Genetic structure of Culex tritaeniorhynchus (Diptera: Culicidae) based on COI DNA barcodes.

Culex tritaeniorhynchus Gile is a major vector of Japanese encephalitis in China. The population genetics study is crucial as it helps understanding the epidemiological aspects of mosquito-brone diseases and improving vector control measures. Here, the genetic population structure of C. tritaeniorhynchus in the mainland China were estimated using the cytochrome c oxidase subunit 1 (COI) DNA barcodes region. 485 individuals of C. tritaeniorhynchus were collected from 38 sampling sites in 21 geographic populations in the mainland China. In total, 485 sequences were used to explore the population structure and genetic diversity. The results showed that the populations of C. tritaeniorhynchus had high haplotype diversity (Hd = 0.98, with 303 haplotypes), low nucleotide diversity (p = 0.02245) and high gene flow (Nm = 47.11) with two maternal lineages and four groups. An AMOVA indicated that 98.8% of the total variation originated from variation within populations. In addition, the population genetic structure exhibited by C. tritaeniorhynchus filling the vacant of the genetic structure in the mainland China. Human activities may also assist mosquito movement and migration. Gene flow among the populations of C. tritaeniorhynchus can facilitate the spread of insecticide resistance genes over geographical areas, and it will be a challenging for controlling the populations.

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

BACKGROUND: Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-saving and cost-effective procedure to monitor the alleles in large-scale studies. METHODS: Based on the mutation genotypes of the 1534 locus in the kdr gene, F/F, F/S, F/C, F/L, S/S, C/C, L/L and S/C, we designed specific forward and reverse primers and optimized the reaction conditions for establishing of the allele-specific PCR(AS-PCR) detection technique. DNA sequencing in this study was taken as the gold standard, and used to determine the accuracy of AS-PCR. RESULTS: The designed AS-PCR technique showed high specificity for distinguishing the mutations at the 1534 locus, as the accuracy for F/F, F/S, F/C, F/L, S/S, C/C and S/C were 100%, 95.35%, 100%, 100%, 100%, 100% and 100%, respectively. CONCLUSIONS: The designed AS-PCR technique effectively distinguished individual genotypes for the mutations at the 1534 locus in the kdr gene, which could facilitate the knockdown resistance surveillance in Ae. albopictus in large-scale studies.

Susceptibility Status and Resistance Mechanisms in Permethrin-Selected, Laboratory Susceptible and Field-Collected Aedes aegypti from Malaysia.

This study is intended to provide a comprehensive characterization of the resistance mechanisms in the permethrin-selected (IMR-PSS) and laboratory susceptible (IMR-LS) Aedes aegypti strain from Malaysia. Both IMR-PSS and IMR-LS provide a standard model for use in assessing the pyrethroid resistance in field-collected strains collected from three dengue hotspots: the Taman Seri Bayu (TSB), the Flat Camar (FC), and the Taman Dahlia (TD). Two established methods for determining the resistance mechanisms of the pyrethroid are the quantification of detoxification enzymes via enzyme microassay and the nucleotide sequencing of the domain 2 region from segment 1 to 6 via classical polymerase chain reaction (PCR) amplification-were employed. Enzyme activities in IMR-LS served as the resistance threshold reference, providing a significant standard for comparison with IMR-PSS and other field-collected strains. The amino acids in the domain 2 region of voltage-gated sodium channel (Vgsc) of IMR-LS were served as the reference for detection of any changes of the knockdown resistance (kdr) alleles in IMR-PSS and field-collected strains. Studies clearly indicated that the IMR-LS was highly susceptible to insecticides, whilst the IMR-PSS was highly resistant to pyrethroids and conferred with two resistance mechanisms: the elevated oxidase enzyme activity and the altered target-site mutations. Mutations of V1023G alone, and the combination mutations of V1023G with S996P in IMR-PSS, as well as the in field-collected Aedes aegypti strain, indicate the spread of the (kdr) gene in Aedes aegypti, particularly in dengue-endemic areas in Malaysia.

Comparison of Two New Traps To the Biogents BG-Sentinel Trap For Collecting Aedes albopictus In North Florida.

Multiple surveillance traps are currently available for adult mosquito surveillance. In this study, 2 new types, the Maxttrac™ Uno and Maxttrac™ Breeze mosquito traps, were compared against the BioGents™-Sentinel trap (BGS) for overall success in collecting Aedes albopictus in a 1,027-m2 vehicle enclosure and in the field. The enclosure test results showed both traps collected significantly fewer Ae. albopictus, compared to the BGS trap. The modification of using all BG lures for each trap did not increase the number of mosquitoes collected in the 2 new traps, when compared to collection with the original lures tested in the vehicle enclosure. Field test results showed that BGS trap collected higher numbers of Ae. albopictus than the 2 new traps, regardless of switching lure or not. Overall, the BGS trap was the most effective trap and the 2 new traps could be used as additional tools for the collection of Ae. albopictus.

[Molecular characteristics of the full-length genome of two new Japanese encephalitis virus isolated from mosquitoes in Hubei province].

OBJECTIVE: To sequence and analyze the complete genome of two new Japanese encephalitis virus (JEV) strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area. METHODS: RT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of Clustal X (1.83), MegAlign, Mega (4.0) and Genedoc (3.2). RESULTS: The genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein, the homology of nucleotide and amino acid sequence between two isolates were 98.2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype I. Two new strains were most closely related to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08-55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV. CONCLUSION: Two new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.

[Study on Bartonella vinsonii berkhoffii isolated from blood of native dogs in China].

OBJECTIVE: To isolate and identify Bartonella strains from native dogs in Shandong province in China. METHODS: EDTA-anticoagulated blood samples were collected from 71 native dogs in Yanggu county of Shandong province in March 2005. All isolates were grown on brain heart infusion agar plates containing 5% defibrinated rabbit blood. The agar plates were incubated at 37 degrees C in a humidified with 5% CO2 environment for 4 weeks or longer. All Bartonella-like isolates were examined by routine Gram and Giménez staining and then followed by polymerase chain reaction (PCR) and PCR-RFLP analysis for identification and differentiation of the isolates. Sequencing 16S rRNA, citrate synthase (gltA) gene and 16S-23S rRNA ITS were carried out and sequential similarities were calculated using the DNASTAR5 software package. The phylogenetic tree was inferred from each bootstrap sample, using the neighbor-joining methods as executed in the MEGA 3.1 software. The translation from DNA to protein were determined by DNASIS 2.5. RESULTS: The two Bartonella-like organisms (strains Q52SHD and Q64SHD) were isolated from the blood of 71 dogs. Light microscopic examination of the Gram and Giménez-stained micro-organisms showed small, short and slightly curved pleomorphic gram-negative bacilli. Amplified products of the three pairs of Bartonella genus-specific primers carried the same size as the predicted of those Bartonella species. Data from PCR-RFLP analysis showed that the two strains that having the same profiles were all different from the B. henselae type strain-16S rRNA, gltA and 16S-23S rRNA ITS sequences from the two isolates were 100.0%, 99.7% and 97.2% homologous to B. vinsonii berkhoffii. CONCLUSIONS: Based on these findings, the two isolates Q52SHD and Q64SHD were demonstrated as B. vinsonii berkhoffii. To our knowledge, this was the first report on the presence of Bartonella infection in native dogs from China, which constituted a large reservoir of Bartonella species in this country.