Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.
BackgroundPeste des petits ruminants (PPR) is a highly contagious infectious disease of goats, sheep and small wild ruminant species with high morbidity and mortality rates. The Peste des petits ruminants virus (PPRV) expresses a hemagglutinin (H) glycoprotein on its outer envelope that is crucial for viral attachment to host cells and represents a key antigen for inducing the host immune response.Methodology/Principal FindingsTo determine whether H can be exploited to generate an effective PPRV vaccine, a replication-competent recombinant canine adenovirus type-2 (CAV-2) expressing the H gene of PPRV (China/Tibet strain) was constructed by the in vitro ligation method. The H expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the BGH early mRNA polyadenylation signal, was inserted into the SspI site of the E3 region, which is not essential for proliferation of CAV-2. Infectious recombinant rCAV-2-PPRV-H virus was generated in transfected MDCK cells and used to immunize goats. All vaccinated animals produced antibodies upon primary injection that were effective in neutralizing PPRV in vitro. Higher antibody titer was obtained following booster inoculation, and the antibody was detectable in goats for at least seven months. No serious recombinant virus-related adverse effect was observed in immunized animals and no adenovirus could be isolated from the urine or feces of vaccinated animals. Results showed that the recombinant virus was safe and could stimulate a long-lasting immune response in goats.Conclusions/SignificanceThis strategy not only provides an effective PPR vaccine candidate for goats but may be a valuable mean by which to differentiate infected from vaccinated animals (the so-called DIVA approach).
Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.
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