Genetic and phylogenetic characterization of structural genes from non-B HIV-1 subtypes in Italy

A molecular and phylogenetic characterization on env and gag subgenomic regions has been performed in our laboratory on HIV-1 variants identified in seropositive individuals residing in Italy, infected in the 1999-2001 period, and five non-B-subtype HIV-1 isolates have been described. To confirm the phylogenetic characterization and to determine the genomic organization of three non-B HIV-1 isolates (A, G, and CRF02- AG), the complete gag, pol, and gp120 ORFs (approx. 6900 bp) have been sequenced for each of them. The phylogenetic tree analyses performed on the whole sequence or on individual genes suggested, for the A and G isolates, the identification of divergent strains that do not cluster into any of the known subsubtypes. This has been further validated by pairwise distance analysis. On the contrary, the phylogenetic classification of the CRF02-AG isolate has been confirmed and an overall typical pattern of intragenomic breakpoints has been observed by a Simplot analysis. These results confirm the constant HIV-1 molecular evolution and indicate the relevance of a continuous molecular monitoring of HIV-1 isolates for the development of appropriate vaccine candidates.     

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.     

Antigenic characterization of Tettnang virus: complications caused by passage of the virus in mice from a colony enzootically infected with mouse hepatitis virus

Neutralization assays were undertaken for the purpose of antigenically characterizing three strains of Tettnang virus from two geographic regions. The previously reported relationship of Tettnang virus strains to mouse hepatitis virus (MHV) was confirmed. However, the precise relationship of the Tettnang strains to prototype MHV strains was obscured in our study by the finding that the isolates had been passaged in mice from a colony subclinically infected with MHV. An Egyptian strain of Tettnang which had not been passaged in that colony was reciprocally related to the neurotropic JHM strain of MHV. Our data stress the importance of microbiological monitoring of apparently healthy laboratory animals used for virologic research.     

Natural variation in replicative and chronological life spans of Saccharomyces cerevisiae

Natural variation in the lifespan of natural yeast populations has not been systematically investigated. Here, we have quantified the variation in the replicative and chronological life spans (RLS and CLS) in natural isolates of Saccharomyces cerevisiae and found that genotypic variation accounts for about 22% of the total variation of RLS. Strikingly, the average RLS of 14 natural isolates is about 30% longer than that of 13 laboratory strains (32 versus 21 cell divisions). As is the case for aging in mammals, there is a negative correlation between the logarithmic transformation of the initial mortality rate and the Gompertz coefficient for RLS. Thus this characteristic feature of aging is conserved from yeast to mammals. The average CLS of the natural isolates is about 7 days, significantly shorter than that of the laboratory strains. There is no correlation between RLS and CLS in natural isolates. Possible reasons for the differences between natural and laboratory strains are discussed.     

Efficacy of culture filtrate protein preparations from Indian isolates of M. tuberculosis to activate T cells derived from healthy donors.

SETTING: While culture filtrate proteins (CFPs) of Mycobacterium tuberculosis appear to be good vaccine candidates for tuberculosis, only CFPs derived from certain popular laboratory strains of M. tuberculosis have been studied for this purpose. OBJECTIVE: To compare the relative efficacies of CFP preparations from two laboratory strains and four contemporary clinical isolates of M. tuberculosis to induce T-cell activation. DESIGN: CFPs were isolated from six strains of M. tuberculosis and were used to induce 1) T-cell proliferation, 2) IFN-gamma secretion, and 3) IL-12 secretion from peripheral blood derived mononuclear cell (PBMC) preparations from 33 healthy donors. RESULTS: Significant amounts of IL-12 were spontaneously secreted by PBMC preparations; CFP preparations from two clinical isolates (JNU-7 and JNU-51) significantly boosted this response. All six CFP preparations induced IFN-gamma secretion by PBMCs, but those from two contemporary strains of M. tuberculosis (JNU-7 and JNU-22) were most effective in this regard. The effect of CFPs from JNU-7 and JNU-22 was significantly better than those from the laboratory strains (H37Ra and Erdman). Similar results were obtained with the T-cell proliferation parameter. CONCLUSION: These results suggest that CFPs derived from selected clinical isolates of M. tuberculosis may outperform those of standard laboratory strains, and may therefore be a better source of potential candidates for a tuberculosis vaccine.     

