Number Of Nucleotide Substitutions Research Articles

Cytokine profile and viral diversity in the early seronegative stage of community-acquired hepatitis C virus (HCV) infection

Most Hepatitis C virus (HCV)-infected subjects develop chronic infection, whereas a minority clear the virus in the early phase of infection. We analyzed factors associated with outcome (chronicity vs clearance) during the preclinical seronegative phase of community-acquired HCV infection. Among 17.5 million blood donations in the years 2000–2016, 124 blood donors were found to be HCV RNA-positive/anti-HCV-negative. All were contacted after 0.5–12.7 years and 40 responded and provided blood sample. Hypervariable region 1 was analyzed by ultradeep pyrosequencing and cytokines in serum were quantified by Luminex (R&D Systems) multiplex immunoassay. Twenty-one (52.5%) donors were found to be HCV-RNA-positive, while 19 (47.5%) were HCV RNA negative (none received antiviral treatment). All but one seroconverted to anti-HCV. Donors with resolving hepatitis did not differ significantly from donors with chronic infection with respect to age, genotypes, IL28B polymorphisms, number of viral variants, nucleotide diversity per site or the overall number of nucleotide substitutions. However, the former group had significantly higher levels of IL-1beta, IL-1RA, IL-6, IFN-gamma and FGF-2 in serum. In our study of community-acquired acute hepatitis C approximately half of all subjects eliminated the virus spontaneously, and this clearance was associated with marked cytokine response in the early seronegative stage of infection.

Determinants of Persistent Patterns of Pepino Mosaic Virus Mixed Infections.

Pepino mosaic virus (PepMV) has become a pandemic virus in tomato crops, causing important economic losses worldwide. In Spain, isolates of the EU and CH2 strains co-circulate, with PepMV-EU predominantly found in mixed infections. Simultaneous in planta mixed infections result in an asymmetric antagonism against PepMV-CH2, but the outcome of over-infections has never been tested. PepMV-EU and PepMV-CH2 time-lagged inoculations were performed, and viral accumulation was measured 10 days after challenge inoculation. PepMV-EU had a protective effect over PepMV-CH2; in contrast, the accumulation of PepMV-EU increased in plants pre-inoculated with PepMV-CH2 as compared to single infections. We also studied the effect of the type of infection on viral transmission. Independently of the nature of the infection (single or mixed), we observed a strong positive correlation between virus accumulation in the source plant and transmission, excluding mixed infection effects different than modulating viral accumulation. Finally, in order to determine the genetic variability of PepMV strains in single and mixed infections, a 430 nucleotide region was RT-PCR amplified from samples from a serial passages experiment and deep-sequenced. No significant differences were found in the number of nucleotide substitutions between single and mixed infections for PepMV-EU; in contrast, significant differences were found for PepMV-CH2, which was more variable in single than in mixed infections. Comparing PepMV-EU with PepMV-CH2, a higher nucleotide diversity was found for PepMV-CH2. Collectively, our data strongly suggest that PepMV mixed infections can impact the virus epidemiology by modulating in planta virus strain accumulation and diversification.

Resistance to pyridaben in Canadian greenhouse populations of two-spotted spider mites, Tetranychus urticae (Koch)

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

Hepatitis C virus (HCV) genotype 1b displays higher genetic variability of hypervariable region 1 (HVR1) than genotype 3

Hepatitis C virus (HCV) is characterized by high genetic variability, which is manifested both at the inter-host and intra-host levels. However, its role in the clinical course of infection is less obvious. The aim of the present study was to determine the genetic variability of HCV HVR1 (hypervariable region 1) of genotype 1b and 3 in plasma of blood donors in the early seronegative stage of infection (HCV-RNA+, anti-HCV−) and in samples from chronically infected patients using next-generation sequencing. Sequencing errors were corrected, and haplotypes inferred using the ShoRAH software. Genetic diversity parameters (intra-host number of variants, number of nucleotide substitutions and diversity per site) were assessed by DNA SP and MEGA. During the early infection, the number of variants were significantly lower in subjects infected with genotype 3 than with genotype 1b (p < 0.02). Similarly, intra-host number of variants, number of nucleotide substitutions and diversity per site were lower in genotype 3 chronic infection (p < 0.0005). In addition, early infection was characterized by significantly lower HVR1 variability values (p < 0.04) when compared to chronic infection for both genotypes. It seems that the observed differences in HVR1 variability represent an inherent property of particular viral genotypes.

