Mutations and CpG islands among hepatitis B virus genotypes in Europe

Background

Hepatitis B virus (HBV) genotypes have a distinct geographical distribution and influence disease progression and treatment outcomes. The purpose of this study was to investigate the distribution of HBV genotypes in Europe, the impact of mutation of different genotypes on HBV gene abnormalities, the features of CpG islands in each genotype and their potential role in epigenetic regulation.

Results

Of 383 HBV isolates from European patients, HBV genotypes A-G were identified, with the most frequent being genotype D (51.96%) in 12 countries, followed by A (39.16%) in 7 countries, and then E (3.66%), G (2.87%), B (1.57%), F (0.52%) and C (0.26%). A higher rate of mutant isolates were identified in those with genotype D (46.7%) followed by G (45.5%), and mutations were associated with structural and functional abnormalities of HBV genes. Conventional CpG island I was observed in genotypes A, B, C, D and E. Conventional islands II and III were detected in all A-G genotypes. A novel CpG island IV was found in genotypes A, D and E, and island V was only observed in genotype F. The A-G genotypes lacked the novel CpG island VI. “Split” CpG island I in genotypes D and E and “split” island II in genotypes A, D, E, F and G were observed. Two mutant isolates from genotype D and one from E were found to lack both CpG islands I and III.

Conclusions

HBV genotypes A-G were identified in European patients. Structural and functional abnormalities of HBV genes were caused by mutations leading to the association of genotypes D and G with increased severity of liver disease. The distribution, length and genetic traits of CpG islands were different between genotypes and their biological and clinical significances warrant further study, which will help us better understand the potential role of CpG islands in epigenetic regulation of the HBV genome.     

一株乙肝病毒的基因组克隆及系统学分析

乙型肝炎病毒HBV的基因分型在基础研究和临床治疗中具有重要意义。PCR扩增并克隆了来自蒙古的乙肝病毒HBV199的全长基因组。分析表明HBV199基因组全长为3 145 bp,属于基因型D。HBV199与乙肝病毒基因型D代表菌株x02496的基因组序列差异为1.9%;与参比菌株AF280817和AY161157具有更高的亲缘关系,它们的基因组序列差异分别为1.1%和1.5%。不同于所有其他HBV基因组,HBV199基因组缺失了HBc-ORF处nt 2021~nt 2057之间的37个碱基。     

Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.     

A novel genotype of GB virus C: its identification and predominance among injecting drug users in Yunnan, China

GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5'UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5'UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a "non-pathogenic virus".     

Population genetic analysis of Enterocytozoon bieneusi in humans

Genotyping based on sequence analysis of the ribosomal internal transcribed spacer has revealed significant genetic diversity in Enterocytozoonbieneusi. Thus far, the population genetics of E. bieneusi and its significance in the epidemiology of microsporidiosis have not been examined. In this study, a multilocus sequence typing of E. bieneusi in AIDS patients in Lima, Peru was conducted, using 72 specimens previously genotyped as A, D, IV, EbpC, WL11, Peru7, Peru8, Peru10 and Peru11 at the internal transcribed spacer locus. Altogether, 39 multilocus genotypes were identified among the 72 specimens. The observation of strong intragenic linkage disequilibria and limited genetic recombination among markers were indicative of an overall clonal population structure of E. bieneusi. Measures of pair-wise intergenic linkage disequilibria and a standardised index of association (IAS) based on allelic profile data further supported this conclusion. Both sequence-based and allelic profile-based phylogenetic analyses showed the presence of two genetically isolated groups in the study population, one (group 1) containing isolates of the anthroponotic internal transcribed spacer genotype A, and the other (group 2) containing isolates of multiple internal transcribed spacer genotypes (mainly genotypes D and IV) with zoonotic potential. The measurement of linkage disequilibria and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. The formation of the two sub-populations was confirmed by STRUCTURE and Wright's fixation index (FST) analyses. The data highlight the power of MLST in understanding the epidemiology of E. bieneusi.     

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A-F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.     

Characteristics of hepatitis B virus isolates of genotype G and their phylogenetic differences from the other six genotypes (A through F)

Eight hepatitis B virus (HBV) isolates of genotype G were recovered from patients and sequenced over the entire genome. Six of them had a genomic length of 3,248 bp and two had genomic lengths of 3,239 bp (USG15) and 3,113 bp (USG18) due to deletions. The 10 HBV/G isolates, including the 8 sequenced isolates as well as the original isolate (AF160501) and another isolate (B1-89), had a close sequence homology of 99.3 to 99.8% among themselves (excluding USG18 with a long deletion) but of <88.7% to any of the 68 HBV isolates of the other six genotypes with the full-length sequence known. The eight HBV/G isolates possessed an insertion of 36 bp in the core gene and two stop codons in the precore region, as did the AF160501 and B1-89 isolates. The 10 HBV/G isolates clustered on a branch separate from those bearing the other six genotypes (A through F [A-F]) in the phylogenetic tree constructed from full-length sequences of 78 HBV isolates as well as in those constructed from the core, polymerase, X, and envelope genes. Despite two stop codons in the precore region that prohibited the translation of the HBV e antigen (HBeAg), all of the eight patients with HBV/G infection possessed the HBeAg in serum. By restriction fragment length polymorphism of the surface gene, all of the eight patients were found to be coinfected with HBV of genotype A (HBV/A), which would be responsible for the expression of HBeAg in them. It is worthy of examination to determine how coinfection occurs and whether HBV/G needs HBV/A for replication.     

