The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
Two hundred and forty nucleotides from the pre-M gene region of 10 Japanese encephalitis (JE) virus strains isolated in Malaysia in 1992 were sequenced and compared with the other JE virus strains from different geographic areas in Asia. Our JE virus strains belong to the largest genotypic group that includes strains isolated in temperate regions such as Japan, China, and Taiwan. Our Malaysian JE virus strains differed in 32 nucleotides (13.3%) from WTP/70/22 strain isolated from Malaysia in 1970, which belonged to another distinct genotypic group.
Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.
Isolation of Japanese encephalitis virus (JEV) from mosquitoes in Sabak Bernam, Selangor, Malaysia, was attempted. An aliquot of homogenate from each pool of mosquitoes, 50 per tube, was inoculated into Aedes albopictus clone C6/36 cells for virus isolation. Each cell culture was tested for the presence of viral antigen by immunoperoxidase staining using an anti-JEV polyclonal antibody. Out of 4 Culex sitiens mosquito pools, 2 pools were positive for JEV by cell culture. Presence of JEV genome in the cell cultures for Cx. sitiens was confirmed by using reverse transcriptase-polymerase chain reaction and JEV-specific primers. This is the first report on the isolation of JEV from Cx. sitiens.
Affected members of 73 families with a variety of autosomal dominant late onset cerebellar ataxias (ADCAs) were investigated for the trinucleotide (CAG) repeat expansion which is found in pedigrees exhibiting linkage to the SCA1 locus on chromosome 6. Most of the families were too small for linkage analysis. The mutation was only found in ADCA type I, in 19 out of 38 such kindreds investigated (50%). It was slightly more common in Italian (59%) than British (50%) families, and was also found in Malaysian, Bangladeshi and Jamaican kindreds. Overall, ADCA type I patients with the expansion had a lower incidence of hyporeflexia and facial fasciculation than those without. The trinucleotide expansion was not found in eight families with ADCA and maculopathy or 24 kindreds with a pure type of ADCA, confirming that these syndromes are genetically distinct. It was also not detected in 12 patients with sporadic degenerative ataxias. DNA analysis for the SCA1 mutation is useful diagnostically in single patients or small families, and can be used for presymptomatic testing where appropriate.
Di-nucleotide microsatellites were isolated from a genomic library of a tropical tree species, Dryobalanops lanceolata, in Sarawak, for the purpose of using them as hypervariable genetic markers to study the pollen-mediated gene flow. Among 1600 recombinant clones, in total 20 clones gave positive signals when hybridized with oligonucleotides with the three different repeat motifs, GT, CA and CT. Estimations of abundance of (GT)n/(CA)n and (GA)n/(CT)n dinucleotide repeats in D. lanceolata genome revealed to be one in every 84 kb and 80 kb, respectively. Among six sequenced microsatellite loci, one was selected to synthesize PCR primers to amplify the microsatellite. PCR product size of the locus was variable among different individuals, which is attributed to the different number of di-nucleotide repeats. The same microsatellite genotype was detected in the trunk and canopy of a single large tree, indicating the utility of trunk tissue as the source of DNA for the population genetic study of tropical tree species, the canopy of which is usually difficult to approach.
The distributions of the D1S80 alleles and genotypes in the Chinese, Malays and Indians in Singapore were determined by amplified fragment length polymorphism (AMP-FLP) analysis. The distributions of the observed genotypes for the three races conformed to Hardy-Weinberg expectations. The system was applied to 19 families whose paternity had been established by restriction fragment length polymorphism (RFLP) analysis. In all cases, Mendelian inheritance of the alleles at the D1S80 locus could be demonstrated. D1S80 typing on DNA recovered by differential extraction of forensic specimens which included vaginal swabs, urethral swabs and seminal stains yielded consistent results.
Discriminatory typing methods are invaluable in the investigation of outbreaks of infectious diseases. Single primers were used to generate randomly amplified polymorphic DNA (RAPD) profiles from Klebsiella pneumoniae isolates of various serotype and K. pneumoniae isolates from cases of sepsis at a Malaysian hospital and two English hospitals. RAPD profiles of acceptable reproducibility, a maximum of three minor band variations, were produced using a rapid DNA extraction method. RAPD typing of K. pneumoniae was shown to be as discriminatory as restriction fragment length polymorphism analysis using pulsed field gel electrophoresis yet quicker and less costly. The findings suggest that RAPD typing may be a useful tool for the epidemiological typing of K. pneumoniae.
