Prevalence of respiratory syncytial virus subgroups over six consecutive outbreaks: 1981-1987

Indirect immunofluorescence with strain-specific monoclonal antibodies was used to determine the phenotype of respiratory syncytial virus (RSV) isolates obtained from infants hospitalized in greater Boston over six successive outbreaks from 1981 to 1987. Of 981 isolates, 591 (60%) were classified as subgroup A and 383 (39%) as subgroup B. The prevalence of subgroups varied both between and within yearly outbreaks. In 1983-84 and 1984-85, both subgroups circulated concurrently and in almost equal proportions; in 1981-82, 1982-83, and 1985-86 subgroup A was dominant, accounting for 93% of all RSV isolates; and in 1986-87 subgroup B accounted for 89% of all RSV isolates. In some outbreaks both geographic and temporal clustering of subgroups occurred. No major differences in age, gender, or frequency of nosocomially acquired RSV between infants infected with either subgroup were seen, either overall or between or within yearly outbreaks. An expanded panel of monoclonal antibodies revealed further heterogeneity among subgroup A isolates. Comparison of these results with similar studies from other geographic locations indicated that the pattern of RSV subgroup prevalence is a localized phenomenon.     

Envelope sequence variation and phylogenetic relations of human T cell lymphotropic virus type 1 from endemic areas of Colombia

The HTLV-1 envelope gene of 12 TSP/HAM patients from two endemic areas of southwest Colombia (Tumaco and Buenaventura) was amplified by nested PCR, sequenced, and compared with previously reported HTLV-1 envelope sequences from isolates worldwide. In general, the sequence divergences among all Colombian samples ranged from 0.1 to 1.6%. Some amino acid substitutions, referring to the ATK-1 prototype strain in the surface domain gp46 and in p21, were highly prevalent in southwest Colombia, suggesting a geographical clustering of mutations in the envelope gene. The phylogenetic analysis showed that the Colombian isolates belong to the HTLV-1a lineage with minor subgroups. The genetic distance between Colombian and Japanese isolates ranged from 0.1 to 1.8%; in comparison, the genetic distance between Colombian and Caribbean isolates ranged from 0.4 to 2.2%. Our results strongly suggest that the actual quasispecies populations in southwest Colombia have been generated by separate, differently timed introductions of virus.     

Genetic diversity and molecular epidemiology of the G protein of subgroups A and B of respiratory syncytial viruses isolated over 9 consecutive epidemics in Korea

To study genetic variation and molecular epidemiology of the G protein of respiratory syncytial virus (RSV), 253 strains from a children's hospital in Korea over 9 consecutive epidemics were analyzed. Restriction analysis of the entire G protein gene demonstrated 24 genotypes among 188 subgroup A and 6 among 65 subgroup B isolates. Two to 4 dominant genotypes of subgroup A cocirculated, and different genotypes predominated in each epidemic. Predominant genotypes were replaced with new genotypes during consecutive epidemics. One of 2 dominant genotypes among subgroup B predominated alternately or concurrently. Phylogenetic analysis revealed that there were multiple lineages, with clustering related to their location and time of isolation among strains from Korea and worldwide. Geographic and temporal distinction have been shown more clearly for subgroup B than subgroup A. These results suggest that the G protein of RSV is continuously evolving, with a distinct pattern presumably due to immune selection in a localized region over time.     

Molecular epidemiology of the global and temporal diversity of Candida parapsilosis

We examined the global epidemiology of C. parapsilosis and assessed the discriminatory capabilities of restriction fragment length polymorphism (RFLP) and RAPD typing methods. We used EcoRI digestion of cellular DNA to generate RFLP; RAPD analysis on genomic DNA. Band profiles were used to distinguish and group isolates. From 7 diverse geographic areas, 536 isolates obtained over 35 y were placed into 23 RFLP subgroups. Subtype VII-1 was dominant worldwide (82.4% of isolates). Dividing the isolates into VII-1 versus non-VII-1 showed temporal variation for the USA pre-1995 versus post-1995 (p<0.0001) and versus Europe pre-1995 (p<0.0001). Genotype distribution differed among localities (p<0.0001); Mexico was unique (p<0.05) due to the high proportion of non-VII-1. The prevalence of C. parapsilosis RFLP type VII-1 apparently has risen in the USA and current isolates show some variation in distribution of types in some non-USA localities. There were no differences in distribution of types comparing babies versus adults, or bloodstream isolates versus colonizing or environmental isolates. RAPD typing showed 3 major profiles, but was less discriminatory.     

Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations

While Epstein-Barr virus (EBV) infection is associated with the development of certain lymphoid and epithelial tumors, the role of the virus in the pathogenesis of these malignancies remains unknown. It has been suggested that EBV strain variation may contribute to tumor development. Two major strains of EBV, type 1 and type 2, have been identified on the basis of genetic polymorphisms and other minor genetic variations give rise to distinct EBV isolates. We analyzed EBV strain variation in healthy individuals and compared them with EBV isolates present in lymphoid and epithelial neoplasms from the same geographic regions. In particular, the incidence of the 30-bp latent membrane protein (LMP1) gene deletion, recently implicated in the development of more aggressive forms of virus-positive lymphomas and Hodgkin's disease [HD], was examined in the normal population. While a preferential association of the LMP1 deletion with the type 2 strain of EBV was observed, there was no increased incidence of virus isolates carrying this deletion in HD, Burkitt's lymphoma, or virus-associated carcinomas compared with the appropriate normal population. A polymorphism in the BamHI F region of the EBV genome, previously identified in Chinese populations, was found at increased incidence in European HD biopsies. Overall, we found that most of the EBV gene polymorphisms detected in EBV isolates from healthy virus carriers occurred with similar frequency in virus-associated tumors from the same geographical region.     

Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products.

We have isolated retroviruses from six acquired immunodeficiency syndrome (AIDS) and three lymphadenopathy syndrome (LAS) patients by cocultivation of patients' lymphocytes with phytohemagglutinin-stimulated normal T cells. In an effort to address the extent to which these viruses have identical genetic information or there is divergence in their genomic sequences, we have compared the nine retrovirus isolates by the following criteria: (i) antigenic cross-reactivity by highly specific and sensitive competition radioimmunoassay for the major internal antigen; (ii) restriction site mapping analysis; and (iii) immunoblot analysis and metabolic labeling of viral structural proteins and their analysis by polyacrylamide gel electrophoresis. The data indicate that individual retroviruses have significant restriction site polymorphism in their genome even though they were isolated from patients residing at one geographic location. Furthermore, this polymorphism is reflected in the variation of the apparent size of the gag and env gene-encoded structural proteins. The heterogeneity in AIDS retrovirus-encoded proteins may be due to either substitutions in the primary amino acid sequence of the protein or deletions or additions in the coding regions of proteins. The alterations in viral structural proteins among various AIDS retroviruses could have important implications in antigenic properties and/or pathogenicity in development of the disease, its detection, and ultimately its eradication.     

Clinical and molecular characterization of vancomycin-resistant Enterococcus faecium strains during establishment of endemicity.

To characterize the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Tennessee, VREF isolates that were recovered from patients during a 3-year period at a tertiary care center and throughout the state were typed by means of pulsed-field gel electrophoresis. Clinical characteristics and outcomes of patients colonized or infected with different strain types were also examined. A total of 34 different strain types were identified. A single VREF strain (type O) predominated (63 [61%] of 103 single-patient isolates (i.e., 1 isolate per patient) obtained from 7 different health care institutions). There were no significant differences between patients harboring type O strains and those harboring non-type O strains (P> or =.05). The rate of recovery of type O subtypes and strains other than type O increased over the 3-year study period. Establishment of VREF endemicity was initially characterized by clonal spread of type O strains. Subsequently, polyclonal dissemination may have been due to microevolutionary changes among type O strains.     