Genetic Changeability by Loci COI mtDNA for Different Coloring of Shell Phenotypes of Black Sea Mussel Mytilus galloprovincialis Lam. (Mollusca: Bivalvia: Mytilidae)

The variability of the nucleotide sequences of the fragment of the COI gene of the female genome was studied in four phenotypic groups of the Black Sea mollusk Mytilus galloprovincialis. It was shown that the parameters of genomic diversity (the number of haplotypes, haplotypic and nucleotide diversity, and the number of nucleotide substitutions, including pairwise ones) in mussels with dark shell color are significantly higher than in individuals with light color phenotypes. The revealed differences are related to the peculiarities of ecological preferences of mussels with dark and light shells.

Genetic Characterization of Kazakh Native Sheep Breeds Using Mitochondrial DNA

In this study, we used mitochondrial (mt) DNA to reveal patterns of genetic diversity among six breeds of sheep native to Kazakhstan, including the Chu and Kalchengel types of Kazakh mutton-semifine-wool breed of sheep, Akzhayik, Edilbay, Kazakh fat-rumped semi-course-wool and South Kazakh Merino breeds. Mitochondrial DNA D-loop was studied in 64 samples to determine the genetic diversity of the local sheep breeds. The analysis identified 54 haplotypes and 113 polymorphic loci of mtDNA D-loop. For the studied breeds, haplotype diversity and nucleotide diversity were 0.993±0.005 and 0.039±0.001, respectively. The results indicated higher level of nucleotide diversity in the South Kazakh Merino (SKM) breed (0.047), while the lowest diversity was observed in the Edilbay breed (0.029). In addition, the average number of nucleotide differences (Kxy) and the average number of nucleotide substitutions (Dxy) were determined among the studied breeds. The minimal value of genetic distance was observed between Akzhayik mutton-wool and Kazakh fat-rumped semi-course-wool breeds (Kxy: 16.956 and Dxy: 0.039), while the maximum value was observed between the Edilbay and SKM breeds (Kxy: 24.600 and Dxy: 0.040). Phylogenetic analysis revealed the presence of three haplogroups, namely, A, B and C in the collected sample, while the haplogroups D and E were not found. The results indicated that 28 of 64 tested animals belong to haplogroup A (43.75%), whereas 25 of investigated animals were attributed to haplogroup B (39.06%) and 11 animals to haplogroup C (17.19%).

Non-coding regions of the Ebola virus genome contain indispensable phylogenetic and evolutionary information.

We compared the numbers of nucleotide substitutions occurring in the non-coding regions and coding regions of Ebola virus genomes and found that non-coding regions contain indispensable phylogenetic and evolutionary information. The omission of genetic data from non-coding regions can lead to unreliable phylogenies and inaccurate estimates of evolutionary parameters.

Comparative analysis of the complete nucleotide sequences of mitochondrial DNA of rat strains Wistar and oxys of the institute of cytology and genetics, Siberian Branch, Russian Academy of Sciences

Complete nucleotide sequences of the mitochondrial DNA of Wistar rats and premature aging rats OXYS, sensitive to cataractogenic effect of galactose, from the Center for collective usage «Gene pool of laboratory animals» of the Institute of Cytology and Genetics (ICG), Siberian Branch, Russian Academy of Sciences, were investigated (nucleotide sequences were submitted to GenBank). It was shown that two additional unique nucleotide substitutions between rats OXYS (ICG) and Wistar (ICG) are not found in other known nucleotide sequences of mitochondrial DNA of rats and do not result in amino acid substitutions, so that all differences in phenotypic data of these lines are determined only by differences in the genomic DNA. Comparative analysis showed that Wistar (ICG) rats have a significant number of nucleotide substitutions compared to rat strains derived from outbred rat Wistar colony, which contradicts possible relationship of Wistar (ICG) rats to baseline Wistar rat line or its descendants. It was assumed that the Sprague Dawley rat line is the progenitor of Wistar (ICG) rats.

