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Genome size variation among recent human isolates of Salmonella typhi

Thong KL, Puthucheary SD, Pang T

Res. Microbiol., 1997 Mar-Apr;148(3):229-35.
PMID: 9765803

We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.

Matched MeSH terms: Base Sequence
Nucleotide sequence and phylogenetic analysis of Newcastle disease virus isolates from recent outbreaks in Taiwan

Yang CY, Chang PC, Hwang JM, Shieh HK

Avian Dis, 1997 Apr-Jun;41(2):365-73.
PMID: 9201401

Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.

Matched MeSH terms: Base Sequence
Plasmodium inui is not closely related to other quartan Plasmodium species

Kissinger JC, Collins WE, Li J, McCutchan TF

J Parasitol, 1998 Apr;84(2):278-82.
PMID: 9576499

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.

Matched MeSH terms: Base Sequence
Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences

Romstad A, Gasser RB, Nansen P, Polderman AM, Chilton NB

Int J Parasitol, 1998 Apr;28(4):611-5.
PMID: 9602384

The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.

Matched MeSH terms: Base Sequence
Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs

Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH

Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
PMID: 9565688

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Matched MeSH terms: Base Sequence
Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18

Barloy F, Lecadet MM, Delécluse A

Gene, 1998 May 12;211(2):293-9.
PMID: 9602158

Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.

Matched MeSH terms: Base Sequence
Molecular analysis of Dichelobacter nodosus isolated from footrot in sheep in Malaysia

Zakaria Z, Radu S, Sheikh-Omar AR, Mutalib AR, Joseph PG, Rusul G

Vet Microbiol, 1998 Jul;62(3):243-50.
PMID: 9791871

Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5'GGGCCC3'), SfiI (5'GGCCNNNNNGGCC3') and SmaI ('5CCCGGG3') enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.

Matched MeSH terms: Base Sequence
Detection of mutations and polymorphism of N-acetyltransferase 1 gene in Indian, Malay and Chinese populations

Zhao B, Lee EJ, Yeoh PN, Gong NH

Pharmacogenetics, 1998 Aug;8(4):299-304.
PMID: 9731716

The xenobiotic metabolizing enzymes N-acetyltransferases (NATs) are important for the biotransformation and/or bioactivation of drugs and carcinogens. NATs are coded for in humans by two distinct genes, designated NAT1 and NAT2. NAT1, which was originally thought to be monomorphic, was recently reported to exhibit variation in human populations. Recent studies suggested that a genetic polymorphism of NAT1 may be associated with colorectal cancer risk. The distributions of NAT1 allele and genotype frequencies in unrelated individuals among Indian (n = 140), Malay (n = 122) and Chinese (n = 181) populations in Singapore were characterized by polymerase chain reaction-restriction fragment length polymorphism and allele-specific-polymerase chain reaction. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Indians were 0.3, 0.51, 0.17 and 0.02, respectively. The corresponding NAT1 allelic frequencies in Malays were 0.29, 0.30, 0.39 and 0.02, respectively, and were similar to those in Chinese in the region. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Chinese were 0.33, 0.35, 0.30 and 0.02, respectively. These findings are of importance for the determination of cancer risk in these populations. In addition, nucleotide changes at positions 350-351 (GG to CC) and 497-499 (GGG to CCC) of the NAT1 gene were not found in the alleles of the populations studied.

Matched MeSH terms: Base Sequence
Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia

Zhu XQ, Jacobs DE, Chilton NB, Sani RA, Cheng NA, Gasser RB

Parasitology, 1998 Aug;117 ( Pt 2):155-64.
PMID: 9778638

The ascaridoid nematode of cats from Kuala Lumpur, Malaysia, previously identified morphologically as Toxocara canis, was characterized using a molecular approach. The nuclear ribosomal DNA (rDNA) region spanning the first internal transcribed spacer (ITS-1), the 5.8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. The sequences for the parasite from Malaysian cats were compared with those for T. canis and T. cati. The sequence data showed that this taxon was genetically more similar to T. cati than to T. canis in the ITS-1, 5.8S and ITS-2. Differences in the ITS-1 and ITS-2 sequences between the taxa (9.4-26.1%) were markedly higher than variation between samples within T. canis and T. cati (0-2.9%). The sequence data demonstrate that the parasite from Malaysian cats is neither T. canis nor T. cati and indicate that it is a distinct species. Based on these data, PCR-linked restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) methods were employed for the unequivocal differentiation of the Toxocara variant from T. canis and T. cati. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and zoonotic potential of this parasite.

