Search

The aim of this study was to investigate the phytochemical composition of dried Roselle calyx (Hibiscus sabdariffa L.) using both ethanolic and aqueous extracts. We report the antimicrobial activities against a wide range of bacteria, yeast, and fungi. The antioxidant activities were tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and 2–2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assays. We report also for the first time the effect of the swarming motility in Pseudomonas aeruginosa PAO1. Our results showed that the tested two extracts were a rich source of phenols, flavonoids, and tannins with different degrees. Additionally, eleven phytoconstituents were identified by LC/MS technique (Hibiscus acid: 3-caffeoylquinic acid, 5-caffeoylquinic acid, 5-feruloylquinic acid, cyanidin 3-o-glucoside, myricetin, quercetin 7-o-rutinoside, quercetin 3-o-glucoside, delphinidin 3-o-sambubioside, and kaempferol 3-o-p-coumaroyl-glucoside). Also, it was shown that the calyx extract can scavenge 86% of the DPPH radical, while the rate of 53% and 23% of inhibition of the DPPH was obtained only at the concentration of 125 and 50 µg/mL, and a small inhibition was made at a concentration of 5 μg/mL. Roselle extracts inhibited the growth of the selected microorganisms at low concentrations, while higher concentrations are needed to completely kill them. However, no activity against CVB-3 was recorded for both extracts. In addition, the obtained extracts reduced the swarming motility of P. aeruginosa at 2.5 mg/ml. The docking simulation showed acceptable binding affinities (up to −9.6 kcal/mol) and interaction with key residues of 1JIJ, 2QZW, and 2UVO. The obtained results highlighted the potential use of Roselle extract as a source of phytoconstituents with promising antimicrobial, antioxidant, and anti-quorum sensing activities.

AimsThis study aimed to determine the antibacterial and antileishmanial potential of Micromeria nervosa extracts. The identification of the antileishmanial compound and the study of its molecular mechanism of action have also been undertaken.Methods and resultsEthanol extract showed high polyphenol content and diethyl ether extract exhibited high DPPH scavenging and low beta-carotene bleaching activity (IC50 = 13.04 ± 0.99 and 200.18 ± 3.32 μg mL−1, respectively). However, diethyl ether extract displayed high antibacterial activity against Gram-positive strains including methicillin-resistant Staphylococcus aureus (MIC = 31.25 μg mL−1), Staph. aureus ATCC6538 (MIC = 62.5 μg mL−1), and Listeria monocytogenes ATCC 19115 (MIC = 125 μg mL−1), as well as high antileishmanial activity against the promastigote forms of L. infantum and L. major (IC50 = 11.45 and 14.53 μg mL−1, respectively). The active compound was purified using bioassay-guided fractionation and thin layer chromatography, and identified as ursolic acid using high-performance liquid chromatography coupled with a photodiode array and mass spectrometry. The purified compound was strongly inhibitory against the promastigote and amastigote forms of L. infantum and L. major (IC50 = 5.87 and 6.95 μg mL−1 versus 9.56 and 10. 68 μg mL−1, respectively) without overt cytotoxicity against Raw 264.7 macrophage cells (SI = 13.53 and 11.43, respectively). The commercial compound (ursolic acid) showed similar activity against amastigotes and promastigotes forms of L. infantum and L. major. Moreover, its molecular mode of action against leishmaniasis seems to involve the expression of the ODC and SPS genes involved in thiol pathway.ConclusionExtracts of M. nervosa can be considered as a potential alternative to antimicrobial and antileishmanial drugs.

Eleven bacterial strains, isolated from various Tunisian biotopes, were characterized for their lignin degradation activities and were screened for Indigo Carmine (IC) decolorization for the first time. Results revealed that these isolated strains were able to decolorize IC. Based on this property, the newly isolates crude filtrates (NICF) showed that eight ones prove the effectiveness for IC degradation (100 mg). More than 80% of IC could be decolorized within 12 h under non-optimised conditions. The UV–Visible absorption spectra and FT-IR analysis of the degradation products showed an IC decolorization and/or transformation, proving the enzyme’s role in dye decolorization. Furthermore, the phytotoxicity and the cytotoxicity using Vero and erythrocyte cells were performed to evaluate the acute toxicity of the treated and untreated dye. The results showed that some NICF can decrease and sometimes detoxify IC. In fact, NICF decolorize the toxic IC into non-toxic products. These NICF, acting as powerful tools, could be effectively used to decolorize and to bioremediate rich-dye-textile effluents and found worthy of investigation for potential applications in restoration work and other biotechnological uses.

