Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.

Nucleotide sequence analysis of human T-cell lymphotropic virus type I pX and LTR regions from patients with sicca syndrome

Human T-cell lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). Other inflammatory disorders may occur in HTLV-I-infected patients, such as sicca syndrome resembling Sjogren's syndrome. The sicca syndrome may be the unique clinical manifestation of HTLV-I infection, but is associated frequently with TSP/HAM, which could suggest that sicca syndrome might be an early event in disease progression to TSP/HAM in some cases. We investigated whether peculiar pX and LTR mutations could be related to sicca syndrome, or might argue the existence of clinical progression to TSP/HAM. pX, especially pX(I), pX(II), and pX(IV) ORFs corresponding to Tax cytotoxic T-lymphocyte epitopes, and LTR regions from Caribbean patients who have sicca sydrome with or without TSP/HAM, ATL patients, and healthy carriers were sequenced. The sequences were aligned and compared with ATK-1 prototype and published sequences. LTR sequences exhibited 1.5-2.4 percent of divergence with ATK-1. pX-sequenced regions showed a lower homology within p12(I) encoding sequences. Only few mutations were found within functionally important regions, but were not associated specifically with the clinical status. Finally, no existence of clinical progression to TSP/HAM were found. It would be of interest to study the clinical evolution of HTLV-I-sicca syndrome in patients and to determine HTLV-I sequences from peripheral blood and salivary glands at different stages. Copyright 1999 Wiley-Liss, Inc.(Au)

Population structure of the primary malaria vector in South America, Anopheles darlingi, using isozyme, random amplified polymorphic DNA, internal transcribed spacer 2, and morphologic markers

A genetic and morphologic survey of Anopheles darlingi populations collected from seven countries in Central and South America was performed to clarify the taxonomic status of this major malaria vector species in the Americas. Population genetics was based on three techniques including isozyme, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), and internal transcribed spacer 2 (ITS2) markers. The results of the isozyme analysis indicated moderate differences in the allele frequencies of three putative loci (glutamate oxalaoacetate transaminase-1, isocitrate dehydrogenase-1, and phosphoglucomutase) of the 31 analyzed. No fixed electromorphic differences separated the populations of An. darlingi, which showed little genetic divergence (Nei distances = 0.976-0.995). Fragments produced by RAPD-PCR demonstrated evidence of geographic partitioning and showed that all populations were separated by small genetic distances as measured with the 1 - S distance matrix. The ITS2 sequences for all samples were identical except for four individuals from Belize that differed by a three-base deletion (CCC). The morphologic study demonstrated that the Euclidean distances ranged from 0.02 to 0.14, with the highest value observed between populations from Belize and Bolivia. Based on these analyses, all the An. darlingi populations examined demonstrated a genetic similarity that is consistent with the existence of a single species and suggest that gene flow is occurring throughout the species' geographic range.(Au)

Diversity and affinities among species and strains of Lipomyces

Phylogenetic relationships of the yeast genus Lipomyces were studied using sequences from fragments of 5.8S rRNA gene and from internal transcribed spacer region ITS2 of 13 strains (7 type strains included) representing five species and subtaxa, and originating from different geographical locations (Japan, Trinidad, Nigeria, North America, Western Europe, Russia, South Africa, Mauritius). Parsimony and distance analyses were performed. Tree topology from the parsimony and distance analyses of the sequence confirmed the results of nDNA reassociation. Results segregate the 13 isolates of Lipomyces into five major clades.(Au)

Angiotensinogen and blood pressure among blacks: findings from a community survey in Jamaica

OBJECTIVE: To examine the association between blood pressure, angiotensinogen levels, angiotensin converting enzyme activity and polymorphisms of the angiotensinogen and angiotensin converting enzyme genes in a population-based sample. METHOD: Five hundred participants were recruited in a house-to-house survey of three communities in metropolitan areas of Kingston and St. Andrew, in Jamaica. Demographic data, anthropometric and blood pressure measurements were obtained for each participant during a brief clinic visit. Circulating levels of angiotensinogen and angiotensin converting enzyme genes were determined. RESULTS: A weak association between angiotensinogen level, angiotensin converting enzyme activity and blood pressure was identified in this population, but substantial joint effect of angiotensin converting enzyme activity and agiotensinogen level on blood pressure was apparent. Variants of the angiotensinogen gene had inconsistent effects on blood pressure and on the risk of hypertension. Angiotensinogen level and angiotensin converting enzyme activity were significantly related to several measures of obesity, including body mass index, waist circumference and skin fold thickness. CONCLUSION: The angiotensinogen and angiotensin converting enzyme genetic variants which were studied appear to have only a modest relationship with blood pressure and associated anthropometric risk factors among blacks. (AU)

