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

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.
Animals ; Anopheles/classification/*genetics ; Base Sequence ; DNA, Ribosomal Spacer/genetics ; *Genetic Variation ; Insect Vectors/classification/genetics ; Iran ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 28S/genetics ; Sequence Alignment ; Sequence Analysis, DNA

To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili (Yunnan Province, China), we collected 2 149 mosquitoes (17 species) in August 2010. Virus isolation was undertaken by the cul- ture of baby hamster kidney cells (BHK-21 cells). Two virus-like isolates were obtained: DHL10M117 was isolated from collected in Mangshi; DHL10M110 was obtained from Anopheles vagus collected in Rui- li. Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route. Two positive amplicons, 702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus (AKV). Phylogenetic analysis suggested that these two virus stains had a distant relation- ship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea, and Taiwan. However,they were separate from other Asian strains and grouped into a small branch. The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan (96.6% and 99.6% for DHL10M117 and 96.7% and 100% for DHL10M110,respectively). Com- parison of the M segment showed they shared the highest amino acid identity with CY-77 (99.6% and 100%, respectively), whereas the highest nucleotide identity was found with the Iriki strain from Japan (99.6% and 100%, respectively). Compared with the MP496 strain from Kenya,they displayed lower lev- els of sequence homology, at 69.7% and 70.0% for nucleotide sequences of the two loci,and 91. 0% for a- mino acids. Our results identified that DHL10M117 and DHL10M110 were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province. These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia. Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.
Amino Acid Sequence ; Animals ; Anopheles ; virology ; Base Sequence ; Bunyaviridae Infections ; virology ; China ; Cricetinae ; Female ; Humans ; Insect Vectors ; virology ; Male ; Mice ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; physiology ; Phylogeny ; Sequence Homology ; Viral Proteins ; chemistry ; genetics

We wished to sequence the full-length genomes of the DHL10M110 strain of the Akabane virus (AKV) isolated from mosquitoes in Yunnan Province, China, in 2010. We also wished to analyze the characteristics of these complete nucleotide sequences. The complete genomic sequence of the DHL10M110 strain from Yunnan Province was obtained by reverse transcription-polymerase chain reaction and direct sequencing. We found that the length of the L, M and S gene nucleotide sequences of the DHL10M110 strain were 6 869-bp, 4 309-bp and 856-bp, respectively, including the open reading frame (ORF) nucleotide sequences of 6 756-bp (L), 4 206-bp (M) and 702-bp (S), encoding 2252, 1402 and 234 amino-acid polyproteins, respectively. Phylogenetic analyses based on L-fragment ORF showed that the DHL10M110 strain had a close relationship with the OBE-1 strain of the AKV from Japan and AKVS-7/SKR/2010 strain of the AKV from South Korea. Phylogenetic analyses based on M- and S-fragment ORF showed that the DHL10M110 strain had a close relationship with the epidemic strains of the AKV from Japan, South Korea and Taiwan, but that the DHL10M110 strain had a lone evolutionary branch. In terms of nucleotide (amino acid) homology, the similarity of L-, M- and S-fragment ORFs of the DHL10M110 strain to the OBE-1 strain from Japan was 92.6% (98%), 88.5% (94%) and 96.4% (99.1%), respectively. When comparing the DHL10M110 strain with the OBE-1 strain, we noted 45, 84, and 2 different sites in the amino acids of L, M and S fragments, respectively. Homology and phylogenetic analyses also suggested that the DHL10M110 strain had a distant relationship with the epidemic strains of the AKV from Kenya and Australia. Also, we confirmed by complete genomic sequence analyses that the DHL10M110 strain was clade-Asia of the AKV. However, differences between the DHL10M110 strain compared with strains from Japan and South Korea were also noted. These results suggest that the DHL10M110 strain harbored relatively stable genetic characteristics and distinct regional features. This is the first time that full-length genomic sequences of the DHL10M110 strain of the AKV in mainland China have been obtained.
Amino Acid Sequence ; Animals ; Base Sequence ; Bunyaviridae Infections ; transmission ; virology ; China ; Culicidae ; virology ; Female ; Genome, Viral ; Humans ; Insect Vectors ; virology ; Male ; Molecular Sequence Data ; Open Reading Frames ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

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