Quantitation of HHV-7 genome by real-time polymerase chain reaction assay using MGB probe technology

A real-time PCR assay was developed to quantify human herpesvirus-7 (HHV-7) genome based on TaqMan technology using the new MGB probe. Primers and probe were chosen in the conserved U100 gene. Plasmid containing the sequence of interest was constructed for the standardisation of the method and to assess its sensitivity. This HHV-7 genomic quantitation assay has a threshold sensitivity of fourteen equivalent genome copy number (EqCop) per reaction. This method was applied to the quantitation of HHV-7 in the peripheral blood mononuclear cells (PBMCs) obtained from 31 healthy subjects. Eighty seven per cent had HHV-7 positive detection in the PBMCs with a viral load ranging from 275 to 14545 EqCop per million of cells. This method presents interesting characteristics with a wide range of quantitation, a good sensitivity, and constitutes a new tool for the study of HHV-7 infection in vivo and in vitro.     

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.The human intestinal tract harbors the most abundant, and among the most diverse, microbial community of all body sites (Ley et al. 2008; Costello et al. 2009). As in most mammals, the gut microbiome is dominated by four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (Ley et al. 2008), which represent more than 1000 different molecular species or phylotypes (Dethlefsen et al. 2008; Claesson et al. 2009). Remarkably, this phylotype composition can be specific and stable for each individual. Repeated sampling of the same individuals indicates that samples from the same subject are more similar than samples from different subjects (Costello et al. 2009; Turnbaugh et al. 2009), and in a 2-yr interval an individual conserves over 60% of phylotypes of the gut microbiome (Manichanh et al. 2008).The gut is considered the primary site for cross-talk between the host immune system and microorganisms, in part because of the size and complexity of its microbiota and the presence of specialized lymphoid structures in the mucosa (Guarner et al. 2006). This close relationship is important for maintaining an adequate homeostasis between the individual and the external environment (Backhed et al. 2005; Guarner et al. 2006). Imbalances of the intestinal microbial composition, named dysbiosis, may disturb homeostasis, and therefore lead to a dysfunction or disease state. For instance, specific changes of this microbial ecosystem were recently associated with two of the major inflammatory bowel diseases (IBD) (Ott et al. 2004; Manichanh et al. 2006; Frank et al. 2007; Dicksved et al. 2008). A large reduction of microbial diversity was found in patients with Crohn''s disease (Manichanh et al. 2006; Dicksved et al. 2008), and a selective reduction of Faecalibacterium prausnitzii, a member of the Firmicutes phylum, was reported in patients with ulcerative colitis (Sokol et al. 2009). Remarkably, in both inflammatory bowel diseases, most bacteria that decrease in abundance relative to healthy controls are producers of butyrate, which has strong anti-inflammatory effects (Nancey et al. 2002; Hamer et al. 2009). Therefore, although the mechanisms underlying these disorders are yet unclear, it is now well accepted that intestinal microorganisms play a key role in the initiation and maintenance of IBD (Round and Mazmanian 2009).Experimental manipulation has great potential to go beyond observational studies and allow us to decode the physiological roles of the gut bacterial community, and also define new therapeutic strategies based on altering this microbiome. In principle, the stability of the gut microbiome could be disrupted by the use of prebiotics, probiotics, and antibiotics. Intake of prebiotics (i.e., specific nondigestible food ingredients) is expected to stimulate the growth and/or bacterial activity in the gut. So far, however, no prebiotic has been shown to have a persistent effect in modifying the gut microbial composition. Similarly, intake of probiotics (i.e., live microorganisms) confers only transient effects on digestive physiology, and long-term persistent alteration of the indigenous gut microbial composition remains controversial. Attempts to manipulate the composition of the intestinal microbiome by fecal bacteriotherapy have now become the focus of an extensive body of clinical case reports with promising results (Borody et al. 2003; You et al. 2008; Khoruts et al. 2009; Shanahan 2009). For instance, it has recently been shown that fecal transplantation from a healthy donor restored both gut microbiota composition and function in a human patient that suffered from recurrent Clostridium difficile–associated diarrhea (Khoruts et al. 2009). Finally, in contrast to prebiotic and probiotic intake, antibiotics have been shown to produce drastic short- and long-term alterations of the human indigenous microbiota. In these studies, microbial compositions were examined using DNA fingerprint techniques (Lofmark et al. 2006; Jernberg et al. 2007), microarrays (Palmer et al. 2007), and, more recently, by taking advantage of DNA pyrosequencing (Dethlefsen et al. 2008; Antonopoulos et al. 2009). All of the above studies indicated that after antibiotic intake there is a drastic disruption of the intestinal microbiota, resulting in a long-term decrease of its overall diversity.The above observations clearly anticipate that experimental manipulation of the gut bacterial community should be feasible to some extent, for example, in the well-established transplantation of exogenous microbiota into germ-free animals. The result of this procedure is a stable colonization by the transplanted community that keeps most of its original diversity (Rawls et al. 2006; Alpert et al. 2008). Therefore, although host factors probably have a major effect in broadly shaping the intestinal microbial ecosystem, long-term alterations of an indigenous consortium might also be induced, especially at the phylotype level. Such changes can now be uncovered due to the rapid development of genomic approaches and computational methods, which permit more detailed comparisons of the compositions of microbial ecosystems. In the present study, we used recently developed tools for deep sequencing and phylogenetic clustering to examine the degree to which the gut ecosystem could be intentionally manipulated. Using rats as a model system, we compared the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. We tested the hypothesis that antibiotics, by reducing bacterial load, would promote establishment of the transferred microbiota, this outcome would have important implications for clinical practice in situations where the goal is to colonize the gut with a new microbiota. The results were surprising, and indicated that the indigenous gut microbial composition could be reshaped to an extent not anticipated in previous studies.     

