Jessada Denduangboripant

Transmission dynamics of rabies virus in Thailand: Implications for disease control

BackgroundIn Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand.MethodsWe analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information.ResultsAll sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus.ConclusionInter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential.

Sequence Analysis of Rabies Virus in Humans Exhibiting Encephalitic or Paralytic Rabies

Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.

Phylogenetic analysis of Pythium insidiosum Thai strains using cytochrome oxidase II (COX II) DNA coding sequences and internal transcribed spacer regions (ITS)

To investigate the phylogenetic relationship among Pythium insidiosum isolates in Thailand, we investigated the genomic DNA of 31 P. insidiosum strains isolated from humans and environmental sources from Thailand, and two from North and Central America. We used PCR to amplify the partial COX II DNA coding sequences and the ITS regions of these isolates. The nucleotide sequences of both amplicons were analyzed by the Bioedit program. Phylogenetic analysis using genetic distance method with Neighbor Joining (NJ) approach was performed using the MEGA4 software. Additional sequences of three other Pythium species, Phytophthora sojae and Lagenidium giganteum were employed as outgroups. The sizes of the COX II amplicons varied from 558-564 bp, whereas the ITS products varied from approximately 871-898 bp. Corrected sequence divergences with Kimura 2-parameter model calculated for the COX II and the ITS DNA sequences ranged between 0.0000-0.0608 and 0.0000-0.2832, respectively. Phylogenetic analysis using both the COX II and the ITS DNA sequences showed similar trees, where we found three sister groups (A(TH), B(TH), and C(TH)) among P. insidiosum strains. All Thai isolates from clinical cases and environmental sources were placed in two separated sister groups (B(TH) and C(TH)), whereas the Americas isolates were grouped into A(TH.) Although the phylogenetic tree based on both regions showed similar distribution, the COX II phylogenetic tree showed higher resolution than the one using the ITS sequences. Our study indicates that COX II gene is the better of the two alternatives to study the phylogenetic relationships among P. insidiosum strains.

Molecular phylogeny of banana cultivars from Thailand based on HAT-RAPD markers

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the wild progenitors of the cultivated banana, they are highly variable in Thailand. The genetic system is relatively unknown and complicated due to interspecific hybridization, heterozygosity and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, especially when sterile. The high annealing temperature-random amplified polymorphic DNA (RAPD) technique was used to estimate the genetic relationship between 22 selected banana cultivars, utilizing 14 random primers. Phylogenetic relationship was determined by unweighted pair group method with arithmetical averages cluster analysis. The dendrogram constructed from the similarity data showed that all the 22 cultivars analysed were closely related with a narrow genetic base. There were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram grouped all the AA, BB, AAA, AAB and ABB genomes into a major cluster. Several subgroups are recognized within the major clade. As expected, Ensete glauca Roxb. (Musaceae) and Strelitzia reginae Banks (Strelitziaceae) were clearly differentiated from the analysed edible bananas. Our study showed that RAPD markers are sufficiently abundant to classify and readily dissect genetic differences between the closely related Musa germplasm and provide a basis for the selection of parents for improvement of this germplasm.

Correlation of camptothecin-producing ability and phylogenetic relationship in the genus Ophiorrhiza.

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.

Rabies virus strains circulating in Bhutan: implications for control.

We report a molecular epidemiological study of rabies virus (RABV) strains circulating in animal populations in Bhutan, and investigate potential origins of these viruses. Twenty-three RABV isolates originating from dogs and other domestic animals were characterized by sequencing the partial nucleoprotein (N) gene (395 bp). Phylogenetic analysis was conducted and the Bhutanese isolates were compared with rabies viruses originating from other parts of the world. Phylogenetic analysis showed that Bhutanese isolates were highly similar and were closely related to Indian strains and South Asian Arctic-like-1 viruses. Our study suggests that the rabies viruses spreading in southern parts of Bhutan have originated from a common ancestor, perhaps from the Indian virus strain.

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn(2+) and Co(2+) resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595(T)=BCC 40155(T)).

DNA barcoding of Aristolochia plants and development of species-specific multiplex PCR to aid HPTLC in ascertainment of Aristolochia herbal materials

The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai “Krai-Krue”. “Krai-Krue” herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination.