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Fulltext The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: a 6-cysteine protein family member

Ahmed MA, Chu KB, Quan FS

PeerJ, 2018;6:e6141.
PMID: 30581686 DOI: 10.7717/peerj.6141

Introduction: The zoonotic malaria parasite Plasmodium knowlesi has currently become the most dominant form of infection in humans in Malaysia and is an emerging infectious disease in most Southeast Asian countries. The P41 is a merozoite surface protein belonging to the 6-cysteine family and is a well-characterized vaccine candidate in P. vivax and P. falciparum; however, no study has been done in the orthologous gene of P. knowlesi. This study investigates the level of polymorphism, haplotypes and natural selection of pk41 genes in clinical isolates from Malaysia.
Method: Thirty-five full-length pk41 sequences from clinical isolates of Malaysia along with four laboratory lines (along with H-strain) were downloaded from public databases. For comparative analysis between species, orthologous P41 genes from P. falciparum, P. vivax, P. coatneyi and P. cynomolgi were also downloaded. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. Phylogenetic relationships between Pk41 genes were determined using MEGA 5.0 software.
Results: Analysis of 39 full-length pk41 sequences along with the H-strain identified 36 SNPs (20 non-synonymous and 16 synonymous substitutions) resulting in 31 haplotypes. Nucleotide diversity across the full-length gene was low and was similar to its ortholog in P. vivax; pv41. Domain-wise amino acid analysis of the two s48/45 domains indicated low level of polymorphisms for both the domains, and the glutamic acid rich region had extensive size variations. In the central domain, upstream to the glutamate rich region, a unique two to six (K-E)n repeat region was identified within the clinical isolates. Overall, the pk41 genes were indicative of negative/purifying selection due to functional constraints. Domain-wise analysis of the s48/45 domains also indicated purifying selection. However, analysis of Tajima's D across the genes identified non-synonymous SNPs in the s48/45 domain II with high positive values indicating possible epitope binding regions. All the 6-cysteine residues within the s48/45 domains were conserved within the clinical isolates indicating functional conservation of these regions. Phylogenetic analysis of full-length pk41 genes indicated geographical clustering and identified three subpopulations of P. knowlesi; one originating in the laboratory lines and two originating from Sarawak, Malaysian Borneo.
Conclusion: This is the first study to report on the polymorphism and natural selection of pk41 genes from clinical isolates of Malaysia. The results reveal that there is low level of polymorphism in both s48/45 domains, indicating that this antigen could be a potential vaccine target. However, genetic and molecular immunology studies involving higher number of samples from various parts of Malaysia would be necessary to validate this antigen's candidacy as a vaccine target for P. knowlesi.
Fulltext A Case Study on the Mortality of Cobia (Rachycentron canadum) Cultured in Traditional Cages

Chu KB, Abdulah A, Abdullah SZ, Bakar RA

Trop Life Sci Res, 2013 Dec;24(2):77-84.
PMID: 24575250 MyJurnal

The mass mortality of cobia (Rachycentron canadum) within 2-3 days was reported by 3 private farms in Bukit Tambun, Pulau Pinang, in February and March 2007. Only cobia with body weights of 3-4 kg were affected. Most diseased cobia swam on the surface and displayed flashing behaviour. All samples were positive for viral nervous necrosis (VNN) with low to medium levels of infection. Infestations by leeches (Zeylanicobdella arugamensis), body monogeneans (Benedenia sp.) and copepods (Caligus sp.) were also found, but no pathogenic bacteria were isolated. All water quality parameters monitored were within optimal ranges for culturing cobia. The main causes of high mortality in cobia remain unclear during the study. However, we believe that the mass mortality of cobia could be probably due to VNN infection and that the rate of mortality will increase further when cobia are subjected to aquaculture-related stresses (e.g., limited space). Traditional cages with a size of 2 (length) × 2 (width) × 1 m (depth) should only be used for rearing cobia below 1 kg in weight given the species' natural behaviours. In addition, cobia fingerlings should be screened for VNN prior to stocking them in cages.
Fulltext Within-population genetic diversity and population structure of Plasmodium knowlesi merozoite surface protein 1 gene from geographically distinct regions of Malaysia and Thailand

Ahmed MA, Chu KB, Vythilingam I, Quan FS

Malar J, 2018 Nov 29;17(1):442.
PMID: 30497496 DOI: 10.1186/s12936-018-2583-z

BACKGROUND: The C-terminal 42 kDa domain of Plasmodium knowlesi merozoite surface protein 1 (PkMSP1) is a potential asexual blood-stage vaccine candidate, however, only a limited number of clinical isolates have been analysed from Malaysia and no inter-country comparative diversity study has been conducted. In the present study, nucleotide diversity, haplotypes and natural selection levels of pkmsp1 in clinical samples from geographically distinct regions of Malaysia and Thailand were investigated. The overall population structure of the parasite from the region was determined.
METHODS: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5.
RESULTS: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain.
CONCLUSIONS: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.
Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian Macrobrachium rosenbergii de Man

Saedi TA, Moeini H, Tan WS, Yusoff K, Daud HM, Chu KB, et al.

Mol Biol Rep, 2012 May;39(5):5785-90.
PMID: 22223294 DOI: 10.1007/s11033-011-1389-7

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.