Morphological and molecular phylogenetic analysis of the high triglyceride‐producing marine diatom, Fistulifera solaris sp. nov. (Bacillariophyceae)

SummaryFistulifera sp. strain JPCC DA0580, a marine pennate diatom, contains extremely high levels of intracellular triglyceride and has been suggested as a promising source of feedstock for biodiesel fuels. JPCC DA0580 was isolated from a mangrove swamp located in Sumiyo Bay below the mouths of the Sumiyo and Yakugachi Rivers in Amami‐Ohshima, Kagoshima, Japan. In this study, the morphology and the 18S rDNA sequence of JPCC DA0580 were compared with those of other Fistulifera strains. JPCC DA0580 possesses morphological characters of the genus Fistulifera, namely lightly silicified frustules, a distinct median costa (raphe sternum), and a wart‐like central pore (fistula). Morphometric analysis revealed that JPCC DA0580 differs from other Fistulifera species by the presence of a valve with coarser striation and coarser areolation. On the basis of 18S rDNA phylogeny, JPCC DA0580 formed a well‐supported clade with other members of the Fistulifera species complex, although the number of nucleotide substitutions was highest in JPCC DA0580. Our results led us to propose the taxonomic name Fistulifera solaris sp. nov. for JPCC DA0580.

DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Nuclear factor-κB (NF-κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-κB, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-κB binding site of the competitor. Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([−]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (−)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.

Comparative Study of Bunt Pathogen Resistance to the Effects of Fungicides in Callus Co-Cultures &amp;lt;i&amp;gt;Triticum aestivum&amp;lt;/i&amp;gt; with Tilletia caries

The morphophysiological and molecular-genetic parameters of T. caries isolates collected from various fields of Southern Urals agrocenoses have been analysed. Isolate 1 of wheat callus had a high growth rate in vitro even in the presence of 0.1 mg/l fungicide Baitan. Isolate 2 of wheat callus had a low growth rate whereas 0.1 mg/l Baitan significantly inhibited its growth. Comparison of nucleotide sequences of 18S RNA gene of the two isolates showed high level of homology between them, but a large number of nucleotide substitutions have been found. The most characteristic excision was five nucleotides at position 461 of the isolate 2, compared with the isolate 1. Our results allow to assume that the environmental stress including high pesticide concentration may cause the resistance of T. caries pathogen to fungicides.

Impact of Genetic Heterogeneity in Polymerase of Hepatitis B Virus on Dynamics of Viral Load and Hepatitis B Progression

ObjectiveThe hepatitis B virus (HBV)-polymerase region overlaps pre-S/S genes with high epitope density and plays an essential role in viral replication. We investigated whether genetic variation in the polymerase region determined long-term dynamics of viral load and the risk of hepatitis B progression in a population-based cohort study.MethodsWe sequenced the HBV-polymerase region using baseline plasma from treatment-naïve individuals with HBV-DNA levels≥1000 copies/mL in a longitudinal viral-load study of participants with chronic HBV infection followed-up for 17 years, and obtained sequences from 575 participants (80% with HBV genotype Ba and 17% with Ce).ResultsPatterns of viral sequence diversity across phases (i.e., immune-tolerant, immune-clearance, non/low replicative, and hepatitis B e antigen (HBeAg)-negative hepatitis phases) of HBV-infection, which were associated with viral and clinical features at baseline and during follow-up, were similar between HBV genotypes, despite greater diversity for genotype Ce vs. Ba. Irrespective of genotypes, however, HBeAg-negative participants had 1.5-to-2-fold higher levels of sequence diversity than HBeAg-positive participants (P<0.0001). Furthermore, levels of viral genetic divergence from the population consensus sequence, estimated by numbers of nucleotide substitutions, were inversely associated with long-term viral load even in HBeAg-negative participants. A mixed model developed through analysis of the entire HBV-polymerase region identified 153 viral load-associated single nucleotide polymorphisms in overall and 136 in HBeAg-negative participants, with distinct profiles between HBV genotypes. These polymorphisms were most evident at sites within or flanking T-cell epitopes. Seven polymorphisms revealed associations with both enhanced viral load and a more than 4-fold increased risk of hepatocellular carcinoma and/or liver cirrhosis.ConclusionsThe data highlight a role of viral genetic divergence in the natural course of HBV-infection. Interindividual differences in the long-term dynamics of viral load is not only associated with accumulation of mutations in HBV-polymerase region, but differences in specific viral polymorphisms which differ between genotypes.

Complete genome sequence of the NVSL BoHV-1.1 Cooper reference strain

The only complete genome sequence available for bovine herpesvirus 1 (BoHV-1) is a composite sequence derived from four different BoHV-1.1 strains and one BoHV-1.2 strain. Such a chimeric genome sequence is problematic for molecular genetic studies on this virus. We report here the complete genome sequence for the BoHV-1.1 NVSL reference strain Cooper. Although similar to the published chimeric genome sequence, there are a number of nucleotide substitutions and deletions/insertions across the genome, many of which affect coding sequences.