Three species of Shorea (S. leprosula, S. acuminata and S. cursitii) were collected from a natural forest reserve of Malaysia and analyzed for genetic variation using the technique of random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). The average number of nucleotide substitutions was estimated. The nucleotide diversities within species were very similar and larger than those found in Drosophila melanogaster. The nucleotide divergences between these species are about 1.5 times the nucleotide diversities within the species, indicating that these species diverged from a common ancestor relatively recently.
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.
In this study, we have sequenced the C-terminal part of the Epstein-Barr virus (EBV)-BNLF-1 gene encoding for the latent membrane protein-1 from tissues of EBV-positive Danish Hodgkin's disease (HD) and of Danish and Malaysian peripheral T-cell lymphomas (PTLs) and from tonsils of Danish infectious mononucleosis (IM). Our study showed that some of the 7 single-base mutations and the 30-bp deletion previously detected between codons of amino acid 322 and 366 in the BNLF-1 gene of the nasopharyngeal carcinoma cell line CAO were present in all Malaysian PTLs and in 60% of the Danish PTLs. In HD and the IM cases, the mutations were present in about 30%. The 30-bp deletion and the single base mutations occurred independently, and mutations were detectable in the majority of EBV type B-positive cases. These findings suggest that the 30-bp deletion and the 7 single-base mutations in the C-terminal part of the CAO-BNLF-1 gene do not characterize a new EBV type A substrain. Rather, some of the positions of single base mutations and the 30-bp deletion are hot spots that may have mutated independently through the evolution of EBV strains.
By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.
Members of the Semai group of Orang Asli ('aborigines') in peninsular Malaysia were examined for apolipoprotein E (apo E) variants in relation to plasma total cholesterol (TC), high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDLC), triglycerides (TG), apolipoprotein AI and apolipoprotein B (apo B). The e2 and e4 alleles were found to be higher than in most other groups as reported. The sample as a whole was normotriglyceridaemic (mean plasma TG, 1.5 mmol/l) and very markedly hypocholesterolaemic (mean plasma TC 1.7 mmol/l). The distribution of apo E variants was not related to any of the plasma lipids or apolipoprotein fractions using results from all subjects, but if a distinctly hypertriglyceridaemic sub-section was omitted (TG > 1.7 mmol/l) then apo E variants were determinants of plasma TC, LDLC, and apo B concentrations, the lower values of these being associated with the 2-2 and 2-3 genotypes, and the higher with 3-4, and 4-4.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
Specific human papillomavirus (HPV) types have been implicated in the development of cervical carcinoma worldwide. Novel molecular techniques have facilitated the detection and typing of HPV in cervical lesions. DNA preparations from a series of 23 histopathologically confirmed cervical carcinoma patients were analyzed by polymerase chain reaction (PCR) using degenerate primers for the presence of HPV DNA sequences. A total of 22 of 23 cases studied (95.7%) were found positive for HPV DNA sequences. Further studies by DNA hybridization with viral specific probe and restriction enzyme analysis demonstrated the presence of HPV 16 in 73.9% (17/23) and HPV 18 in 65.2% (15/23) of the cases examined. Interestingly, the uncommon HPV 31 and 33 were also found but with a lower percentage (16.9%). It was noted that HPV 16 frequency in the carcinoma increased with age but HPV 18 was evenly present at all ages investigated. We found that HPV was frequently associated with the majority of the cervical carcinomas, and in all but one case, oncogenic high risk HPV genotypes were present. We conclude that HPV infection of the genital tract has an important role in the development of the disease in Malaysia.
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.
Detection and isolation of Japanese encephalitis (JE) virus were attempted from female mosquitoes collected in Kampong Pasir Panjang, Sabak Bernam, Selangor, from May to November 1992. A total of 7,400 mosquitoes consisting of 12 species in 148 pools were processed and inoculated into Aedes albopictus clone C6/36 cell cultures. Of these, 26 pools showed the presence of viral antigens in the infected C6/36 cells by specific immunoperoxidase staining using an anti-JE virus polyclonal antibody. Presence of JE virus genome was confirmed in the infected culture fluid for 16 pools by using reverse transcriptase-polymerase chain reaction and JE virus-specific primers. Of these, 3 pools were from Culex tritaeniorhynchus, 4 from Culex vishnui, 3 from Culex bitaeniorhynchus, 2 from Culex sitiens, one from Aedes species, and 3 from Culex species. Isolation of JE virus from Cx. sitiens, Cx. bitaeniorhynchus, and Aedes sp. (Aedes butleri and Ae. albopictus) is reported for the first time in Malaysia.
Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.
A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.