This study screened mastic gum (Pistacia lentiscus L.) for antiviral activity against herpes simplex virus type 2 (HSV-2), coxsackievirus type B3, and adenovirus type 5. The organs of this plant (leaves, stem, and seed) were macerated sequentially using solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and methanol). Only the methanol extract of stem exhibited significant activity against HSV-2. This extract showed anti-HSV-2 activity with a selectivity index of 51 (50% cytotoxic concentration = 186 µg/mL; 50% inhibitory concentration = 3.63 µg/mL), and demonstrated direct inhibition against this virus with a virucidal selectivity index of 620 (50% virucidal concentration = 0.30 µg/mL). A bio-guided assay involving thin-layer chromatography led to the isolation of two active compounds, which have been identified as dammaradienone and dammaradienol using high-performance liquid chromatography-diode array detection coupled with electrospray ionization mass spectrometry. P. lentiscus has been widely studied for other biological activities. However, to our knowledge, this is the first report of P. lentiscus L. exhibiting antiviral activity.

The emergence of multidrug-resistant viruses leads to discover a novel source of drugs or a new strategy to overcome this obstacle. Combined herbal therapies are becoming a promising approach for a better efficiency. In this study, Pistacia lentiscus and Peganum harmala were investigated for their anti HSV-2 activity. Twenty eight organic extracts from different organs were evaluated for their cytotoxicity on Vero cells and their anti HSV-2 activity using the MTT test and the plaque reduction assay, respectively. Peganum harmala methanol seeds extract, Pistacia lentiscus methanol stem and dichloromethane fruit extracts were active on HSV-2 with selectivity index values of 13.19, 51.23 and 10.23, respectively. These active extracts exhibited different mode of action: (1) Peganum harmala methanol seeds extract showed a virucidal action before virus adsorption and penetration, and on the newly formed virions before their release outside infected cells—(2) Pistacia lentiscus methanol stem extract exhibited virucidal effect only before virus adsorption and penetration—(3) Pistacia lentiscus dichloromethane fruit extracts demonstrated virus inhibition up to 7 h post infection. The binary combination of the three active extracts did not improve their individually activity. This report could contribute to prove that combination of active extracts do not necessary increase the initial activity.

The present study was the first to evaluate the phytochemical composition, antioxidant, antimicrobial, antibiofilm, and anti-quorum sensing potential of

Commiphora gileadensis, locally known as becham, is a plant used in traditional Arabian medicine for treating headache, constipation, stomach, joint pain, and inflammatory disorders. Several studies have reported its antibacterial properties; however, no study has demonstrated its antiviral activity. This study aimed to evaluate the antiviral activity of C. gileadensis as well as to isolate its active compound and investigate its mode of action. This activity was evaluated using 4 viruses, herpes simplex virus type 2 (HSV-2), respiratory syncytial virus type B (RSV-B), coxsackie virus B type 3, and adenovirus type 5 by performing the plaque reduction assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for enveloped and nonenveloped viruses, respectively. The methanol extract of C. gileadensis leaves only showed antiviral activity against enveloped viruses with a selectivity index of 11.19 and 10.25 for HSV-2 and RSV-B, respectively. The study of the mechanism underlying antiviral activity demonstrated a virucidal effect by direct contact with these target viruses. The active compound, isolated using bio-guided assays involving TLC, was identified as guggulsterone by HPLC-diode array detection coupled with electrospray ionization mass spectrometry. Guggulsterone is an antagonist of the bile acid receptor and a modulator of cholesterol metabolism; however, its antimicrobial properties have been reported for the first time in this study.