Whole‐genome analysis of influenza A(H1N1)pdm09 viruses isolated in Uganda from 2009 to 2011

We report a whole‐genome analysis of 19 influenza A(H1N1)pdm09 isolates from four Ugandan hospitals between 2009 and 2011. The isolates differed from the vaccine strain A/California/07/2009 by three amino acid substitutions P100S, S220T, and I338V in the hemagglutinin and by two amino acid substitutions V106I and N248D in the neuraminidase proteins with consistent mutations in all gene segments distinguishing isolates from the 2009/2010 to 2010/2011 seasons. Phylogenetic analysis showed low genetic evolution, with genetic distances of 0%–1.3% and 0.1%–1.6% for HA and NA genes, respectively. The amino acid substitutions did not lead to antigenic differences from the reference strains.     

Monoclonal antibodies to human parainfluenza virus type 1 detect major antigenic changes in clinical isolates.

The extent of antigenic diversity within a population of human parainfluenza virus type 1 (HPIV-1) isolates collected over a 26-year period was investigated. Twenty-three monoclonal antibodies (MAbs) made to the hemagglutinin-neuraminidase protein (HN), fusion protein (F), phosphoprotein (P), and nucleoprotein (NP) of a 1957 type strain were compared in their ability to bind to the different clinical isolates in ELISA and hemagglutinin-inhibition (HI) assay. Four HN, one F, and two NP MAbs bound equally to all of the viruses tested, but six of the MAbs demonstrated significant antigenic heterogeneity. Most of these antigenic changes appeared stable over time and noncummulative. Four of the clinical isolates and the type virus had similar reactivity patterns (subtype A) to these MAbs, while the remaining 10 isolates may form a second group (subtype B). Children's sera demonstrated this same subtype specificity in HI assays. One neutralization site was present on the 1957 strain and not on any of the subsequent isolates. The possibility of two or more major subtypes of HPIV-1 should be considered in future epidemiologic, therapeutic, and vaccine-related work.     

Characterization and subgrouping of Campylobacter concisus strains using protein profiles, conventional biochemical testing and antibiotic susceptibility

OBJECTIVE: To characterize and subgroup clinical strains of Campylobacter concisus isolated from patients with gastrointestinal disease. METHODS: A total of 109 C. concisus isolates from 98 patients obtained between June 1997 and December 1998 were analysed using protein profiles, conventional biochemical tube tests, ApiCampy, and susceptibility patterns by Neosensitabs and E-test. RESULTS: Two groups were identified by using protein profiles. One resembled the ATCC 33237 type strain of oral origin, and a second group differing from it, particularly in the high molecular weight zone. Considerable diversity exists in the lower molecular range of the gels, also within assigned subgroups. Biochemical testing showed differences between the groups in the ability to reduce nitrate, ApiCampy testing also yielded differences between the two assigned groups, although reactions were highly heterogeneous. Resistance to erythromycin, ciprofloxacin, ampicillin, ceftriaxone and tetracycline occurred in 3%, 13%, 7%, 11% and 0% of the isolates when using Neosensitabs. The E-test yielded comparable results 7%, 5%, 0%, 2% and 3%, respectively. CONCLUSION: Results indicate that C. concisus can be assigned to two broad groups based on differences in protein profiles. No distinct phenotypic marker was identified. Susceptibility patterns are not suitable for discrimination between the two assigned groups. Further studies using a polyphasic approach including the application of genetic methods are needed to assess the complex taxonomy of this potential pathogen.     

Clinical characteristics of respiratory syncytial virus (RSV) subgroup infections in Japan.

The subgroup characteristics of 130 strains of respiratory syncytial virus (RSV) isolated in Sapporo during 9 epidemic years 1980-1989 were determined. Monoclonal antibodies raised against the RSV Long strains were used. Subgroup A included 77 (59.2%) isolates and subgroup B 52 (40.0%) strains, while 1 strain was considered to be a variant of a subgroup A strain. The distribution by age of infants and children was different for the 2 subgroups: less than 1 year of age infants with subgroup A infection dominated, greater than 1 year of age subgroup A infections were less common than subgroup B infections. These was no difference in type of illness between the subgroups. Bronchiolitis was the dominant diagnosis in all patients.     

A population genetic framework for the study of invasive diseases caused by serotype b strains of Haemophilus influenzae.

