Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

International audienceHepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis

Molecular epidemiology of Cryptosporidium spp. in North Lebanon

International audienceCryptosporidium spp. are enteroparasites with worldwide distribution that infect the gastrointestinal tract of several vertebrates including humans. Human to human, zoonotic, foodborne and waterborne are reported as the main transmission routes of this parasite. Cryptosporidium spp. have been recognized as the predominant cause of waterborne and foodborne outbreaks. However, the epidemiological situation of cryptosporidiosis is not well known in Lebanon, a developing country with a population often affected by intestinal parasitic infections. This study was devoted to determine the prevalence and the genetic diversity of Cryptosporidium spp. in symptomatic hospitalized patients and in two children populations with different socioeconomic level in North-Lebanon, as well as the risk factors associated with cryptosporidiosis. Fecal samples obtained from these populations were examined microscopically by modified Ziehl-Neelsen staining as well as nested PCR were done for the detection of Cryptosporidium oocysts. Out of 163 symptomatic hospitalized patients and 249 children, Cryptosporidium was present in 11% and 10.4% respectively according to microscopy examination and/or molecular tests. The genotyping showed the predominance of Cryptosporidium hominis in both populations. Subgenotype analysis of the isolates at the gp60 locus identified three subtypes IdA19, IbA10G2 and IaA18R3 for C. hominis and two subtypes IIaA15G1R1 and IIaA15G2R1 for C. parvum. Moreover, cryptosporidiosis was correlated with having meals outside home and presence of gastrointestinal symptoms especially diarrhea (p <0.05). This work constitutes the first molecular epidemiology study outlining risk factors associated with cryptosporidiosis in Lebanon. These findings support a need of a control program to prevent the circulation of this parasite

Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum β-Lactamase (CTX-M-15)-Producing Enterobacteriaceae in Lebanon

A prospective study was carried out to assess the extent of carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected β-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii

First report on the prevalence and subtype distribution of Blastocystis sp. in dairy cattle in Lebanon and assessment of zoonotic transmission

International audienceBlastocystis sp. is frequently identified in a wide range of animal hosts, including bovids. Because of its burden and zoonotic potential, this parasite has been sought in domestic cattle from various countries, since this livestock may also represent a possible reservoir of human infection. However, epidemiological data regarding the prevalence and ST distribution of Blastocystis sp. in this animal group is lacking in Lebanon. Therefore, faecal samples were collected from a total of 254 dairy cattle raised on 55 farms located in the North Lebanon region and screened for the presence of the parasite by quantitative real-time PCR. The overall prevalence of Blastocystis sp. was shown to reach 63.4% in cattle livestock. Sequence analysis of positive samples indicated the presence of seven STs, with predominance of ST10 (44.0%) and ST14 (36.8%) and lower proportions of ST2 (8.0%), ST1 (7.2%), ST5 (2.4%), ST3 and ST7 (0.8% each). This survey was the first conducted worldwide reporting ST2 and ST7 in domestic cattle and confirmed that ST10 and ST14 represent cattle-adapted STs in view of their high prevalence. Faecal samples from in-contact dairy farmers and patients hospitalised in the same Lebanese gov-ernorate who reported no contact with cattle livestock were also analysed for the presence of Blastocystis sp. The same three STs were identified in both human cohorts, with predominance of ST3, followed either by ST1 or ST2 depending of the group. No other STs, including ST10 or ST14, have been reported. Moreover, even though ST1, ST2 and ST3 were found to be common to dairy cattle and farmers cohorts, only one ST3 isolate showed 100% sequence identity between both hosts. Consequently, the presence and low prevalence of ST1, ST2, ST3, ST5 and ST7 identified herein in domestic cattle, most of which exhibit low host specificity, could be derived from occasional direct exposure to faecal material from human and non-human hosts or by ingestion of contaminated drinking water or food in the enclosure of the farms. Together with the absence of ST10 and ST14 in the human population, these data suggest that cattle play a negligible role as zoonotic reservoirs of Blastocystis sp

N-acetylcysteine potentiates diclofenac toxicity in Saccharomyces cerevisiae: stronger potentiation in ABC transporter mutant strains

International audienceDiclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast

Prevalence and subtype distribution of Blastocystis sp. isolates from poultry in Lebanon and evidence of zoonotic potential

International audienceBackground: Blastocystis sp. is a common protozoan parasite frequently identified in the digestive tract of humans and a large variety of animal hosts worldwide, including birds. It exhibits a large genetic diversity with the identification of 17 subtypes (STs), most of them with low host specificity. ST6 and ST7 were identified in birds and suggested to represent avian STs only in the context of scarce small-scale epidemiological surveys. Moreover, these two STs also account for a significant proportion of human infections whose zoonotic origin has never been clearly confirmed. Therefore, molecular screening of Blastocystis sp. was conducted by quantitative real-time PCR for fecal samples from poultry farms and their in-contact humans from slaughterhouses in Lebanon. In parallel, a control group consisting of patients hospitalized in the same geographical area and reporting no contact with poultry was also screened for the presence of the parasite.Results: The overall prevalence of Blastocystis sp. was shown to reach around 32% in chicken samples and 65% in the farms screened. All the avian isolates were subtyped and belonged to either ST6 or ST7, with a large predominance of ST6. Fifty-four percent of slaughterhouse staff members were positive for Blastocystis sp. compared with a similar prevalence of 56% in hospitalized patients. ST3 was predominant in both human cohorts followed by either ST1 then ST2 among slaughterhouse staff or by ST2 then ST1 among hospitalized patients. ST6 was also identified in two slaughterhouse workers and not in the group of hospitalized patients. Gene sequence identity was observed between chicken and human ST6 isolates from the same slaughterhouse.Conclusions: Our data revealed a high prevalence of Blastocystis sp. in chicken samples and confirmed that ST6 and ST7 represented avian-adapted STs. Among both human cohorts, Blastocystis sp. infection was shown to exceed 50% with a predominance of ST3. The identification of ST6 in slaughterhouse staff members confirmed the zoonotic transmission of this ST through repeated and direct contact between chickens and their handlers

Synthesis of N-pyridyl azoles using a deprotometalation-iodolysis-N-arylation sequence and evaluation of their antiproliferative activity in melanoma cells

International audienceN-Arylation of pyrrole with 3-iodo-4-methoxypyridine was investigated by copper catalysis under different conditions. The best conditions, that proved to be protocol A (CuI, DMEDA or TMEDA, K 3 PO 4 , DMF at 110 C) and above all protocol B (Cu 2 O, Cs 2 CO 3 , DMSO at 110 C), were applied to the synthesis of various N-(methoxypyridyl) pyrroles, indoles and benzimidazoles. The behavior of the different iodinated methoxypyridines was rationalized by evaluating the partial positive charge on the carbon bearing iodine from the 1 H NMR chemical shift of the corresponding deiodinated substrates. The reaction was next connected with the deprotometalation-iodolysis step generating iodinated methoxypyridines: straight involvement of the crude iodo intermediates in pyrrole N-arylation afforded the expected N-(methoxypyridyl) pyrroles in good yields. Several synthesized N-(methoxypyridyl) azoles exerted low to moderate antiproliferative activity in A2058 melanoma cells