Differences in neurovirulence among isolates of Herpes simplex virus types 1 and 2 in mice using four routes of infection

Differences in neurovirulence between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated using recent clinical isolates and laboratory-passaged strains in intravaginal, intranasal, intraperitoneal, and intracerebral infections of mice. The HSV-2 isolates caused higher death rates in all four infections. No differences in death rate were observed between recent and passaged isolates of either HSV-1 or HSV-2. After intravaginal inoculation, HSV-1 isolates replicated to higher titers in the vaginal mucosa, but HSV-2 isolates produced a higher death rate and a greater frequency of latent infection in lumbosacral ganglia of surviving animals. After intranasal inoculation, HSV-2 isolates again produced a higher death rate, but the frequency of latent infection in trigeminal ganglia was higher with HSV-1 isolates. The data suggest that the HSV-2 isolates have an enhanced capacity to enter and replicate in the central nervous system of mice but that latency is influenced by both virus type and route of inoculation.     

Codetection of a mixed population of candHPV62 containing wild-type and disrupted E1 open-reading frame in a 45-year-old woman with normal cytology

We have cloned, sequenced, and characterized the complete genome of a novel human papillomavirus (HPV), candHPV62. During cloning, 2 candHPV62 viral isolates were recovered from a single cervical sample; 1 had all anticipated HPV open-reading frames (ORFs) intact, whereas the other exhibited an E1 frame-shift mutation. Further experiments indicated that the 2 strains were equivalent in abundance. It appears that an early mutation occurred within the E1 ORF, which was transcomplemented by an intact E1 protein. A search of the HPV database identified disruption of the E1 ORF in the cloned reference isolates of HPV16, HPV53, HPV56, and HPV72. These data suggest that disruption of the E1 ORF in genital HPVs is not uncommon.     

Evaluation of 7SL RNA gene sequences for the identification of Leishmania spp

We evaluated the use of 7SL RNA gene sequences for the identification of Leishmania spp. A fragment (approximately 137 basepairs) of the 7SL RNA gene from 13 reference strains and 18 clinical isolates of 11 different Leishmania species was amplified and sequenced using conserved primers. Reference strains from each Leishmania spp. complex showed unique sequences. The nucleotide sequences were compared pairwise and a range of 81.0-99.3% intercomplex similarity was observed. Clinical isolates of the same species had sequences identical to the corresponding reference strains; thus, the intraspecies similarity was 100%. A phylogenetic tree derived from the 7SL RNA gene partial sequences was constructed and is in agreement with accepted phylogenetic schemes.     

Typing of Human Cytomegalovirus Clinical Isolates from Saudi Patients by PCR-RFLP

Abstract Background: Information on strain types of human cytomegalovirus (HCMV) isolates from Saudi Arabian patients is lacking. Materials and Methods: 80 clinical isolates of HCMV from Saudi Arabian patients were analyzed by PCR amplification of three regions (DNA polymerase, glycoprotein B, and glycoprotein H) of the virus genome. The resultant amplicons (2.0–2.7 kb) were further studied by restriction fragment length polymorphism (RFLP) using four enzymes (HaeIII, HhaI, MspI, and RsaI). Results: Combined analysis of the cleavage patterns generated by the enzymes identified five strains, S1–S5, and several mixed and unique strains. 18 isolates belonged to S1 strain and were similar to laboratory strain AD169. Eight isolates were present in each of S2 and S3 strains. Six isolates and four isolates were found in S4 and S5 strains, respectively. 12 isolates contained a mixture of S3 and S5, which may have resulted from a dual infection. Each of the 24 remaining isolates had a different strain pattern. Conclusion: Our findings show that 80 HCMV clinical isolates were distributed into 30 different strains using PCRRFLP analysis of multiple viral subgenomic regions. However, the number of isolates is not uniformly distributed among strains (p < 0.02).     

Genetic characterization of three newly isolated CRF07_BC near full-length genomes in China

Though HIV-1 CRF07_BC rapidly spread in China, there have been few reports about this subtype since its first genetic characterization nearly 10 years ago. It was urgent and necessary to know the current gene variation of circulating CRF07_BC strains. Xinjiang was the main region for the CRF07_BC epidemic and also an ideal region for research on the viral gene evolution. The strains of Ulumuqi and Yili in Xinjiang were isolated, cloned, and sequenced in this study. Analyses of phylogenetic, potential CTL epitopes and N-glycosites were preformed simultaneously. New CRF07_BC isolates showed higher genetic diversity and more potential N-glycosites than old isolates. It was interesting that although the env and nef genes are highly variable, highly conserved potential CTL epitopes and N-glycosites were found in deduced gp120 V3 and Nef product of all CRF07_BC isolates. The analysis of the sequences provides some valuable information on the investigation of the epidemiology and on vaccine development.     

