The Japanese encephalitis (JE) serocomplex of flaviviruses comprises 10 members, 9 of which: Alfuy (ALF); Koutango (KOU); Kokobera (KOK); Kunjin (KUN); Murray Valley encephalitis (MVE); JE; Stratford (STR); Usutu (USU); and West Nile (WN) have been isolated from Africa, southern Europe, Middle East, Asia, and Australia. The tenth member, St. Louis encephalitis (SLE) virus, is confined to North, Central, and South America. For ALF, KOK, KOU, STR, and USU, no sequence data have as yet been reported, and little molecular phylogeny has been determined for this complex as a whole. Using a rapid, one-step RT-PCR and universal primers, we have amplified and sequenced a 450-600 base pair region of the virus genome encompassing the N terminus of the nonstructural protein NS5 and the 5' end of the 3' noncoding region, for several strains of all of these viruses, except USU and SLE viruses. These data, as well as published sequence data for other flaviviruses, were analyzed with the ClustalW and Phylip computer packages. The resultant phylogenetic data were consistent with some of the current flavivirus serological classification, showing a close relationship between ALF and MVE viruses and between KOK and STR viruses, but suggested that KOK and STR are distantly related to the other viruses and should perhaps be reclassified in their own serocomplex. The data also confirmed the close relationship between KUN and WN viruses and showed that an isolate of KUN virus from Sarawak may represent a "link" between these two virus species. In addition, the primary sequence data revealed a polymorphic region just downstream of the stop codon in the 3' end of the viral genomes.
DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.
Over the past year, many reports have been published on a variety of clinical manifestations related to antiphospholipid antibodies. The low prevalence of anticardiolipin antibodies with the rare occurrence of thrombosis and a low rate of fetal loss in studies in Malaysia and China showed a potential role for local factors. A study of cross-reactive idiotype of the anticardiolipin antibody suggested that anticardiolipin antibodies are derived from a set of natural autoreactive clones. Regarding the pathogenic role of the antiphospholipid antibody, evidence has been presented that the epitopes formed between cardiolipin and beta 2 glycoprotein I are the targets of the antiphospholipid antibody. Complement activation, abnormalities of natural anticoagulants such as protein S deficiency, and genetic association with DR4, DR7, and DRw53 have also been studied.
The spectrum of beta-thalassemia mutations in Malaysia has been determined in 45 beta-thalassemia chromosomes using dot blot hybridization of the polymerase chain reaction amplified DNA and direct DNA sequencing. Eleven different molecular defects, including those previously detected in Chinese, Asian Indians, and American blacks, and a novel frameshift mutation causing beta zero-thalassemia were detected. Since this novel mutation, a T deletion in codon 15 creates a new restriction site for EcoRII enzyme; the mutation could be detected by EcoRII digestion of the appropriate amplified fragment. The results of the present study provide additional information on the molecular heterogeneity of beta-thalassemia in this population. We also demonstrated the nonradioactive detection method of the beta-thalassemia mutation based upon the digoxigenin-labeled oligonucleotide probes.
Forty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
The distribution in Thailand of antibody to a recently discovered variant of circumsporozoite proteins of Plasmodium vivax was determined by enzyme-linked immunosorbent assay (ELISA). The ELISA capture antigens were a synthetic peptide of the principal variant sequence ANGAGNQPG and a candidate P vivax vaccine that contained the predominant repeat sequence GDRAA/DGQPA. Serological evidence of recent inoculation with the variant was found throughout Thailand and in migrants from Cambodia, Malaysia, and Burma. IgG antibody to the two P vivax circumsporozoite proteins was detected in 217 of 804 test sera (27%). Within the regions studied the proportion of positive sera specific for the variant epitope ranged from 28% to 66%. A vaccine against the predominant repeat domain may rapidly select for the variant, which already appears to be widespread within Thailand.
