Polymorphism analysis of propeller domain of k13 gene in Plasmodium ovale curtisi and Plasmodium ovale wallikeri isolates original infection from Myanmar and Africa in Yunnan Province, China
Authors: Chen Mengni , Dong Ying , Deng Yan , Xu Yanchun , Liu Yan , Zhang Canglin , Huang Herong 
Published: February 2020

Abstract
Background: More than 20 cases of imported ovale malaria patients have been diagnosed and reported in Yunnan Province over the past eight years. By applying morphological examination and 18ss RNA gene analysis of P. ovale , more than 90% of the cases have achieved definite diagnosis. Nevertheless, some cases with observation of typical P. ovale morphology could be identified as the other Plasmodium species based on 18ss RNA gene test, thusly posing challenges on the accurate diagnosis of malaria. To help establish a more sensitive and specific method for the detecting of P. ovale genes, the present study performs sequence analysis on k13-propeller polymorphisms in P. ovale.Methods: The blood samples of ovale malaria patients in Yunnan Province were collected from January 2013 to August 2019, and the infection sources were confirmed according to epidemiological investigation. The DNAs of P. ovale were extracted, and the coding region (from 206 th aa to 725 th aa) in k13 gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain cDNA sequence. The haplotypes and mutation loci of the cDNA sequence were analyzed, and the spatial structure of the amino acid peptide chain of k13 gene propeller domain was predicted by using SWISS-MODEL database ( www.swissmodel.expasy.org/interactive ).Results: Of the 18 collected blood samples of ovale malaria patients, the coding region from 224 th aa to 725 th aa of k13 gene from P. ovale in 83.3% of the samples (15/18) were amplified and the amplified products were around 1732 bp in length. 15 cDNA sequences were obtained. Three haplotypes were observed in these sequences, and the values of Ka / Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250, respectively. Curtisi haplotype, Wallikeri haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15), respectively. The predominant haplotypes of P. ovale curtisi were determined in all six Myanmar isolates yielded. Of the nine African isolates, six were identified as P. ovale curtisi ; and three were P. ovale wallikeri . Base substitutions between the sequences of P. ovale curtisi and P. ovale wallikeri were determined at 38 loci, such as c.711. Moreover, the A> T base substitution at c.1426 is a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476 th position. Compared with sequence of P. ovale wallikeri , the double nonsynonymous mutations of G> A and A> T at the sites of c.1186 and c.1426 leads to the variations of D396N and T476S for the 396 th and 476 th amino acids positions, respectively. For P. ovale curtisi and P. ovale wallikeri , the peptide chains in the coding region from 224 th aa to 725 th aa of k13 gene merely formed a monomeric spatial model, whereas the double-variant peptide chains of D396N and T476S formed homodimeric spatial model.Conclusion: The propeller domain of k13 gene in the Plasmodium ovale isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to P. ovale curtisi , while the sequences of African isolates showed the sympatric distribution from P. ovale curtis i, P. ovale wallikeri and mutant isolates. The cDNA sequence with a double base substitution formed a dimeric spatial model to encode the peptide chain, which is completely different from the monomeric spatial structure to encode the peptide chain from P. ovale curtisi and P. ovale wallikeri .