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: July 2020

Abstract
Background
Eighteen imported ovale malaria cases imported from Myanmar and various African countries have been reported in Yunnan Province, China from 2013 to 2018. All of them have been confirmed by morphological examination and 18S small subunit ribosomal RNA gene (18S rRNA) based PCR in YNRL. Nevertheless, the subtypes of Plasmodium ovale could not be identified based on 18S rRNA gene test, thus posing challenges on its accurate diagnosis. To help establish a more sensitive and specific method for the detection of P. ovale genes, this study performs sequence analysis on k13-propeller polymorphisms in P. ovale.

Methods
Dried blood spots (DBS) from ovale malaria cases were collected from January 2013 to December 2018, and the infection sources were confirmed according to epidemiological investigation. DNA was extracted, and the coding region (from 206th aa to 725th aa) in k13 gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain CDS. The haplotypes and mutation loci of the CDS were analysed, and the spatial structure of the amino acid peptide chain of k13 gene propeller domain was predicted by SWISS-MODEL.

Results
The coding region from 224th aa to 725th aa of k13 gene from P. ovale in 83.3% of collected samples (15/18) were amplified. Three haplotypes were observed in 15 samples, and the values of Ka/Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250. Curtisi haplotype, Wallikeri haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15). The predominant haplotypes of P. ovale curtisi were determined in all five Myanmar isolates. Of the ten African isolates, six were identified as P. o. curtisi, three were P. o. wallikeri and one was mutant type. Base substitutions between the sequences of P. o. curtisi and P. o. wallikeri were determined at 38 loci, such as c.711. Moreover, the A > T base substitution at c.1428 was a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476th position. Compared with sequence of P. o. wallikeri, the double nonsynonymous mutations of G > A and A > T at the sites of c.1186 and c.1428 leads to the variations of D396N and T476S for the 396th and 476th amino acids positions. For P. o. curtisi and P. o. wallikeri, the peptide chains in the coding region from 224th aa to 725th 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 P. ovale isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to P. o. curtisi, while the sequences of African isolates showed the sympatric distribution from P. o. curtisi, P. o. wallikeri and mutant isolates. The CDS 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. o. curtisi and P. o. wallikeri.