Polymorphisms analysis of the Plasmodium ovale tryptophan-rich antigen gene (potra) from imported malaria cases in Henan Province

Plasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed. A total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011–2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country. Totally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa. This study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.


Background
Plasmodium ovale was first described in 1922 [1], as the fourth malaria parasite of humans [2,3]. Generally, a P. ovale infection is of low parasitaemia, and the morphology of the parasite is similar to Plasmodium vivax. Also, it frequently presents as a mixed infection with the other Plasmodium species [4][5][6][7][8][9]. As a result, P. ovale attracted less attention compared to other species, and its prevalence has apparently been underestimated. It has long been considered predominantly found in Africa and some islands of Western Pacific [10,11], with confirmed cases occasionally found in other endemic regions [12,13].
Currently, P. ovale spp. may be classified into two different subspecies by molecular genotyping: P. o. curtisi (classic type) and P. o. wallikeri (variant type) [14]. The nuclear genome sequences further confirmed that the two species were genetically different, but morphologically indistinct [15], and their duration of latency were seemly different [16]. Both species were considered to exist sympatrically in Africa and Asia, and even both parasites were infected simultaneously [17][18][19][20][21].
Because of the generally low parasitaemia of P. ovale infections, sensitive molecular methods to detect and identify the two subspecies must be used in future investigations, with polymorphic markers as a method to discriminate the different strains. Many protocols showed that the SS rRNA genes [17,22,23] were suitable for identification but not for genotyping. The recent study showed that the gene encoding P. ovale tryptophan-rich antigen (potra) could be used to distinguish the two P. ovale subspecies [14]. The sequence and size of the tryptophan-rich antigen gene was variable among the P. ovale subspecies (poctra and powtra) [14]. A nested PCR detection assay was exploited to discriminate the species by the size of the amplified fragments (299 or 317 bp for poctra; 245 bp for powtra), where the conserved sequences were chosen as primers for these two genes [19]. Additionally, a semi-nested PCR protocol was developed by Tanomsing et al. [24] with which the two P. ovale subspecies could be discriminated efficiently, and more fragment sizes were found comparing with previous studies, the 299 bp fragment was overlapping between the two subspecies. This would invalidate amplified fragment size difference, as a means of distinguishing between P. o. curtisi and P. o. wallikeri. The amplified fragment size variations resulted from differences in the number of repeated units, which suggested that a broader range of size variants might occur. In this study, more variations of potra gene were observed.

Sample collection and DNA extraction
Dried blood spots on filter paper (Whatman 3M) were collected from patients returned from Africa with P. ovale infection before treatment. All the patients were diagnosed by nested PCR and microscopy. All the dried blood spots were labelled with a unique identification number, air-dried and individually placed in plastic bags with desiccant and stored at − 20 °C until laboratory analysis. DNA was extracted from the dried blood spots using a QIAamp DNA mini kit (Qiagen, Germany).

Nested PCR amplification and DNA sequencing
The fragments of potra was amplified with nested PCR using the primers as described previously [14,19]. The amplified products were identified by agarose gel electrophoresis. Bidirectional sequencing was performed for the secondary potra PCR products using the secondary primers by Sangon Biotech Co Ltd (Shanghai, China).

Sequencing alignments and analysis
All the genes sequences were analysed with multiple sequence alignment using the Clustal X software. HM594180-HM594183 [19], KF018430-KF018433 [24] and KX417700-KX417704 [25] from the GenBank would be as the reference sequences of P. ovale spp. Phylogenetic trees were constructed using the Molecular Evolutionary Genetics Analysis (MEGA) 6.06.

Amplification of the potra gene of Plasmodium ovale spp.
A total of 110 dried blood samples, from patients returned from Africa to Henan Province with P. ovale infection, were collected. The amplified nested PCR products of the potra gene of 110 samples were blasted in the GenBank. The blast data showed that 67 samples infected with P. o. wallikeri and 43 samples infected with P. o. curtisi. More fragment sizes of the potra gene from this study were found comparing with the previous reports. P. o. wallikeri had five different sizes including 227, 245, 263, 281 and 299 bp, while P. o. curtisi had three polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.
Keywords: Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium ovale tryptophan-rich antigen (potra), Amino acid unit, Subtype sizes including 299, 317 and 335 bp. Also, the amplified fragment size differed as a result of differences in 18 bases of units, with the overlap of 299 bp between the two species (Fig. 1) Table 1).

Genotypes of potra of Plasmodium ovale spp.
There were 8 genotypes of the potra gene for the 67 isolates infected with P. o. wallikeri and three genotypes of potra for the 43 isolates infected with P. o. curtisi. The sizes of 277, 245 and 263 bp for P. o. wallikeri all had two different subtypes. The sequences of the 11 genotypes of potra gene were deposited in GenBank under accession number MG588144-MG588154. For P. o. wallikeri, the genotype of MG588146 was the same with that of the reference sequences HM594180 and HM594181, but the reference sequences KF018430 and KF018431 were different with any of MG588144-MG588151. For P. o. curtisi, the genotype of MG588152 was the same with that of the reference sequences HM594182 and KF018433, and the genotype of MG588153 was the same with that of the reference sequence HM594183. Combination with the previous studies, there were ten genotypes of potra exited for P. o. wallikeri, and four genotypes for P. o. curtisi. The number of isolates for each genotype was shown in the Table 2.

Alignment of the translated amino acid sequence of potra fragments
Interestingly, the translated amino acid sequence of potra fragments were composed with multiple amino acid units, and six amino acids was considered as a unit.  Table 2. As the same with the genotypes of potra, there were 10 subtypes exiting for P. o. wallikeri and 4 subtypes for P. o. curtisi. The detail information is shown in Fig. 2 and Table 2.

