of flavivirus were constructed. The GenBank accession numbers of NS5 gene were JQ409188, JQ409186, JQ409187, JQ409191, JQ409189 and JQ409190. The GenBank accession numbers of envelope gene were JQ065883, JQ065882, JQ065881, JQ065879,JQ065877 and JQ065878.
Animals ; Base Sequence ; Cell Line ; China ; Culex ; classification ; virology ; Flavivirus ; classification ; genetics ; isolation & purification ; Insect Vectors ; virology ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.
Amino Acid Sequence ; Animals ; Anopheles ; virology ; Cell Line ; China ; Coltivirus ; classification ; genetics ; isolation & purification ; Culex ; virology ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

BACKGROUNDTo determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings.

METHODSLaboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay.

RESULTSInoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml.

CONCLUSIONBy intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.

Animals ; Culex ; classification ; virology ; Encephalitis Virus, Japanese ; genetics ; growth & development ; immunology ; Encephalitis, Japanese ; virology ; Humans ; Insect Vectors ; virology ; Japanese Encephalitis Vaccines ; immunology ; Species Specificity ; Vaccines, Attenuated ; immunology ; Viral Plaque Assay

OBJECTIVETo determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V.

METHODSThe full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis.

RESULTSThe full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V.

CONCLUSIONThe molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Amino Acid Sequence ; Animals ; Base Sequence ; Culex ; virology ; Encephalitis Virus, Japanese ; genetics ; Genome, Viral ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Tibet ; Young Adult