Identification of human T-cell leukemia/lymphoma virus type I antibodies, DNA, and protein in patients with polymyositis

OBJECTIVE: To investigate a possible association between human T cell leukemia/lymphoma virus type I (HTLV-I) and polymyositis (PM). METHODS: Sera and muscle biopsy samples from 9 Jamaican PM patients were compared with specimens from American HTLV-I positive PM patients and normal controls. Sera were evaluated for HTLV antibodies by enzyme-linked immunosorbent assay and Western blot. The biopsy samples were analyzed for HTLV-I/II DNA by polymerase chain reaction and were also immunohistochemically stained for HTLV gp46 envelope protein. RESULTS: Seven of the 8 Jamaican PM patients from whom sera were available were HTLV-I seropositive. The muscle biopsies of all 9 Jamaican patients demonstrated severe lymphocytic infiltration, cellular degeneration, myofiber atrophy, and fibrosis. Each muscle biopsy specimen contained HTLV-I DNA. Two of 6 samples demonstrated intense staining for HTLV-I gp46 in many of the invading mononuclear cells and weak staining for HTLV-I gp46 in many of the other specimens were weakly positive for gp46 in rare mononuclear cells. All controls specimens were negative for the presence of HTLV-I DNA and protein. CONClUSION: HTLV-I is associated with an inflammatory muscle disease characterized by direct invasion of the affected muscle by HTLV-I-infected mononuclear cells.(AU)

Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (OFRs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch. (AU)

HLA-DQA1 and DQB1 Alleles associated with genetic susceptibility to IDDM in a black population

Transracial analysis provides a method of distinguishing primary associations between insulin-dependent diabetes mellitus (IDDM) and HLA class II alleles from those secondary to linkage disequilibrium. Blacks show DR-DQ relationships that are different from other races and are a useful group in which to investigate HLA-D region associations with IDDM. In this study, the frequencies of HLA-DQA1 and -DQB1 alleles in Afro-Caribbean IDDM and control subjects were compared. Alleles were identified with sequence-specific oligonucleotide probing. The DQA1 allele A3 was positively associated with IDDM (relative risk[RR] = 25.3, corrected P [Pc]<7.0 x 10 -6). THe DQB1 alleles DQw2 and DQw8 were also positively associated (RR = 4.7, Pc<6.5 x 10 -3 and RR = 12.3,Pc = 3.4 x 10 -3, respectively). The A1.2 and DQw6 alleles were negatively associated (RR = 0.16, Pc<3.5 x 10 -3 and RR = 0.15, Pc = 2.4 x 10 -2, respectively). These findings were compared to data from other races. The positive associations with A3 and DQw2 are consistent with all racial groups investigated. The negative association with DQw6 is present in all racial groups in which it is a common allele. These findings suggest that DQ alleles, and hence DQ molecules, may directly affect predisposition to IDDM. (AU)

Screening for prolonged incubation of HTLV-I infection in British and Jamaican relatives of British patients with tropical spastic paraparesis [see comments]