Phylogenetic relationship among potra subtype families
Neighbour-joining was used to cluster the potra gene sequences. The 11 genotypes were classified into two clusters, eight genotypes infected with P. o. wallikeri were  Tanomsing et al. [24] 299 bp (n = The sequences 10-317 and 11-335 had a closer genetic relationship, which formed another sub-cluster with reference sequences, having the same two repeats of TITPIS (Fig. 3).   (Table 3).

Discussion
Malaria elimination is a long-term goal to be achieved worldwide. As one species of human Plasmodium, the identification of P. ovale is more widespread than formerly known. Plasmodium ovale, like P. vivax, has hypnozoites that cause relapses [26,27], and it consists of two different subspecies: P. ovale curtisi and P. ovale wallikeri [14]. Therefore, the differentiation of the two Fig. 3 Genetic relationship of potra among isolates of Plasmodium ovale subspecies. Black triangles represented the isolates in this study. The reference sequences of P. ovale spp., HM594180-HM594183, KF018430-KF018433 and KX417700-KX417704, were obtained from the NCBI database P. ovale species, especially with respect of molecular phylogeny will need to be better understood. In 2011, Oguike et al. [19] published the discrimination of the two P. ovale subspecies by the size of the amplified fragments of the potra gene (299 or 317 bp for P. o. curtisi; 245 bp for P. o. wallikeri), using nested PCR. Although this technique was specific for P. ovale spp., the sizes of the amplified fragment varied with the number of repeat units, which reduced the discrimination between species: for P. o. curtisi, 299, 317 and 353 bp, and for P. o. wallikeri, 245, 299 and 335 bp [24]. Tanomsing et al. [24] suggested the number of potra size variations might be more than those evaluated and this speculation was confirmed in this study. Using the same primes and method, more fragment sizes were identified in this study, while some sizes overlapped between the two subspecies: for P. o. curtisi having sizes 299, 317 and 335 bp, and for P. o. wallikeri having sizes 227, 245, 263, 281 and 299 bp. The results of the three studies [19,24] were also combined, as shown in Table 1. Four different sizes for P. o. curtisi and six sizes for P. o. wallikeri have been reported, and that the fragment sizes of 299 and 335 bp were overlaps between the two species. As more samples were analysed, it was likely that the number of potra size variants would be more than expected. Conceivably, more size variants may be identified in future studies.
Potra gene was used to discriminate the two P. ovale species, because the tryptophan-rich antigen was encoded by a repeat pattern of variable length 3-amino acid [14]. Sutherland et al. [14] and Oguike et al. [19] had also proposed that the potra gene of P. o. curtisi (poctra) could be identified by the pattern of the six amino acids of the repeat region, TITPIS, while the potra gene of P. o. wallikeri (powtra) were different in two non-synonymous positions. By alignment of the amino acid sequence of potra fragments, 11 genotypes of potra were found from the 110 isolates in this study. Combination with the previous studies of Oguike et al. [19] and Tanomsing et al. [24], showed ten subtypes of potra gene for P. o. wallikeri and four subtypes for P. o. curtisi, and 14 genotypes of potra gene were under analysis. This study showed that the amino acid sequence of potra fragments were composed with multiple amino acid units, six amino acids were as one unit. There was different dominant amino acid repeat of potra for the two P. ovale species, which could be used to discriminate the subtype of P. ovale  spp. The repeat of six amino acids as one unit to analyse the difference of the genotypes between the two P. ovale species was first reported, which could make the results more clear and simple. The sizes of 277, 245 and 263 bp for P. o. wallikeri all had two different subtypes, and this phenomenon did not find in the P. o. curtisi. The sizes of the reference sequences KX417700-KX417704 [25] from Genbank were short for discriminating the differences of dominant amino acid repeats. In our study, the genetic relationship of the P. ovale spp. was analysed by the neighbour-joining tree mainly based on the number of the dominant amino acid repeats including MANPIN, AITPIN, TINPIN or TITPIS.
The distribution of the two P. ovale spp. was different. The isolates of P. o. wallikeri was more than that of P. o. curtisi, and the genotype of the 245 bp size was the predominant type for P. o. wallikeri in most Africa countries, but the other genotypes were less. For P. o. curtisi, the genotypes of the 299 and 317 bp size were commonly in Africa and the genotype of 335 bp size was less.
Molecular epidemiological studies on genetic diversity of Plasmodium vivax have been based mainly on single copy polymorphic genes which code for parasite surface antigens such as circumsporozoite protein (csp), merozoite surface protein-1 (msp-1) and merozoite surface protein 3 alpha (msp 3α) [28]. Pvcsp comprises of central domain of tandem repeated sequences flanked by two non-repeated conserved sequences [29][30][31][32]. Two types of repeat elements, either VK210 or VK247 types were detected in clinical isolates of P. vivax and thus pvcsp serves as a useful tool for genotyping [33,34]. Potra has the similar characteristics with pvcsp, which also could be used for parasite genotyping.

Conclusions
Considering the change of malaria epidemiology and the approaching of malaria elimination, P. ovale spp. deserves more attention. Molecular techniques are a good tool for detecting and identifying the two P. ovale subspecies and their relative distribution.

Authors' contributions
RMZ was responsible for the molecular genetic analysis and data interpretation and drafted the manuscript. YL participated in sample detection and data analysis. HWZ and BLX conceived the study and revised the manuscript. FH revised the manuscript. SUL participated in sample collection and sample detection. YLZ, YD and DLL provided the administrative coordination. CYY and DQ participated in the data collection and analysed the data. All authors read and approved the final manuscript.