Reconstruction of the Transmission History of RNA Virus Outbreaks Using Full Genome Sequences: Foot-and-Mouth Disease Virus in Bulgaria in 2011

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.

Genetic polymorphism at 5′ flanking region of the prolactin gene and its effect on egg quality traits in naked neck chickens

A study was conducted to explore polymorphism at 5′ flanking region of prolactin gene and to estimate the effect of polymorphism on egg quality traits in naked neck chickens. Single-stranded conformation polymorphism technique was employed to detect polymorphism in the gene. Three genotypes (AA, AB and AC) were observed in naked neck chicken of which the predominant genotype was AB. The predominant and least frequent alleles in the population were A and C, respectively. The sequence alignment study revealed a number of nucleotide substitutions at different positions of the alleles. Genotypes showed significant effect (P < 0.05) on Haugh unit and yolk index where birds with AB and AC genotypes were the best performers. In conclusion, it may be inferred that 5′ flanking region of prolactin gene was polymorphic with having significant effect on some egg quality traits in the naked neck chicken.

Sequence variation in ITS spacers and 5.8S rDNA and relationship of E, St, P, Ns, Xm, and H genomes in the genera of Agropyron, Elytrigia, Leymus, Pascopyrum, Psathyrostachys, and Hordeum

Understanding species evolution and improvement requires information of their genome origin and differentiation. Among the species in the family Gramineae, genome identities of Agropyron-Elytrigia-Leymus group are still ambiguous. In order to delineate the genome relationship, nucleotide sequence analysis in the rDNA ITS regions was carried out among the species in the genera Elytrigia, Agropyron, Psathyrostachys, Leymus, and Psacopyrum containing E, St, P, Ns, and Xm genomes. The ITS-1 and ITS-2 showed a narrow range of variation in length except for the presence of a pentanucleotide, TGGGG, in/del in some haplotypes, whereas higher numbers of nucleotide substitutions were observed in most genera. There were 187 variable sites in the ITS-1, 5.8S, and ITS-2 regions, in which a few genome specific mutations were observed. While the level of variation was similar between ITS-1 and ITS-2, the rate of transition mutation versus transversion mutations was different among the ITS-1, 5.8S, and ITS-2 segments. GC contents of the ITS regions ranged between 55–65% between genomes and the haplotypes of P and H genomes were slightly higher than others. In phylogenetic analysis, the ITS haplotypes were classified into two groups; one containing H, Ns, NsXm genomes, and another containing P, St, and E genomes, which are congruous to the genome affinities from other studies. Among the four genomes in Pascopyrum smithii (2n=8x=56, StStNsNsHHXmXm), the haplotypes of H and St genomes were identified with the reference diploid species, but the haplotypes having Ns and Xm genomes were not found in the present analysis.

Polymorphism of the capsid L1 gene of human papillomavirus types 31, 33, and 35

The L1 gene encodes for the major capsid protein of human papillomaviruses (HPV). There is limited information on the polymorphism of L1 for types related to HPV-16. This report explores the polymorphism of L1 in phylogenetically related types 31, 33, and 35 compared to HPV-16. Genital specimens collected from 732 HIV-seropositive and 323 HIV-seronegative women were screened for HPV DNA with consensus L1 PCR. Cervical samples positive for HPV-16 (n = 74), HPV-31 (n = 78), HPV-33 (n = 37), and HPV-35 (n = 58) were further characterized by PCR-sequencing of the complete L1 gene. The number of nucleotide substitutions within L1 ranged from 19 for HPV-33 to 52 for HPV-31. The ratio of the number of variants/number of isolates tested was higher for HPV-31 (56.4%, P = 0.05) and HPV-35 (60.3%, P = 0.04) compared to HPV-16 (40.5%), while this ratio was lower for HPV-33 (24.3%), although not significantly (P = 0.14). The maximal distance between HPV variants was greater in the five putative surface-exposed loops of L1 than in sequences outside the loops (P < 0.01). Synonymous variations were encountered in 1.7% (95% CI 1.1-2.3) of nucleotides inside the L1 loops and 2.4% (95% CI1.2-3.7) of nucleotides outside the L1 loops. Non-synonymous variations were encountered in 1.8% (95% CI 1.1-2.5) of nucleotides within the L1 loops and 0.2% (95% CI 0-0.4) of nucleotides outside the loops. dN/dS ratios were below 1.0 in extra-loop and intra-loop regions, but they were lower in extra-loop regions. These results suggest that sequences within and outside the hypervariable loops of L1 were under selective constraint.