Coronavirus disease (COVID-19) is caused by SARS-CoV-2 and represents the causative agent of a potentially lethal disease. COVID-19 has been described as a significant global public health pandemic by the World Health Organization due to its high mortality rate, rapid spread, and the lack of drugs and vaccines for it. Active antiviral drugs are desperately needed to combat the potential return of severe acute respiratory syndrome (SARS).In this study, we selected 39 natural compounds present in plants, algae, and sponges with antiviral activity. Molecular docking was used to screen the compounds’ activity on SARS- CoV-2 RNA-dependent-RNA polymerase, receptor-binding domain (RBD), and the human ACE2 receptor. Compounds with binding energy ≤ -6.5 kcal/mol enter pre-clinical testing using in silco ADME/Tox (absorption, distribution, metabolism, excretion, and toxicity).We found eight potential SARS-CoV-2 inhibitors: (glycyrrhizin, rutin, baicalin, 1, 6-di-O- galloyl-beta-D-glucose, pyropheophorbide A, pheophorbide A, beta-Sitosterol, and vitexin). These outcomes indicate that these compounds could be potential candidates to be utilized in lead optimization for the design and production of the anti-SARS-CoV-2 drug.

Parasporins (PS), a class of non-insecticidal and non-hemolytic crystal proteins of Bacillus thuringiensis (Bt), are being explored as promising anti-cancer agents due to their specific toxicity to cancer cells. This work is considered as a first initiative aiming at investigating Algerian soil Bt isolates’ activity and cytotoxic potential against cancer cells. A total of 48 Bacillus spp. were isolated from different sites in Algeria. Phenotypic and biochemical tests, 16S rDNA molecular identification, and microscopic observation of crystal have confirmed the identification of Bt for ten strains. A screening for non-hemolytic crystalline proteins was performed. Extraction, purification, and activation of non-hemolytic proteins by chromatographic analysis yielded several polypeptides of different molecular weights. A purified PS1, with pro-protein of 81 kDa and several peptides with different molecular weights (18–58 kDa) after activation by trypsin, has been identified from the strain BDzG. The NH2-terminal sequence deciphered in BLAST analysis showed homology to a Bt PS1 protein. Moreover, the screening of parasporin-1 (PS1) gene has also been performed. Cytocidal activity against human epithelial type 2 (HEp2) cells, considered to originate from a human laryngeal carcinoma, was observed with an IC50 equal to 2.33 μg/ml, while moderate cytotoxicity against adenocarcinomic human alveolar basal epithelial (A549) cells has been shown with IC50 equal to 18.54 μg/ml. No cytotoxicity against normal cells was noted. Fluorescence microscopy revealed a condensed or fragmented chromatin indicating the apoptotic death of HEp2 cells. Thus, Bt PS-producer isolated from Algerian soil might have a potential to join the arsenal of natural anti-cancer drugs with high therapeutic potential.

The use of synthetic food additive and the appearance of antibiotic resistance are at the basis of important human health problems. The substitution of synthetic compounds with new natural substances extracted from plants or microorganisms is therefore the ideal solution to this scourge. The objective of this work was to evaluate the phyto-constituents (polyphenols, flavonoids and condensed tannins), and to test the biological activities (antioxidant, antibacterial and antiviral) of the Ajuga iva (L) aerial part extracts. The antioxidant activity assayed by DPPH method showed an IC50 of 0.43 ± 0.03 mg/mL. Antibacterial activity of aqueous and hydro methalonic extracts was tested against seven pathogenic bacteria (Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRS), Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli and Salmonella enteritidis) using the diffusion method. A Thin Layer Chromatography-bioautotography-guided was performed, and the isolated antibacterial fraction was identified by CG-MS analysis. Antiviral effect of methanolic extract performed on 4 viruses: Coxsackie Virus type B-3 (CVB-3), Adenovirus type 5 (ADV-5), Respiratory Syncytial Virus type B (RSV-B) and Herpes Simplex Virus type 2 (HSV-2) showed an activity against Coxsackie Virus. As a result of this study, the aerial parts of Ajuga iva (L) extract could be used in the food, cosmetic, medical and health sectors.

A Trichoderma orientale strain LSBA1 was isolated from the Mediterranean marine sponge Cymbaxinella damicornis. The crude extract of T. orientale mycelium showed inhibitory activity against growth of Gram-positive and Gram-negative bacteria as well as clinical isolates of Candida albicans. Purification of the anti-Candida component was performed using a combination of open silica gel-60 column and reverse phase high performance liquid chromatography. The active compound called hyporientalin A has been identified as a peptaibol analogue of longibrachin-A-II using mass spectrometry. It exhibited fungicidal activity against clinical isolates of C. albicans with minimal inhibitory concentrations (MICs) ranging from 2.49 to 19.66 µM, comparable to that of the antifungal agent amphotericin B. Our data support the use of hyporientalin A as a promising new and efficient antifungal drug in the treatment of candidiasis while controlling toxicity.