Genetic variability of hepatitis B virus isolates among population of Shuryskarsky area of Yamal-Nenets autonomous region

The purpose of this work was to determine occurrence of serological markers of hepatites B and to describe subtypes of a superficial antigen and genotypes of hepatitis B virus (HBV) isolates among indigenous population of Yamal-Nenets Autonomous Region (YNAR), Russia. METHODS: We investigated 657 serum samples from inhabitants of Shuryskarsky area of YNAR. ELISA method was used to define the hepatitis B markers: HBsAg, anti-HBs (total) and anti-HBc (IgG and IgM). The HBsAg-positive samples were PCR-tested for the presence of HBV DNA. Genotyping of isolates was by sequencing of the Pre-Sl/Pre-82/S region of HBV genome and phylogenetic analysis. Definition of HBsAg subtypes was executed by two methods: ELISA with subtype-specific monoclonal antibodies and S-gene nucleotide sequence analysis. RESULTS: The following occurrence of hepatitis B markers was observed: HBsAg - 3.2%, anti-HBs (total) - 36.2%, anti-HBc IgG - 30.3%, anti-HBc IgM - 1.6%. Frequency of carrying even one of the markers in the observed population was 47.5%. HBV DNA was found in 17 HBsAg-positive samples. Pre-SI, Pre-S2 and S regions sequences were determined for all HBV DNA-positive samples. The phylogenetic analysis showed an accessory of all investigated HBV isolates to genotype D. HBsAg subtypes distribution appeared the following: ayw2 - 23.5%, ayw3 - 70.6%, adw2 - 5.9%. Results of definition of the subtype ELISA method and by the analysis of S gene nucleotide sequences have coincided in 10/11 (90.1%) cases. CONCLUSIONS: The indigenous population of Shuryskarsky area of YNAR belongs to groups with average HBV carrying. Absolute domination of genotype D (subtypes ayw2, ayw3 and adw2) was revealed. High percentage of concurrence of HBsAg subtypes detected by the ELISA method and method of the analysis of S gene primary structure (90%) was observed. Sequencing of HBV S-gene is preferable to define HBsAg subtypes.     

High Diversity of Hepatitis B Virus Genotypes in Panamanian Blood Donors: A Molecular Analysis of New Variants

Hepatitis B Virus (HBV) is an infectious agent that causes more than half of the cases of liver disease and cancer in the world. Globally there are around 250 million people chronically infected with this virus. Despite 16% of the cases of liver disease in Central America are caused by HBV, the information regarding its genetic diversity, genotypes and circulation is scarce. The purpose of this study was to evaluate the genetic variability of the HBV genotypes from HBV-DNA positive samples obtained from screening blood donors at the Social Security System of Panama and to estimate its possible origin. From 59,696 blood donors tested for HBV infection during 2010–2012, there were 74 HBV-DNA positive subjects. Analysis of the partial PreS2-S region of 27 sequences shows that 21% of the infections were caused by genotype A, 3% by genotype D and 76% by genotype F. In addition, we were able to confirm circulation of six sub-genotypes A1, A2, A3, D4, F3, F1 and a proposed new sub-genotype denominated F5pan. We found a confinement of sub-genotypes F1 and F5pan to the western area of Panama. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location.     

Specific mutations of basal core promoter are associated with chronic liver disease in hepatitis B virus subgenotype D1 prevalent in Turkey

The role of hepatitis B virus (HBV) genetics in the clinical manifestations of infection is being increasingly recognized. Genotype D is one of eight currently recognized major HBV genotypes. The virus is ubiquitous worldwide, but shows different features in different regions. One hundred and ninety‐eight patients with chronic HBV infection were enrolled in this study, 38 of whom had been diagnosed with cirrhosis of the liver and/or hepatocellular carcinoma. HBV DNA was isolated from the patients' blood samples and the entire genome and/or the basal core promoter/core promoter region sequenced. Phylogenetic analysis of the complete genomes revealed that subgenotype D1 is the most prevalent subgenotype in Turkey, but there was no definite phylogenetic grouping according to geography for isolates from different regions within Turkey, or for isolates in Turkey relative to other parts of the world. Turkish isolates tended to be genetically similar to European and central Asian isolates. Overall, HBV‐infection in Turkey appears to be characterized by early HBeAg seroconversion, a high incidence of the A1896 core promoter mutation and a small viral load. Genotype D characteristic mutations A1757 and T1764/G1766 were found in the BCP region. T1773 was associated with T1764/G1766 and a larger viral load. In conclusion, infection with HBV genotype D in Turkey has a similar clinical outcome to that of Europe and central Asia. Genotypic mutations in genotype D may be linked with disease prognosis in Turkey, but further studies with higher sample numbers and balanced clinical groups are needed to confirm this.     