Matched MeSH terms: Base Sequence
Outbreak of fatal childhood viral infection in Sarawak, Malaysia in 1997: inocula of patients' clinical specimens induce apoptosis in vitro

Abubakar S, Shafee N, Chee HY

Malays J Pathol, 1998 Dec;20(2):71-81.
PMID: 10879266

Identification of the aetiologic agent(s) associated with an outbreak of fatal childhood viral infection in Sarawak, Malaysia, in mid 1997 remains elusive. It is reported here that African green monkey kidney (Vero) and human monocytic (U937) cells treated with inocula derived from clinical specimens of some of these fatal cases showed the presence of cellular genomic DNA degradation when the extracted DNA was separated by pulsed field gel electrophoresis (PFGE), oligonucleosomal DNA ladders characteristic of apoptotic cells when the infected cells' DNA was separated by agarose gel electrophoresis, and apoptotic cellular DNA fragmentation when cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). These results suggest that inocula derived from the patients' clinical specimens contain factors which stimulate apoptotic cellular responses in vitro.

Matched MeSH terms: Base Sequence
Molecular epidemiology of Malaysian dengue 2 viruses isolated over twenty-five years (1968-1993)

Fong MY, Koh CL, Lam SK

Res. Virol., 1998 Nov-Dec;149(6):457-64.
PMID: 9923022

The limited sequencing approach was used to study the molecular epidemiology of 24 Malaysian dengue 2 viruses which were isolated between 1968 and 1993. The sequences of a 240-nucleotide-long region across the envelope/non-structural 1 protein (E/NS1) gene junction of the isolates were determined and analysed. Alignment and comparison of the nucleotide and deduced amino acid sequences of the isolates revealed that nucleotide changes occurred mostly at the third position of a particular codon and were of the transition (AG, CU) type. Five nucleotide changes resulted in amino acid substitutions. Pairwise comparisons of the nucleotide sequences gave divergence values ranging from 0 to 9.2%. At the amino acid level, the divergence ranged between 0 and 3.8%. Based on the 6% divergence as the cut-off point for genotypic classification, the isolates were grouped into two genotypes, I and II. Comparison of the nucleotide sequences of the Malaysian dengue isolates with those of the dengue viruses of other regions of the world revealed that members of genotypes I and II were closely related to viruses from the Indian Ocean and Western Pacific regions, respectively.

Matched MeSH terms: Base Sequence
Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein

Marmey P, Bothner B, Jacquot E, de Kochko A, Ong CA, Yot P, et al.

Virology, 1999 Jan 20;253(2):319-26.
PMID: 9918890

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.

Matched MeSH terms: Base Sequence
Characterization of a novel HLA-A2 variant, A*02012, in the Southern Thai-Muslim population

Scheltinga SA, van der Zwan AW, Mongkolsuk T, Sujirachato K, Chiewsilp P, Tilanus MG

Tissue Antigens, 1999 May;53(5):507-9.
PMID: 10372546

Southern Thai Muslims (STM)--from Nakon Si Thammarat, whose ancestors come mainly from Malaysia--constitute more than half of all Thai Muslims which, in total, represent approximately 10% of the country's population. The most common A2 subtypes in STM were A*0203 (n=15), A*0201 (n=8) and A*0207 (n=7). In this study, samples with unexpected amplification patterns were sequenced. Three individuals were indicative of a novel A2 allele, now known as A*02012.