The emergence of the antibiotic resistance is an ongoing problem in public health, and therefore the search for new natural molecules represents an alternative to synthetic drugs. The aim of this study was to test the antibacterial activity of ten Mediterranean plants ( Diplotaxis harra , Ecballium elaterium , Pergularia tomentosa , Myrtus communis , Solanum villosum , Solanum sodomaeum , Peganum harmala , Lepidium sativum , Pistacia lentiscus , and Calendula arvensis ) against seven pathogenic bacteria ( Staphylococcus aureus, Escherichia coli , Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella sp., Salmonella enteritidis , and Bacillus cereus ) using the disk diffusion method, in order to isolate and identify the active compound(s). Dichloromethane extract of D. harra flower showed the best activity against S. aureus and L. monocytogenes (MIQ = 30 μg/disk and 15 μg/disk, respectively). This extract was submitted to a bio-guided purification using Thin Layer Chromatography (TLC)-bioautography, and an antibacterial fraction (MIQ = 2 μg/disk) was isolated. The active fraction was characterized by RP-HPLC-DAD-ESI-MS n and GC–MS. Sulforaphane, an isothiocyanate known for its anti-cancer, antioxidant, and anti-inflammatory properties, was identified as antibacterial agent in D. harra for the first time. Due to its high antibacterial activity, sulforaphane could be considered a good candidate for the selection of new natural antibacterial molecules.

Genital herpes is a sexually transmitted disease caused by herpes simplex virus type 2 (HSV-2). Nucleoside analogues such as acyclovir (ACV) are the usual therapy for treating HSV infection. However, the overuse of this drug has led to the emergence of resistant strains. Therefore, the search for new alternative or complementary molecules to overcome this obstacle is needed. In this objective, Peganum harmala was investigated for its HSV-2 activity. The organic extracts of the different plant organs were evaluated for their cytotoxicity on Vero cells by the MTT test and anti HSV-2 activity by plaque reduction assay. Only the methanol seeds extract was active with a 50% inhibitory concentration (IC50) and a selectivity index (SI) of 161 and 13.2 μg/mL, respectively. In addition, the study of the antiviral mode of action revealed that this extract exerts a virucidal action both during the entry of viruses and the release of the newly formed virions, whereas no cell protection effect was observed. The active compound was isolated by bio-guided purification using thin layer chromatography (TLC) and identified by GC-MS and HPLC-DAD-ESI-MSn as harmine. The combination of harmine standard compound with ACV showed a combination index (CI) of 0.5 indicating that these two compounds have a synergic effect. This data suggests that harmine could be associated to ACV to improve the treatment of genital herpes essentially for the immunocompromised patients.

The present study describes the strains of hepatitis C virus (HCV) isolated from Tunisian hemodialysis patients. Thirty-three HCV strains isolated from different dialysis centers in Tunis City were amplified by RT-PCR in a region of the NS5b gene, genotyped by sequencing, and compared to international sequences by phylogenetic analysis. The phylogenetic tree showed that 16 HCV isolates have been identified as subtype 4k (48.5%), 7 as unspecified HCV-4 subtype (21.2%), 5 as subtype 4a et 1b (each 15.2%). The analysis of this tree revealed that the HCV-1b strains were closely related to Anglo-Saxon and European isolates, while the HCV-4 isolates are genetically similar to Egyptian and African strains. Phylogenic analysis of 33 Tunisian isolates with international HCV strains on a region of the NS5b gene demonstrated that the subtype 4k submerged the Tunis city and a new subtype of HCV4 seems to be suspect in this area.

The Potato virus Y (PVY) is one of the most damaging viruses in cultivated potatoes and otherSolanaceous crops. In this work, we studied the molecular phylogeny and the genetic variability of 107 PVY isolates selected from “GeneBank” which classification and country origins have been mentioned. The general phylogenetic profile reveals six main genetic clusters of PVY. However, the tree did not highlight a distribution of the PVY according to his geographic isolation. The genetic divergence within and between the deduced groups and subgroups shows that the minimum inter-group genetic divergence is 11%. The alignments of the deduced amino-acids sequence define consensus for each strain group as well as specific differences between them. The specific amino-acids residues of each strain group were highlighted.

Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.

Background In this study, we evaluated the prevalence of the most common mutations occurring in Enhancer II (EnhII), Basal Core Promoter (BCP), Precore (PC), and Core (C) regions of hepatitis B virus (HBV) genome. Objectives We also investigated the correlation between HBV variants, their genotypes, and patients’ HBe antigen (HBeAg: soluble shape of the capsid antigen) status. Patients and Methods We retrieved viral DNA from 40 serum samples of Tunisian patients positive for hepatitis B surface antigen (HBsAg) and HBV DNA, amplified the above mentioned regions using specific primers, and sequenced the corresponding PCR (polymerase chain reaction) products. For further analysis purpose, the patients were divided into two groups: Group1 including 34 HBeAg-negative patients and Group2 with 6 HBeAg-positive patients. Results Twenty-one patients (52.5%) showed PC G1896A mutation and 11 (27.5%) carried A1762T/G1764A double mutations. These mutations were more frequent in HBeAg-negative patients than that in HBeAg-positive ones. Indeed, 58.8% of patients bearing G1896A mutation were HBeAg-negative while 16.7% were positive. In patients bearing T1762/A1764 double mutation, 29.4% were positive and 16.7% were negative. In addition, the A1896 mutation was restricted to HBV isolates that had wild-type T1858, while C1858 was rather linked to the occurrence of T1762/A1764 mutation. Interestingly, this study revealed a high frequency of genotype E. This frequency was important as compared to that of genotype D known to be predominant in the country as delineated in previous studies. Conclusions Previous results supported and showed that HBV strains present in Tunisia belonging to genotype D and, to a lesser extent, to genotype E, were prone to mutations in BCP/ PC regions. This observation was more obvious in HBV isolates from asymptomatic chronic carriers (AsC). The high mutational rates observed in our study might result from a mechanism of viral escape that plays an important role in the loss of HBeAg.

The barks and leaves of Juglans regia L. are widely used in Tunisia to maintain good oral hygiene and are known to possess high therapeutic effects. In this paper, and for the first time, we report the reversed phase-high performance liquid chromatography (RP-HPLC) identification of polyphenols in the ethyl acetate extract obtained from the barks of J. regia L.and we tested its antioxidant properties. The results showed that the ethyl acetate extract possess high antioxidant activities comparatively to the butylated hydroxytoluene (BHT) (DPPH IC50, 3 µg/ml; reducing power EC50, 99 mg.ml-1 and IC50 = 280 µg/ml for the β-carotene bleaching test). The total antioxidant activity was estimated at 329 mg GAE.g-1 DW, the total phenolic content was 33,833 mg GAE.g-1 DW and the total condensed tannin were 16.167 mg EC.g-1 DW. The RP-HPLC showed the presence of 69 compounds: 24 with a percentage ranging from 1.37 to 15.02% including the caffeic acid (15,02%), the rutin trihydrate (12.71%), the syringic acid (3.44%), the gallic acid (2.58%) and 55 compounds with a percentage varying from 0.02 to 0.93% including the chlorogenic acid (0.8%), the resorcinol (0.78%), the vanillic acid (0.77%), the naphtho-resorcinol (0.68%), the quercetin dihydrate (0.57%), the p-coumaric acid (0.56%), the trans-cinnamic acid (0.53%) and the catechine hydrae (0.18%). In conclusion, we confirm the presence of natural compounds in the bark of Tunisian tree of J. regia with great interest, especially the polyphenols which confer to this plant a wide range of biological activities and reinforce the use of walnut bark in the developing countries in order to maintain oral hygiene. Key words : Juglans regia, reversed phase-high performance liquid chromatography (RP-HPLC), polyphenols, antioxidant activities.

In this study, we evaluated the inhibitory effect of ethanol and aqueous extracts from a stem of Opuntia ficus indica on replication of three kinds of viruses: two enveloped viruses [herpes simplex virus type 2 (HSV-2), influenza A virus (IFV-A)], and one non-enveloped virus [poliovirus type 1 (PV-1)]. Only ethanol extract from the cactus stem showed significant antiviral activity in vitro. Two chlorophyll derivatives, pheophorbide a and pyropheophorbide a, were isolated as active substances exhibiting potent virucidal effects on HSV-2 and IFV-A, but no activity against PV-1 was observed. These findings suggest that these active compounds might recognize specific glycoproteins of enveloped viruses, precluding their binding to host cell receptors and inhibiting viral infections.