One hundred seventy-seven isolates of serotype b Haemophilus influenzae recovered largely from children with invasive disease in the United States were characterized by the electrophoretic mobilities of 16 metabolic enzymes, the NaDodSO4/PAGE pattern of outer-membrane proteins (OMP), and biotype. Thirty-two distinctive multilocus genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Twenty-eight OMP types and five biotypes were identified, but only 55 distinctive combinations of ET, OMP type, and biotype were represented. The strong nonrandom associations of characters and the recovery of isolates with identical properties in widely separated geographic regions and over a 40-year period suggest that the population structure of H. influenzae is basically clonal. Examination of nonserotype b isolates indicated that clones of serotype b are a restricted subset of the genotypes in the species as a whole. Currently, most of the invasive H. influenzae disease in the United States is caused by serotype b strains of two related ETs, and, more specifically, much of it is attributable to two subclones marked by OMP type. There is evidence that the frequency of the ET-1/OMP 1H/biotype I subclone has increased dramatically in the United States since the 1939-1954 period. The hypothesis that populations of H. influenzae are subject to marked temporal variation in clonal composition is supported by evidence of major differences in the genetic structure of populations in the United States and the Netherlands.     

Molecular characteristics of Neisseria meningitidis strains isolated in Burkina Faso in 2001

In the course of an epidemic of meningitis in Burkina Faso in 2001, 27 cerebrospinal fluid samples from patients in 7 districts were forwarded to Norway for isolation and characterization of the causative agents. Neisseria meningitidis was isolated from 13 (48%) samples. The isolates were analysed using serological and genetic methods. Of the 13 strains, 4 were serogroup A, serotype 21:P1.9, sequence type (ST)-5 and belonged to clonal subgroup III, while the remaining 9 strains were serogroup W135, serotype 2a:P1.5,2, ST-11 and belonged to the electrophoretic type-37 complex. PCR analyses revealed meningococcal DNA in 13/14 culture-negative samples. Sequence analysis of the PCR products demonstrated that at least 3 different meningococcal strains were responsible for these 13 cases. Our results show that the W135 strain associated with the 2000 hajj (Muslim pilgrimage) outbreak was a significant cause of disease in Burkina Faso in 2001. Further studies are warranted to determine whether W135 is about to replace serogroup A in sub-Saharan Africa.     

Respiratory syncytial virus in a community population: circulation of subgroups A and B since 1965

Respiratory syncytial viruses were isolated from residents of Tecumseh, MI, with illnesses of all severities during the periods 1965-1971 and 1976-1981. These isolates were grouped using one monoclonal antibody specific for each subgroup. All were identified as either subgroup A or B. Subgroup A predominated in most years. No differences in age distribution or illness characteristics could be found between the subgroups. This study demonstrated that the currently recognized subgroups have been present in a single community since 1965, and their behavior in the past is similar to that currently described.     

Helicobacter pylori genetic diversity within the gastric niche of a single human host

Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.     

Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.     

间日疟原虫云南分离株传播阻断疫苗候选抗原Pvs 28基因多态性分析

目的分析我国疟疾混合流行区云南分离株间日疟原虫（P．v）传播阻断疫苗候选抗原Pvs28基因特点。方法收集云南省血样17份；提取疟原虫基因组DNA；PCR扩增Pvs28基因；基因测序；DnaSP version4．0软件进行基因多态性分析。结果成功扩增Pvs28全长基因17个序列。与标准株Sal—I比较，检测出7个错义突变，7个基因型和7种氨基酸型。云南P.v分离株核苷酸多态性n值为0．0044。若不考虑重复片段拷贝数的差异，云南P．v分离株和湖北P．v分离株Pvs 28蛋白主导氨基酸型完全一致，即V^14-L^52-L^98-E^105-L^115-S^14-I^122。与泰国P．v分离株比较。我国主导基因型突变位点完全包含在泰国P．v分离株突变位点中。结论以Sal—I Pvs28为基础建立的传播阻断疫苗能够克服云南P．v分离株Pvs28抗原多样性而发挥传播阻断作用，提示间日疟原虫传播阻断疫苗在我国具有良好的应用前景。     