Matched MeSH terms: Base Sequence
Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia

AbuBakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK

Virus Res, 1999 May;61(1):1-9.
PMID: 10426204

Thirteen enterovirus 71 (EV71) isolates were obtained from both fatal and non-fatal infections of patients seen in Peninsula Malaysia and in Sarawak during an outbreak of hand, foot and mouth disease (HFMD) in Malaysia in 1997, with incidences of fatal brainstem encephalomyelitis. The isolates were identified using immunofluorescence staining, neutralization assays, and partial sequencing of the 5' untranslated regions (UTR). Assessment of the potential genetic relationships of the isolates using the partial 5'UTR sequences suggested clustering of the isolates into at least two main clusters. Isolates from Peninsula Malaysia were found in both clusters whereas Sarawak-derived isolates clustered only in cluster II. Isolates derived from fatal infections, however, occurred in both clusters and no distinctive nucleotide sequences could be attributed to the fatal isolates. Examination of the nucleotide sequences revealed at least 13 nucleotide positions in all the isolates which differ completely from the previously reported EV71 5'UTR sequences. In addition, at least 11 nucleotide position differences within the 5'UTR were noted which differentiated cluster I from cluster II. Predicted secondary RNA structures drawn using the nucleotide sequences also suggested differences between isolates from the two clusters. These findings suggest the presence of at least two potentially virulent EV71 co-circulating in Malaysia during the 1997 HFMD outbreak.

Matched MeSH terms: Base Sequence
Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994

Kobayashi N, Thayan R, Sugimoto C, Oda K, Saat Z, Vijayamalar B, et al.

Am J Trop Med Hyg, 1999 Jun;60(6):904-9.
PMID: 10403318

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Matched MeSH terms: Base Sequence
Effects of intragenic variability at 3 polymorphic sites of the apolipoprotein B gene on serum lipids and lipoproteins in a multiethnic Asian population

Choong ML, Sethi SK, Koay ES

Hum Biol, 1999 Jun;71(3):381-97.
PMID: 10380374

We determined the allelic (X+/X-, M+/M-, and E+/E-) distribution frequencies of the XbaI, MspI, and EcoRI restriction fragment length polymorphisms (RFLPs) in the apolipoprotein B gene in a control group of 374 healthy Chinese, Malays, and Indians and in a hyperlipidemic cohort of 131 Chinese patients. Covariability between the RFLPs and serum lipid, lipoprotein, and apolipoprotein concentrations was also studied. We found a lower frequency (average 0.0829) of the X+ allele and higher frequencies of the E+ (average 0.9452) and M+ (average 0.9772) alleles in our study population compared with frequencies reported in other populations. The 3 polymorphic sites did not contribute to significant variations in lipid levels (p > 0.1 in all cases). Also, there was no significant variation in genotype frequencies between the control subjects and the hyperlipidemic subjects. Despite their relative close proximity within the APOB gene sequence, the 3 polymorphic sites did not show any significant linkage disequilibrium. However, the presence of the X+ cutting site was in linkage disequilibrium with the Del allele of the 5' insertion-deletion polymorphism and the E-allele was in linkage disequilibrium with the 3' VNTR located near the 3' end of the coding region of the APOB gene.

Matched MeSH terms: Base Sequence
Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences

Chansiri K, Kawazu S, Kamio T, Terada Y, Fujisaki K, Philippe H, et al.

Vet Parasitol, 1999 Jun 15;83(2):99-105.
PMID: 10392966

Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.

Matched MeSH terms: Base Sequence
Sequence changes in six variants of rice tungro bacilliform virus and their phylogenetic relationships

Cabauatan PQ, Melcher U, Ishikawa K, Omura T, Hibino H, Koganezawa H, et al.

J Gen Virol, 1999 Aug;80 ( Pt 8):2229-37.
PMID: 10466823

The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.

Matched MeSH terms: Base Sequence
Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida

Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al.

J Allergy Clin Immunol, 1999 Nov;104(5):1084-92.
PMID: 10550757

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.
OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

Matched MeSH terms: Base Sequence
Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998

Brown BA, Oberste MS, Alexander JP, Kennett ML, Pallansch MA

J Virol, 1999 Dec;73(12):9969-75.
PMID: 10559310

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.

Matched MeSH terms: Base Sequence

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