Recurrent isolation of poliovirus 3 strains with chimeric capsid protein Vp1 suggests a recombination hot-spot site in Vp1

Five oral poliovirus vaccine (OPV) strains carrying an intertypic PV3/PV2 recombination in VP1 capsid protein were isolated during poliovirus surveillance. These five PV3 strains had altogether four diverse recombination crossover points near the 3′ end of the VP1 coding region. The complete antigenic site IIIa was replaced by PV2-specific amino acids in four of the studied PV3 strains. Low overall number of nucleotide substitutions in VP1 indicated that the predicted replication time, “age”, of the PV3 strains was short, 6 months or less. The nucleotide 472-T in the 5′ non-coding region, associated to the attenuated phenotype of PV3/Sabin, was reverted to wild-type C in all studied PV3/PV2 recombinant strains. Three of the PV3 strains had at least a tripartite genome deduced from the partial 3D polymerase-coding region sequences. Our results suggest that there exists a PV3/PV2 recombination hot-spot site in the 3′ partial region of the VP1 capsid protein and that the recombination may occur within weeks or a few months after the administration of OPV.

Response to Yang et al.: Problems with Bayesian methods of detecting positive selection at the DNA sequence level

It is unfortunate that Yang et al. (1) misrepresent the contents and conclusions of our recent study (2). Our study was motivated by the recent publication of papers reporting the detection of positive selection in many genes of the human lineage by using the branch-site method (BSM) and others, where the ratio (ω) of nonsynonymous to synonymous nucleotide substitutions is used as a measure of positive selection (e.g., ref. 3). Because the number of nucleotide substitutions per gene (n) in the human lineage after separation from the chimpanzee is very small (n ≈ 4) and BSM depends on the large sample theory, we examined the theoretical basis of BSM in comparison with the small-sample method (SSM) using Fisher's exact test (4), which gives accurate statistical tests for small samples.

Selective pressure on the merozoite surface protein-1 genes of Plasmodium vivax, P. knowlesi and P. cynomolgi

Background: The merozoite surface protein 1 (msp-1) of malarial parasites is a promising target for malaria vaccine development. Because antigenic polymorphism of this protein occurs in both Plasmodium falciparum and P. vivax, it is important to know the extent of sequence variation of this gene in nonhuman primate malarias that can infect humans. Objective: To determine the complete nucleotide sequences of the msp-1 genes of Plasmodium knowlesi (Pkmsp-1) and P. cynomolgi (Pcymsp-1) and their evolutionary history comparing with an orthologous gene of P. vivax (Pvmsp-1). Method: The msp-1 genes of P. knowlesi and P. cynomolgi were amplified by the polymerase chain reaction and their nucleotide sequences were determined directly from the amplified products. Sequences were aligned using the Clustal X program. The extent of polymorphism is expressed in terms of nucleotide diversity (π) and the average number of nucleotide substitutions per site between populations (Dxy) in pair-wise comparison. The ratio of nucleotide substitutions at synonymous and nonsynonymous sites was computed by the McDonald and Kreitman test to detect evidence of departure from neutrality. The phylogenetic tree was constructed by the neighbor-joining method using the maximum composite likelihood with 1,000 bootstrap iterations. Results: The deduced amino acid sequences of the msp-1 of P. knowlesi, P. cynomolgi and P. vivax exhibit sequence similarity, ranging from 56.2 to 60.8 %. Variable blocks detected in Pkmsp-1 and Pcymsp-1 are located in regions corresponding to variable blocks of Pvmsp-1. Sliding window plots of Dxy along the entire coding regions of the msp-1 genes gave similar profiles for each pair of comparison. Pvmsp-1 is more closely related to Pcymsp-1 than that of Pkmsp-1 based on a phylogenetic analysis. However, the Tajima’s relative rate test suggests that the merozoite surface protein 1 (msp-1) of each species has not evolved at the same rate. The McDonald-Kreitman test detects positive selection on regions equivalent to blocks 1 and 9 of Pvmsp-1. Conclusion: The msp-1 genes of P. knowlesi, P. cynomolgi and P. vivax exhibit a similar pattern of sequence variation. Regions equivalent to conserved blocks 1 and 9 of Pvmsp-1 have evolved under positive selective pressure, suggestive of functional importance probably as targets for host immune responses.