OBJECTIVE -- To compare the prevalence of antibody to and proviral DNA of the retrovirus HTLV-I in relatives of 11 British patients with tropical spastic paraparesis who migrated from Jamaica before they developed symptoms, and to examine factors possibly related to transmission of HTLV-I. DESIGN -- Migrant family study. Antibody state was determined by several methods and confirmed by western blotting; the polymerase chain reaction was used to detect proviral DNA. SETTING -- Britain and Jamaica. SUBJECTS -- All available first degree relatives: those born and still resident in Jamaica (group 1); those born in Jamaica who migrated to Britian (group 2); and index patients' children who were born and resident in Britian (group 3). All had been breast fed and none had had blood transfusions. RESULTS -- Of the 66 living relatives, 60 were traced. Seroprevalence among those born in Jamaica (irrespective of current residence) was 22 percent (10/46; 95 percent confidence limits 9 to 34 percent) compared with zero among British born offspring (0/14) and was higher in group 2 at 33 percent (7/21; 12 to 55 percent) than in group 1 at greatest mean age.) Proviral DNA was not detected in any subject negative for HTLV-I antibody, making prolonged viral incubation in those negative for the antibody unlikely. CONCLUSION -- In this sample factors related to place of birth and early residence were more important in transmission of HTLV-I than naternal or age effects. In areas with a low to moderate prevalence policies of preventing mothers who are carriers of the virus from breast feeding would be premature (AU)

A comparitive analysis of RNA coliphages: structural and regulatory features

Describes a comparative analysis of RNA coliphages with special emphasis on regulatory and structural features. Phylogenetic phage sequence comparison reveals kinship between different phages and facilitates deduction of their secondary structure. Complete sequences from phage fr and partial sequences from group I phages M12, R17 and f2 were determined. The nucleotide sequence from a serological intermediate JP34 was partially elucidated, Only the fr sequence proved suitable for comparison with MS2 (similarity 77 per cent). A "core" structure (3' terminal region) of phage RNA proposed a certain structural resemblance with tRNA. Comparison of the MS2 and fr nucleotide sequences revealed absence of an AUG initiator codon for the fr lysis (L) gene. However, 4 codons further downstream there is a UUG codon which proved to be the start codon for the fr lysis gene. This UUG start codon proved crucial for L-gene regulation. It is suggested that terminated but not released ribosomes mediate the L-gene activation. A general eubacterial scanning model is proposed. It seems that ribosomes can scan the mRNA in both directions and reinitiate at the first encountered restart site. This eubacterial scanning model parallels the eukaryotic translation initiation mechanism

Molecular cloning and complete nucleotide sequence of an adult T cell leukaemia virus/human T cell leukaemia virus type I (ATLV/HTLV-I) isolate of Caribbean origin:Relationship to other members of the ATLV/HTLV-I subgroup

We report the first complete nucleotide sequence of an adult T cell leukaemia virus/human T cell leukaemia virus type I (ATLV/HTLV) isolate from a British patient of Caribbean origin. Sequence comparisons of our proviral clone (HS-35) with other molecular clones are shown. We note the strong sequence conservation between isolates of Caribbean and Japanese origin (2.3 percent divergence), but demonstrate the higher homologies existing between isolates originating from similar geographical areas (approximately 1 percent divergence). Implications for the origin, evolution and dissemination of the ATLV/HTLV-I subgroup are discussed. Analysis of defective proviral clones isolated from the same genomic library is also reported,and suggests a pattern of proviral sequence deletions during the biogenesis of defective proviruses. (AU)

Genetic and epidemiological studies of dengue type 2 viruses by hybridization using synthetic deoxyoligonucleotides as probes

A rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to common and unique RNase T1 oligonecleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type- and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the labourious T1 oligonucleotide fingerprinting.(AU)

G gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the G gamma gene

Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which the normal shutoff of fetal hemoglobin (Hb F) production fails to occur. In the G gamma beta+ type of HPFH, erythrocytes of adult heterozygotes contain approximately equal to 20 percent Hb F, which is almost exclusively of the G gamma-globin variety, without increased levels of gamma-globin chains from the nearby A gamma-globin gene. Unlike some forms of HPFH, no major deletions in the globin gene cluster have been found by genomic blotting in the G gamma beta+ variety. We report here a family with this condition, from which cosmid clones of the beta-globin gene cluster from the G gamma beta+ HPFH allele have been obtained. Sequencing around the fetal genes has identified a point mutation 202 base pairs 5' to the G gamma-globin gene that is present in genomic DNA of 3/3 unrelated individuals with G gamma beta+ HPFH but in none of more than 100 non-HPFH individuals. Although the mutation could represent a tightly linked polymorphism, its location in a region suggested by recent data to be important in tissue-specific control of gene expression suggests the possibility that the -202 mutation accounts for the phenotype. The sequence created resembles elements of other eukaryotic promoters known to be important for efficient transcription. (AU)