High genetic diversity and fine-scale spatial structure in the marine flagellate Oxyrrhis marina (Dinophyceae) uncovered by microsatellite loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1-6 and 7-23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (H(e)) of 0.00-0.30 and 0.81-0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional F(ST) values indicated weak to moderate population sub-division (0.01-0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms.     

Abundant sequence variation around the mitochondrial origin of replication in the human opportunistic yeast pathogen Candida albicans from a tropical island in China

Due to its haploid nature and its predominantly uniparental mode of inheritance, the mitochondrial genome has been analyzed extensively in population and evolutionary genetic studies of eukaryotes. Among the mitochondrial DNA (mtDNA) fragments, the region surrounding the origin of replication is the most commonly studied. However, most of the studies have focused on animals and little is known about the extent and patterns of sequence variation around the mtDNA origin of replication (mtOri) in fungi. In this study, we found abundant variation in a 597bp fragment surrounding the mtOri for 53 isolates of the pathogenic yeast Candida albicans obtained from a diverse group of hosts in Hainan, a tropical island of China. Within this DNA fragment, a total of 17 haplotypes were found for the 53 isolates. The extent of sequence variation for this group of strains was similar to that for 24 strains that represented the global nuclear genotype diversity. In contrast to those in animals where there were significant biases in favor of transitional mutations (e.g. the transition to transversion ratio is about 20 in human mtDNA), our data showed a transition to transversion ratio of approximately 0.5 around mtOri in C. albicans. Our analysis revealed no apparent geographic pattern of sequence variation based on the birthplaces of the analyzed hosts. However, the sample from patients had a lower genotypic diversity than those from healthy hosts borne either in Hainan or elsewhere in China. Our results suggest that C. albicans mtDNA has a base substitution pattern different from its nuclear genome and that sequences from the mtOri region could enhance our understanding of C. albicans genome evolution and population structuring.     

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing

AIM: Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. METHODS AND RESULTS: Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. CONCLUSIONS: Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.     

Phytophthora infestans Field Isolates from Gansu Province,China are Genetically Highly Diverse and Show a High Frequency of Self Fertility

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self‐fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within‐area and among‐area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.     

Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70

Background

The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages.

Methods

The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage.

Results

The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1.

Conclusions

These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest an independent evolutionary history for each type.     

乙型肝炎病毒逆转录酶区基因序列准种与变异特点

乙型肝炎病毒(Hepatitis B Virus,HBV)P基因编码产物从功能上分为末端蛋白(1～178aa)、间隔区(179～336aa)、逆转录酶区(337～682aa)和RNA酶H区(683～816aa),各区有相应的生物学功能;逆转录酶区包含S基因主蛋白编码区.近年来的研究提出HBV感染者体内存在有准种[1,2]的假说.我们以逆转录酶区序列为研究靶区域,应用聚合酶链反应(PCR)技术扩增慢性乙型肝炎患者血清中的靶基因序列,随机选择克隆测序,比较其结果,证明了HBV准种特点的存在,并发现多种基因突变形式.     

Virulence Patterns in a Murine Sepsis Model of ST131 Escherichia coli Clinical Isolates Belonging to Serotypes O25b:H4 and O16:H5 Are Associated to Specific Virotypes

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.     

河北省番茄上致病疫霉Phytophthora infestans群体结构的分析

番茄晚疫病是河北省番茄生产上最具毁灭性的病害之一,对引起该病害的致病疫霉群体结构进行分析有利于病害的防治。利用对峙培养法和菌落直径法对2007-2008年采自河北省保定、沧州和唐山分离自番茄的49个致病疫霉菌株进行了交配型和甲霜灵抗性的表型测定,结果表明该群体所有菌株均为A1交配型,以甲霜灵敏感菌株为主,抗性菌株仅7株。利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)、简单序列重复(SSR)和扩增片段长度多态性(AFLP)等分子技术对该群体的基因型进行了分析,结果表明供试菌株线粒体基因型均为Ia型,共鉴定出了Ⅰ、Ⅱ和Ⅲ3种SSR基因型,AFLP聚类分析在相似系数0.87时可以形成α、β和γ等3个不同的分支。河北省所有番茄上致病疫霉菌株均分布在α分支上,该分支又可进一步分为7个亚分支。AFLP亚分支与甲霜灵抗性和地理来源均无明显相关性,但Ⅱ型SSR与甲霜灵抗性和地理来源有明显的相关性。综合表型和基因型数据说明河北省番茄上致病疫霉群体结构比较单一,遗传多样性程度较低。     

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.