Hendra virus (HeV) and Nipah virus (NiV) are members of a new genus, Henipavirus, in the family paramyxoviridae. Each virus encodes a phosphoprotein (P) that is significantly larger than its counterparts in other known paramyxoviruses. The interaction of this unusually large P with its nucleocapsid protein (N) was investigated in this study by using recombinant full-length and truncated proteins expressed in bacteria and a modified protein-blotting protein-overlay assay. Results from our group demonstrated that the N and P of both viruses were able to form not only homologous, but also heterologous, N-P complexes, i.e. HeV N was able to interact with NiV P and vice versa. Deletion analysis of the N and P revealed that there were at least two independent N-binding sites on P and they resided at the N and C termini, respectively. Similarly, more than one P-binding site was present on N and one of these was mapped to a 29 amino acid (aa) C-terminal region, which on its own was sufficient to interact with the extreme C-terminal 165 aa region of P.
The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-encephalitogenic dengue 3 virus strains isolated in Malaysia were determined and compared. Multiple sequence alignment revealed a high degree of similarity in the E protein of the strains suggesting that neurovirulence of these four encephalitogenic strains is not attributed to this protein.
Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.
Biogeographic connections between Australia and other continents are still poorly understood although the plate tectonics of the Indo-Pacific region is now well described. Eupetes macrocerus is an enigmatic taxon distributed in a small area on the Malay Peninsula and on Sumatra and Borneo. It has generally been associated with Ptilorrhoa in New Guinea on the other side of Wallace's Line, but a relationship with the West African Picathartes has also been suggested. Using three nuclear markers, we demonstrate that Eupetes is the sister taxon of the South African genus Chaetops, and their sister taxon in turn being Picathartes, with a divergence in the Eocene. Thus, this clade is distributed in remote corners of Africa and Asia, which makes the biogeographic history of these birds very intriguing. The most parsimonious explanation would be that they represent a relictual basal group in the Passerida clade established after a long-distance dispersal from the Australo-Papuan region to Africa. Many earlier taxonomic arrangements may have been based on assumptions about relationships with similar-looking forms in the same, or adjacent, biogeographic regions, and revisions with molecular data may uncover such cases of neglect of ancient relictual patterns reflecting past connections between the continents.
The taxonomic status of two actinomycetes isolated from the wall of a hypogean Roman catacomb was established based on a polyphasic investigation. The organisms were found to have chemical and morphological markers typical of members of the genus Amycolatopsis. They also shared a range of chemical, molecular and phenotypic markers which served to separate them from representatives of recognized Amycolatopsis species. The new isolates formed a branch in the Amycolatopsis 16S rRNA gene sequence tree with Amycolatopsis minnesotensis NRRL B-24435(T), but this association was not supported by a particularly high bootstrap value or by the product of the maximum-parsimony tree-making algorithm. The organisms were distinguished readily from closely related Amycolatopsis species based on a combination of phenotypic properties and from all Amycolatopsis strains by their characteristic menaquinone profiles, in which tetra-hydrogenated menaquinones with 11 isoprene units predominated. The combined genotypic and phenotypic data indicate that the isolates merit recognition as representing a novel species of the genus Amycolatopsis. The name proposed for this novel species is Amycolatopsis nigrescens sp. nov., with type strain CSC17Ta-90(T) (=HKI 0330(T)=DSM 44992(T)=NRRL B-24473(T)).
Plasmodium inui (Halberstaedter and von Prowazek, 1907), a malarial parasite of Old World monkeys that occurs in isolated pockets throughout the Celebes, Indonesia, Malaysia, and the Philippines, has traditionally been considered to be related more closely to Plasmodium malariae of humans (and its primate counterpart Plasmodium brasilianum), than to other primate Plasmodium species. This inference was made in part because of the similarities in the periodicities or duration of the asexual cycle in the blood, the extended sporogonic cycle, and the longer period of time for development of the pre-erythrocytic stages in the liver. Both P. inui and P. malariae have quartan (72 hr) periodicities associated with their asexual cycle, whereas other primate malarias, such as Plasmodium fragile and Plasmodium cynomolgi, are associated with tertian periodicities (48 hr), and Plasmodiumn knowlesi, with a quotidian (24 hr) periodicity. Phylogenetic analyses of portions of orthologous small subunit ribosomal genes reveal that P. inui is actually more closely related to the Plasmodium species of the "vivax-type" lineage than to P. malariae. Ribosomal sequence analysis of many different, geographically isolated, antigenically distinct P. inui isolates reveals that the isolates are nearly identical in sequence and thus members of the same species.