Genetic characterisation of influenza C viruses detected in Singapore in 2006

In an earlier study on respiratory infections in Singapore military recruits, four influenza C virus (FLUCV) infections were detected out of the 1354 samples collected. All four isolates were detected in 2006, and their whole genome was completely sequenced and analysed. Phylogenetic analysis of the hemagglutinin esterase fusion (HEF) gene revealed that all four Singapore isolates belonged to the C/Japan‐Kanagawa/1/76‐related lineage. However, the genes of the four FLUCV isolates had origins from several different lineages, and the genome composition resembles that of the C/Japan‐Miyagi/9/96‐like strains that had been circulating in Japan between 1996 and 2000.     

不同地区间日疟原虫环子孢子蛋白基因两侧翼DNA序列的比较

用多聚酶链反应（ＰＣＲ）技术、引物标记周期反应测序法和自动核酸序列分析仪直接测定我国和３个西太平洋国家共１８个间日疟分离株的环子孢子蛋白（ＣＳＰ）基因两侧翼ＤＮＡ序列，并与ＢＺＬ、Ｂｅｌｅｍ、Ｓａｌ－１、Ｎ．Ｋ．和Ｔｈａｉ等地区株进行比较。结果表明不同地区间日疟ＣＳＰ基因Ｎ端和Ｃ端非重复序列高度保守；１８个分离株与５个地区株的上述序列基本一致。但在Ｃ端可变区则有较多变异，包括一些尚无记载的地理局限性变异。     

布氏菌分离株16SrDNA基因遗传进化分析

目的检测内蒙古布氏菌临床分离株的16SrDNA基因序列,构建16SrDNA遗传进化树,确定该菌株的分类地位。方法用BCSP31-PCR对临床分离株检测,再进行16SrDNA双向测序;从GeneBank下载标准参考菌株和与布氏菌有共同抗原细菌的16SrDNA基因序列;用Mega6.0进行序列比对并构建遗传进化树。结果BCSP31-PCR结果显示均有223 bp扩增条带,16SrDNA测序得到单一基因序列;比对后发现有4处特异片段,经Blast比对,位于844-1224包括380 bp的片段为布氏菌独有;进化树显示布氏菌临床分离株与标准菌株聚在1个末端分支上,遗传距离接近0,无法精细区分相互关系;与人苍白杆菌聚集在1个亚分支上,遗传距离0.019;与其它试验菌株如霍乱弧菌、小肠结肠炎耶尔菌遗传进化距离较远,遗传距离>0.02。结论从遗传进化角度对布氏菌分离株与标准菌株的亲缘关系进行分析,布氏菌所有种型遗传距离接近,提示布氏菌16SrDNA为高度保守序列,不宜做遗传进化分析的靶基因;布氏菌与人苍白杆菌有较近的亲缘关系;布氏菌属16SrDNA特有的380 bp的基因片段可作为布氏菌快速鉴定的靶序列。     

Improvement of growth of Plasmodium falciparum fresh clinical isolates by using an established serum-free medium, GIT

In the present study, we have tried to establish continuous cultures of fresh clinical isolates of P. falciparum by using a serum-free medium, GIT. To examine the ability of GIT to support the parasite growth, the growth of various P. falciparum isolates including two laboratory strains of P. falciparum, FCR3 and K1 was compared in both of GIT and RPMI 1640 medium supplemented by 10% human serum (RPMI-HS). Growth rates of various P. falciparum expressed as fold increases were compared in GIT and RPMI-HS, and the maximum growth rates of P. falciparum were 72 in GIT and 35 in RPMI-HS during the culture for 8 days. Growth rate of the clinical isolates varied individually in both culture media, with average growth rates of parasites being 15.9 in GIT and 8.8 in RPMI-HS, respectively (not significant). Growth rates of FCR3 and K1 strains were 28.0 and 6.6 in GIT, and 10 and 7.5 in RPMI-HS. After 30 days culture of P. falciparum in GIT, 9 of 12 clinical isolates still continuously propagated but other three isolates disappeared. Despite variation of the P. falciparum isolates in their abilities to multiply in GIT, our experiments suggested that GIT is useful for culture of fresh clinical isolates of P. falciparum that are derived from geographically distinct areas as well as laboratory strains used commonly in laboratory research.     

Potential for antimicrobial resistance in Chlamydia pneumoniae

Antimicrobial resistance has not yet been described in wild type Chlamydia pneumoniae isolates, nor has selective emergence of resistance in the laboratory after exposure to subinhibitory concentrations of antibiotic. However, few clinical isolates have been tested for resistance, especially strains with resistance phenotypes (i.e., those associated with clinical failure or persistence). More widespread testing of such strains is needed. Further understanding of antimicrobial resistance in chlamydiae would benefit from the development of standardized methods. Further, more physiologic testing methodologies that more closely mimic the chronic intracellular infection usually being treated in vivo would be of value. Animal models demonstrate persistence of C. pneumoniae after antimicrobial therapy and could be used to better define the clinical correlates of in vitro testing.     

