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Background Few studies have been conducted to investigate the distribution of mosquito vectors and the population structure of secondary vectors in the border region of Cambodia-Laos. The aim of this work was to study the mosquito diversity and molecular phylogeny of secondary vectors, i.e., Anopheles nivipes in this area. Methods 1440 adult mosquitoes were trapped in the Cambodia-Laos border. mtDNA-COII were amplified and sequenced from 53 An. nivipes DNA samples. Together with COII sequences deposited in GenBank, a total of 86 COII sequences were used for examining population variations, genetic differentiation, spatial population structure, population expansion, and gene flow patterns. Results The adult mosquitoes were classified into 5 genera and 27 species in this border region. The predominant genera were Culex (60.07%, 865/1440) and Anopheles (31.25%, 450/1440), and the major Anopheles species were An. nivipes (73.56%, 331/450) and Anopheles maculatus (14.22%, 64/450). Based on sequences analysis of COII, a high level of genetic differentiation was reported in two Northwest India (Cheema and Bathinda, Punjab) and Cambodia-Laos (Siem Pang, Stung treng) populations (FST = 0.97824, 0.97343, P An. nivipes populations. Phylogenetic analysis and SAMOVA revealed a gene barrier restricting gene flow among three An. nivipes populations. Mantel test suggested a significant correlation between geography and gene distance in all An. nivipes populations (Z = 44,983.1865, r = 0.5575, P = 0.0070). Neutrality test and Mismatch distribution revealed a recent population expansion of An. nivipes in the Cambodia-Laos population. Conclusions Anopheles nivipes was one of the major Anopheles species in the Cambodia-Laos border. Based on sequences analysis of COII, a genetic barrier between Cambodia-Laos and two Indian populations was found, and a recent population expanding or selecting of An. nivipes occurred in the Cambodia-Laos population, suggesting that COII might be an effective marker for describing the molecular phylogeny of An. nivipes. Further investigation and continuous surveillance of An. nivipes are warranted in this region.

Background Mutations in the Plasmodium falciparum k13 gene are associated with artemisinin (ART) resistance. However, it is unclear whether the F446I mutation, the most prevalent allele at the China–Myanmar border and north of Myanmar, is associated with ART resistance. Therefore, the aim of this study was to investigate the role of this mutation in ART resistance by generating transgenic parasites expressing the F446I mutant allele. Methods The transgenic parasites carrying the F446I or C580Y mutation in both 3D7 and FCC1/HN isolates were generated by single crossing-over recombination and verified using PCR and gene sequencing. The ring-stage survival assay of 0–3 h (RSA0–3 h) was used to evaluate ART susceptibility of the transgenic parasites in vitro. Results Four transgenic parasite lines named 3D7F446I mut, 3D7C580Y mut, FCC1/HNF446I mut and FCC1/HNC580Y mut were successfully generated. These parasite lines showed no changes in the expression level of k13 when compared with their parent parasite isolates. However, introduction of the F446I mutation in k13 of the 3D7 and FCC1/HN isolates led to elevated ring survival rates detected using RSA0–3 h when subjected to both 700 and 20 nM concentrations of dihydroartemisinin. The survival rates were similar to those detected in the parasite lines with the C580Y mutation. Conclusions Insertion of the F446I mutation in k13 led to increased ring survival, suggesting that this mutation may be associated with ART resistance and could be used as a molecular marker for monitoring ART-resistant parasites. The results also highlights the importance of surveillance of F446I mutants for containing the resistant parasite.

Background Antigenic variation in Plasmodium falciparum involves switching among multicopy var gene family and is responsible for immune evasion and the maintenance of chronic infections. Current understanding of var gene expression and switching patterns comes from experiments conducted on long laboratory-adapted strains, with little known about their wild counterparts. Methods Genome sequencing was used to obtain 50 var genes from a parasite isolated from the China-Myanmar border. Four clones with different dominant var genes were cultured in vitro in replicates for 50 generations. Transcription of the individual var gene was detected by real-time PCR and then the switching process was analysed. Results The expression of multicopy var genes is mutually exclusive in clones of a wild P. falciparum isolate. The activation of distinct primary dominant var genes leads to different and favoured switching patterns in the four clones. The on/off rates of individual var genes are variable and the choice of subsequent dominant var genes are random, which results in the different switching patterns among replicates of each clonal wild P. falciparum isolate with near identical initial transcription profiles. Conclusions This study suggests that the switching patterns of var genes are abundant, which consist of both conserved and random parts. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0565-y) contains supplementary material, which is available to authorized users.