运用多元性基因库方法揭示克罗恩病患者粪便中微生物种群多样性减少

背景与目的：已知肠道微生物群参与了克罗恩病（CD）的发生及慢性过程，然而，即便是应用无需培养的分子生物学方法研究这样一个复杂的生态系统也是比较困难的。方法：该首次采用综合的多元性基因库方法来研究肠道微生物的多样性。以fosmid质粒载体构建2个基因组DNA库，其DNA来自6例健康供体和6例CD患的粪便样品。通过筛选DNA库并以DNA杂交方式寻找16SrRNA以分析细菌多样性，每个DNA库由25000个克隆组成。结果：1190个备选克隆中，125个非重复核糖型主要代表拟杆菌和厚壁菌。在厚壁菌中，从健康的微生物群中确定了43个独立的核糖型，而CD中仅为13个（P〈0．025）。     

Real-time PCR as a versatile tool for investigating the susceptibility of human herpesvirus 6 to antiviral agents

A quantitative real-time PCR assay was developed for the determination of antiviral drug susceptibility and growth kinetics of human herpesvirus 6. The susceptibility and fitness of a sensitive strain, HST, and its ganciclovir-resistant derivative, GCVR1, were then characterized, leading us to conclude that the mutations of this latter virus did not alter its fitness significantly.     

Role of the human herpesvirus 6 u69-encoded kinase in the phosphorylation of ganciclovir

The human herpesvirus 6 (HHV-6) U69 gene product (pU69) is the presumed functional homolog of the human cytomegalovirus (HCMV) UL97-encoded kinase (pUL97), which converts ganciclovir to its monophosphate metabolite in HCMV-infected cells. It has been reported that insertion of U69 into baculovirus confers sensitivity to ganciclovir in insect cells (J Virol 73:3284-3291, 1999). Our metabolic studies in HHV-6-infected human T-lymphoblast cells indicated that the efficiency of ganciclovir phosphorylation induced by HHV-6 was relatively poor. Recombinant vaccinia viruses (rVVs), expressing high levels of pU69 from two HHV-6 strains (representing the A and B variant), were constructed and used to compare the ganciclovir-phosphorylating capacity of pU69 and pUL97 in human cells. Metabolic studies with [8-(3)H]ganciclovir showed that ganciclovir was phosphorylated in human cells infected with pU69-expressing rVVs, although the levels of phosphorylated ganciclovir metabolites were approximately 10-fold lower than those observed with pUL97. We also demonstrated that pU69, like pUL97, is expressed as a nuclear protein. Our results indicate that the limited phosphorylation of ganciclovir by pU69 may contribute to its modest antiviral activity against HHV-6 in certain cell systems.     

Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach

BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.