Neutralization of multiple HIV-1 isolates from a single subject by autologous sequential sera

Titers of neutralizing antibodies to different strains of human immunodeficiency virus type 1 (HIV-1), including five isolates sequentially obtained from one infected subject, were determined using sequential serum samples obtained from that individual. Neutralizing antibodies were detected against the HIV-IIIB laboratory strain of HIV-1 and against a clinical isolate from another HIV-1-infected individual. Sera from the subject under investigation possessed differential ability to enact viral neutralization, depending on which homotypic clinical isolate was used. In general, it appeared that effective neutralization capacity was present in serum against homotypic viral isolates of HIV-1 only if these isolates were obtained at or before serum collection. These data suggest that variants of HIV-1 in infected individuals may not be effectively neutralized by antibodies that have been generated in these same people against previously dominant viral strains.     

Bacterial genome sequencing and its use in infectious diseases

The availability of genome sequences is revolutionising the fields of bacteriology and infectious diseases. By mid-2007, 479 bacterial genomes from 352 distinct species have been sequenced, including representatives of all notable human pathogens. Additionally, the genomes of several strains from each of 55 species have been sequenced. This tremendous amount of genomic data has led to unprecedented advances in pathogen diagnosis, genotyping, detection of virulence, and detection of resistance to antibiotics. We review current achievements in these fields and potential developments in the future for the clinical microbiology laboratory.     

梅毒螺旋体TpN47基因分析及实时荧光定量PCR检测方法的建立

目的 确定不同梅毒螺旋体TpN47基因序列的差异性及其跨膜结构分析,建立基于TpN47基因检测梅毒螺旋体的荧光定量PCR方法。方法 通过使用NCBI／Blast,Pfam,TMHMM Server v. 2.0等网络资源分析梅毒螺旋体TpN47基因编码蛋白的保守功能结构域、跨膜区域。根据参考序列设计引物,采用PCR技术从梅毒螺旋体基因组DNA中扩增TpN47基因,将其连接入克隆载体pMD18-T进行测序,并用DNASTAR软件比较不同梅毒螺旋体菌株TpN47基因的核苷酸序列。根据梅毒螺旋体TpN47基因保守区域设计引物,建立基于该目的基因的荧光定量分析PCR检测方法并分析其灵敏度、特异性和稳定性。同时,采用基于TpN47基因的荧光定量PCR对梅毒螺旋体感染患者血液样本进行检测。结果 研究表明,梅毒螺旋体TpN47基因为梅毒螺旋体外膜蛋白编码基因,其产物定位于外膜表面。不同梅毒螺旋体菌株中TpN47基因的核苷酸序列保守,相似度分别达99％以上。基于TpN47基因的荧光定量PCR方法可有效地检测梅毒螺旋体感染患者血清及泌尿道分泌物样本中的梅毒螺旋体。结论 TpN47基因为梅毒螺旋体外膜蛋白编码基因,建立的基于TpN47基因的荧光定量PCR方法具有快速、敏感、稳定等优点,适用于梅毒螺旋体临床实验室诊断。     

Intraspecies divergence of 16S rDNA, ITS, rpoB gene and hsp65 gene sequence for Mycobacterium lentiflavum

OBJECTIVE: To clarify the genetic microheterogeneity of Mycobacterium lentiflavum and identify the predominant genotype. MATERIALS AND METHODS: Clinical isolates of M. lentiflavum used in this study were obtained from sixteen patients of lung diseases. In order to assess their intraspecies variability, four gene fragments, from the 16S rDNA (1471 bp), 16S-23S ITS (282 bp), rpoB (306 bp), and hsp65 (401 bp), were sequenced. RESULTS: Intraspecies variabilities were found in all of the four targeting fragments. As multilocus sequence typing with these four targets, 16 clinical isolates were divided into 3 genotypes, i.e., MLST2, MLST3, and MLST4. Among them, MLST2 to which 12 clinical isolates belonged, was a predominant genotype. Three strains belonged to MLST3 and the remaining one strain belonged to MLST4. Drug susceptibility study indicated that there was no clear relation between sequence types and drug susceptibility. CONCLUSION: Multilocus sequence typing could aid in characterization and in better understanding of the epidemiology of M. lentiflavum.