Background The piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening. The identification of genotype of piggyBac mutants in the Plasmodium genome is thus an indispensable step in forward genetic analysis. Several PCR-based approaches have been used to identify the piggyBac insertion sites in Plasmodium falciparum and Plasmodium berghei, but all are tedious and inefficient. Next generation sequencing can produce large amounts of sequence data and is particularly suitable for genome-wide association studies. In this study, the Next generation sequencing technology was employed to efficiently identify piggyBac insertion sites in the genome of P. berghei. Methods Plasmodium berghei parasites were co-transfected with piggyBac donor and helper plasmids. Initially, the classical inverse PCR method was used to identify the existence of piggyBac insertions in the P. berghei genome. The whole genome of post-transfection parasites was subsequently sequenced with a PCR-free paired-end module using the Illumina HiSeq sequencing system. The two distinct methods (‘BLAST method’ and ‘SOAP method’) were employed to identify piggyBac insertion sites in the P. berghei genome with Illumina sequencing data. All the identified piggyBac insertions were further tested by half-nested PCR. Results The inverse PCR method resulted in a very low yield of ten individual insertions identified. Conversely, 47 piggyBac insertions were identified from about 1 Gb of Illumina sequencing data via the two distinct analysis methods. The majority of identified piggyBac insertions were confirmed by half-nested PCR. In addition, 1,850 single nucleotide polymorphisms were identified through alignment of the Illumina sequencing data of the P. berghei ANKA strain used in this study with the reference genome sequences. Conclusion This study demonstrates that a high-throughput genome sequencing approach is an efficient tool for the identification of piggyBac-mediated insertions in Plasmodium parasites.

Background Apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP1) of Plasmodium falciparum are two leading blood-stage malaria vaccine candidates. A P. falciparum chimeric protein 2.9 (PfCP-2.9) has been constructed as a vaccine candidate, by fusing AMA-1 domain III (AMA-1 (III)) with a C-terminal 19 kDa fragment of MSP1 (MSP1-19) via a 28-mer peptide hinge. PfCP-2.9 was highly immunogenic in animal studies, and antibodies elicited by the PfCP-2.9 highly inhibited parasite growth in vitro. This study focused on locating the distribution of epitopes on PfCP-2.9. Methods A panel of anti-PfCP-2.9 monoclonal antibodies (mAbs) were produced and their properties were examined by Western blot as well as in vitro growth inhibition assay (GIA). In addition, a series of PfCP-2.9 mutants containing single amino acid substitution were produced in Pichia pastoris. Interaction of the mAbs with the PfCP-2.9 mutants was measured by both Western blot and enzyme-linked immunosorbent assay (ELISA). Results Twelve mAbs recognizing PfCP-2.9 chimeric protein were produced. Of them, eight mAbs recognized conformational epitopes and six mAbs showed various levels of inhibitory activities on parasite growth in vitro. In addition, seventeen PfCP-2.9 mutants with single amino acid substitution were produced in Pichia pastoris for interaction with mAbs. Reduced binding of an inhibitory mAb (mAb7G), was observed in three mutants including M62 (Phe491→Ala), M82 (Glu511→Gln) and M84 (Arg513→Lys), suggesting that these amino acid substitutions are critical to the epitope corresponding to mAb7G. The binding of two non-inhibitory mAbs (mAbG11.12 and mAbW9.10) was also reduced in the mutants of either M62 or M82. The substitution of Leu31 to Arg resulted in completely abolishing the binding of mAb1E1 (a blocking antibody) to M176 mutant, suggesting that the Leu residue at this position plays a crucial role in the formation of the epitope. In addition, the Asn15 residue may also play an important role in the global folding of PfCP-2.9, as its substitution by Arg lead to reduced binding of most mAbs and abolishing the binding of mAb6G and mAbP5-W12. Conclusions This study provided valuable information on epitopes of PfCP-2.9 vaccine candidate through generation of a panel of mAbs and a series of PfCP-2.9 mutants. The information may prove to be useful for designing more effective malaria vaccines against blood-stage parasites.