Among human rotaviruses, G9 has emerged as the fifth most important genotype circulating globally. Ongoing surveillance of rotavirus in Tunisia during the past 10 years identified the first G9 strains ir. 2004. These strains exhibited the P[8] VP4 genotype and had a long RNA electrophoretype. The G9 strains were characterized by phylogenetic analysis of the VP7 gene sequence and showed high identity with other human rotavirus G9 strains belonging to the rotavirus VP7 lineage group III.

Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.

Enteroviruses were monitored in three wastewater plants that used activated-sludge, trickling-filter, and oxidation-ponds processes, respectively, from October 2000 to September 2001 in the region of Monastir, a tourist zone situated in the center of the Tunisian coast. Isolation and serotyping were conducted as recommended by the World Health Organization. Enteroviruses were present during the whole period of investigation. From February to June, however, enterovirus titers decreased (cytopathic effect45 percent); they increased during summer and autumn and at the beginning of winter. Among the isolates in the 120 wastewater samples that were collected, eight were found to be poliovirus vaccine-related, 30 were echoviruses, and 8 were untypable. Echovirus Type 6 was the serotype most frequently isolated (in 49 percent of samples) during all seasons, Some serotypes appeared occasionally (echovirus types 11, 25, and 13). Isolation of serotypes varied according to the step of wastewater treatment. Poliovirus 1 and Echovirus 6 were the most resistant serotypes.

The sequencing of the VP1 hypervariable region of the human enterovirus (HEV) genome has become the reference test for typing field isolates. This study describes a new strategy for typing HEV at the serotype level that uses a reverse transcription-PCR assay targeting the central part of the VP2 capsid protein. Two pairs of primers were used to amplify a fragment of 584 bp (with reference to the PV-1 sequence) or a part of it (368 bp) for typing. For a few strains not amplified by the first PCR, seminested primers enhanced the sensitivity (which was found to be approximately 10 −1 and 10 −4 50% tissue culture infective dose per reaction tube for the first and seminested assay, respectively). The typing method was then applied to 116 clinical and environmental strains of HEV. Sixty-one typeable isolates were correctly identified at the serotype level by comparison to seroneutralization. Forty-eight of 55 “untypeable” strains (87.3%) exhibited the same serotype using VP1 and VP2 sequencing methods. For six strains (four identified as EV-71, one as E-9, and one as E-30 by the VP2 method), no amplification was obtained by the VP1 method. The last strain, typed as CV-B4 by VP1 and CV-B3 by VP2 and monovalent antiserum, could exhibit recombination within the capsid region. Although the VP2 method was tested on only 36 of the 68 HEV serotypes, it appears to be a promising strategy for typing HEV strains isolated on a routine basis. The good sensitivity of the seminested technique could avoid cell culture and allow HEV typing directly from PCR products.

Objective: Echovirus 11 is one of the most frequently isolated enterovirus serotypes, causing a wide range of clinical diseases. We studied the genetic diversity in the 3′ end of the VP1 gene of strains from different geographical origin in the world. Methods: The sequences in the 3′ end of the VP1 of 11 Tunisian isolates were determined and aligned with the published sequences to establish a phylogenetic profile. Results: The grouping of the sequences was similar to what was previously reported by analyzing the whole VP1 gene with 4 genogroups, designated A–D, and 5 lineages in genogroup D. All Tunisian strains belonged to genogroup D, together with other sequences mainly from the USA and Europe. Contrary to the sequences from the USA isolated during the last 3 decades, which mostly belonged to the D4 lineage, those from Tunisia belonged to different lineages within genogroup D according to their isolation date: isolates from the early 1990s belonged to D3, those of the mid 1990s to D4 and the most recent ones to D5. Conclusion: Our findings further widen the interest of partial sequencing in the VP1 to study the molecular epidemiology of echovirus 11 and indicate that the genetic evolution of circulating strains may differ from one country to another according to the region’s epidemiological specificities.

Background Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes. Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured. Results Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences. Conclusion Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.