Tectonic movements, climatic oscillations, and marine transgressions during the Cenozoic have had a dramatic effect on the biota of the tropical rain forest. This study aims to reveal the phylogeography and evolutionary history of a Peninsular Malaysian endemic tropical timber species, Neobalanocarpus heimii (Dipterocarpaceae). A total of 32 natural populations of N. heimii, with 8 samples from each population were investigated. Fifteen haplotypes were identified from five noncoding chloroplast DNA (cpDNA) regions. Overall, two major genealogical cpDNA lineages of N. heimii were elucidated: a widespread southern and a northern region. The species is predicted to have survived in multiple refugia during climatic oscillations: the northwestern region (R1), the northeastern region (R2), and the southern region (R3). These putative glacial refugia exhibited higher levels of genetic diversity, population differentiation, and the presence of unique haplotypes. Recolonization of refugia R1 and R2 could have first expanded into the northern region and migrated both northeastwards and northwestwards. Meanwhile, recolonization of N. heimii throughout the southern region could have commenced from refugia R3 and migrated toward the northeast and northwest, respectively. The populations of Tersang, Pasir Raja, and Rotan Tunggal exhibited remarkably high haplotype diversity, which could have been the contact zones that have received an admixture of gene pools from the northerly and also southerly regions. As a whole, the populations of N. heimii derived from glacial refugia and contact zones should be considered in the conservation strategies in order to safeguard the long-term survival of the species.
The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169-183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the Caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100-102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incursions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.
In a previous study, we have reported the detection and isolation of dengue virus in Brunei (Osman, O., Fong, M.Y., Devi, S., 2007. A preliminary study of dengue infection in Brunei. JJID 60 (4), 205-208). DEN-2 was the predominant serotype followed by DEN-1. The full genomic sequences of 3 DEN-2 viruses isolated during the 2005-2006 dengue incident in Brunei were determined. Twenty-five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the viral genome. The amplified PCR products were sent for sequencing and their nucleotides and the deduced amino acids were determined. All three DEN-2 virus isolated were clustered in the Cosmopolitan genotype of the DEN-2 classification by Twiddy et al. This work constitutes the first complete genetic characterization of three Brunei DEN-2 virus strains.
Dyskeratosis congenita (DC) and related diseases are a heterogeneous group of disorders characterized by impaired telomere maintenance, known collectively as the telomeropathies. Disease-causing variants have been identified in 10 telomere-related genes including the reverse transcriptase (TERT) and the RNA component (TERC) of the telomerase complex. Variants in TERC and TERT can impede telomere elongation causing stem cells to enter premature replicative senescence and/or apoptosis as telomeres become critically short. This explains the major impact of the disease on highly proliferative tissues such as the bone marrow and skin. However, telomerase variants are not always fully penetrant and in some families disease-causing variants are seen in asymptomatic family members. As a result, determining the pathogenic status of newly identified variants in TERC or TERT can be quite challenging. Over a 3-year period, we have identified 26 telomerase variants (16 of which are novel) in 23 families. Additional investigations (including family segregation and functional studies) enabled these to be categorized into 3 groups: (1) disease-causing (n = 15), (2) uncertain status (n = 6), and (3) bystanders (n = 5). Remarkably, this process has also enabled us to identify families with novel mechanisms of inheriting human telomeropathies. These include triallelic mutations, involving 2 different telomerase genes, and an epigenetic-like inheritance of short telomeres in the absence of a telomerase mutation. This study therefore highlights that telomerase variants have highly variable functional and clinical manifestations and require thorough investigation to assess their pathogenic contribution.