我国旋毛虫地理株遗传变异与系统发生关系的研究

目的通过分析旋毛虫线粒体基因分子标记,研究我国旋毛虫地理株间的遗传变异与系统发生关系。方法应用旋毛虫线粒体核糖体小亚基DNA（mitochondrial small subunit ribosomal DNA,mtSSU）和线粒体细胞色素氧化酶Ⅰ基因（mitochondria cytochrome coxidase,COΙ）为分子标记,分别对我国7个旋毛虫地理株扩增后进行双向测序,利用DNAMAN软件将测序结果和GenBank中相应序列进行比对研究,并使用MEGA4.0程序包构建系统进化树。结果 PCR对7个旋毛虫地理株分别扩增出了649bp（mtSSU）和419bp（COΙ）的DNA片段,7个地理株的DNA片段与GenBank中T.spiralis的相应片段相比存在一定差异,其中mtSSU基因有4个变异位点（248、293、537和539位）,而COI基因仅存在2个变异（273和382位）,所有变异均为T-C或C-T的转换;进化树显示南阳株和云南株位于比较近的分枝,而西安株、广西株及西藏株亲缘关系较近。结论我国7个旋毛虫地理株的遗传变异较小,亲缘关系较近。     

Multicenter study of strains of respiratory syncytial virus

Two major groups of respiratory syncytial virus (RSV) strains, A and B, have been identified and their patterns of isolation determined in different communities but not simultaneously in multiple communities. In this study, we tested 483 RSV isolates from 14 university laboratories in the United States and Canada for the 1984/1985 and 1985/1986 RSV seasons; 303 (63%) isolates were group A, 114 (24%) were group B, and 66 (14%) could not be grouped. Isolates were subdivided into six subgroups within group A and three within group B; up to six and often four or more different subgroups were isolated in the same laboratory during the same RSV season. The pattern of group and subgroup isolations varied among laboratories during the same year and between years for the same laboratory. These differences suggest that RSV outbreaks are community, possibly regional, but not national phenomena. The ability to identify group and subgroup differences in isolates is a powerful tool for epidemiologic studies of RSV.     

Behaviour of Crohn's disease according to the Vienna classification: changing pattern over the course of the disease.

BACKGROUND: Crohn's disease is a heterogeneous disorder with both a genetic and environmental aetiology. Clinical classifications of the disease, such as the newly proposed Vienna classification, may help to define subgroups of patients suitable for studying the influence of specific genetic or environmental factors. AIM: To assess the stability over the course of the disease of its location and behaviour, as determined according to the Vienna classification. PATIENTS AND METHODS: The notes of 297 Crohn's disease patients regularly followed up at our institution were carefully reviewed retrospectively. The behaviour and location of the disease according to the Vienna classification were determined at diagnosis and after 1, 3, 5, 10, 15, 20, and 25 years of follow up. The proportions of the different behaviours and locations of the disease were calculated at these time points. A statistical analysis of the evolution of these characteristics over 10 years was performed on a subgroup of 125 patients with at least 10 years of follow up. The influence of age at diagnosis on location and behaviour of the disease was assessed as well as the influence of location on the behaviour of the disease. RESULTS: The location of the disease remained relatively stable over the course of the disease. Although the proportion of patients who had a change in disease location became statistically significant after five years (p=0.01), over 10 years only 15.9% of patients had a change in location (p<0.001). We observed a more rapid and prominent change in disease behaviour, which was already statistically significant after one year (p=0.04). Over 10 years, 45.9% of patients had a change in disease behaviour (p<0.0001). The most prominent change was from non-stricturing non-penetrating disease to either stricturing (27.1%; p<0.0001) or penetrating (29.4%; p<0.0001) disease. Age at diagnosis had no influence on either location or behaviour of disease. Ileal Crohn's disease was more often stricturing, and colonic or ileocolonic Crohn's disease was more often penetrating: this was already the case at diagnosis and became more prominent after 10 years (p<0.05). CONCLUSIONS: Location of Crohn's disease, as defined by the Vienna classification, is a relatively stable phenotype which seems suitable for phenotype-genotype analyses. Behaviour of Crohn's disease according to the Vienna classification varies dramatically over the course of the disease and cannot be used in phenotype-genotype analyses. The potential influence of genes on the behaviour of Crohn's disease should be studied in subgroups of patients defined by their disease behaviour after a fixed duration of disease.