This report is an overview of enterovirus epidemiology in Tunisia during a 12-year period from 1992 to 2003. A total of 4700 clinical samples were collected as part of the national poliovirus surveillance programme and the routine diagnostic programme for aseptic meningitis. Enterovirus detection was performed by isolation on cell culture according to World Health Organization recommended protocols. Serotype identification was performed by seroneutralization of the cytopathic effect using pools of specific antisera and sequencing in the VP1 region of the genome. Poliovirus isolates were assessed for their wild or vaccine-related origin by standard World Health Organization recommended methods (PCR, probe hybridization and ELISA). The results confirm the interruption of wild poliovirus circulation since 1995. A total of 236 non-polio enterovirus (NPEV) strains were isolated; seroneutralization allowed typing of 93 % (219 out of 236) of them. The antisera used allowed the identification of the most common enterovirus serotypes. The remaining 17 isolates were sequenced; 16 of them belonged to enterovirus serotypes that were not targeted by the antisera pools used. A total of 29 different serotypes of NPEV were detected in the country during the study period. Echoviruses of serotypes 6, 11 and 30 were the most frequently isolated, almost every year; other serotypes had a cyclic occurrence and others were detected during a limited period with very few isolates. The NPEV isolation rate varied from year to year but was steadily under 10 %, suggesting a relatively low prevalence of these viruses in comparison to that in other developing countries. A seasonal variation was also noted; the high transmission period starts in March and peaks in September-November. This study is the first report of the epidemiology of NPEV in Tunisia. These viruses are associated with various diseases and epidemiological data may help to clarify their impact on human health.
To estimate the susceptibility to enterovirus infection and the frequency of long-term poliovirus excreters in Tunisian patients with primary immunodeficiencies (PIDs), enteroviruses were assessed in stool specimens of 82 patients with humoral, combined, and other PIDs. Isolated viruses were typed and intratyped by standard molecular techniques, and the whole VP1 region of poliovirus isolates was sequenced. Polioviruses were detected in 6 patients; all isolates were vaccine related. Five patients rapidly stopped excretion; one excreted a poliovirus type 1 isolate for several months, and the isolate accumulated up to 14 mutations in the VP1 region. Nonpolio enteroviruses were identified in 6 patients; 4 of them kept excreting the same strain for more than 6 months. The rate of enterovirus infection was 13.4% of the PID patients and 20.7% of those with an IgG defect; it greatly exceeded the rates generally found in Tunisian supposed-immunocompetent individuals (4.1% during the study period; P ؍ 0.001 and P < 0.0001, respectively). Interestingly, patients with combined immunodeficiencies were at a higher risk for enterovirus infection than those with an exclusively B cell defect. A major histocompatibility complex (MHC) class II antigen expression defect was found in 54% of enterovirus-positive patients and in the unique long-term poliovirus excreter. The study results also suggest that substitutive immunoglobulin therapy may help clearance of a poliovirus infection and that most PID patients have the ability to stop poliovirus excretion within a limited period. However, the high susceptibility of these patients to enterovirus infection reinforces the need for enhanced surveillance of these patients until the use of oral poliovirus vaccine (OPV) is stopped.
Coxsackie B viruses of serotype 5 are associated frequently with sporadic cases of neurological diseases, epidemics of meningitis, and chronic diseases such as cardiomyopathy and diabetes. In this article, 15 strains of Coxsackievirus B5 isolated from patients with neurological disorders and healthy people were investigated by partial sequencing in the 5' half of the VP1 region and compared to other published sequences of Coxsackievirus B5, in the same genomic region. All Coxsackievirus B5 sequences showed less than 25% nucleotide difference between each other and a minimum of 27.8% of divergence with prototype sequences from other Coxsackievirus B serotypes. Within the Coxsackievirus B5 group of sequences, four clusters were individualized and may correspond to four genotypes: one genotype with large geographical distribution, containing most recent strains that have circulated from 1984 to 2000, another genotype represented by the prototype Faulkner strain, isolated in the early 1950s, and two intermediate genotypes, comprising strains isolated from 1970 to 1999 and closely related to swine vesicular disease virus. This study confirms the ability of partial sequencing in VP1 to determine serotype and to genetically characterize Coxsackievirus B5 field isolates. It gives a first approach on the molecular epidemiology of these viruses, which have a particular importance in human health.
Echovirus 6 (E6) and echovirus 11 (E11) are common causes of meningitis and other human diseases; they are among the most frequently isolated enteroviruses worldwide. In the present work we have studied genetic variability over the entire VP1 gene of selected isolates representing a wide geographical and temporal range. Fifty new sequences from North Africa were included, together with previously published sequences from different countries. The sequence diversity between strains of the same type was high: 22 and 30 % for E6 and E11, respectively. Phylogenetic analysis revealed five genogroups within each type, the genetic diversity within a genogroup generally being ,20 %. Some genogroups were further subdivided into genotypes, most containing isolates that had circulated over a wide geographical (several countries from different continents) and temporal (up to two decades) range. Several genotypes were also shown to co-circulate in a region during the same period of time. These features differ from other enteroviruses that divide into temporal or geographical clusters. This study reports new sequences from North Africa, updates the molecular epidemiology of E6 and E11, and proposes a new genogroup in each type. INTRODUCTIONHuman enteroviruses (HEVs) are small non-enveloped viruses with a worldwide distribution. Laboratory diagnosis of related infections currently is based on virus detection by isolation in cell culture or by direct PCR amplification from clinical samples. HEV types are identified by seroneutralization or partial sequencing of the VP1 region (Caro et al., 2001;Norder et al., 2001;Oberste et al., 1999a Oberste et al., , b, 2000Palacios et al., 2002). Similar to other RNA viruses, HEVs have a high capacity to evolve genetically. Genotypes are identified based on the genetic variability and phylogenetic relationships among isolates. The circulation of these genotypes throughout the world and over time has been studied to increase our understanding of the dynamics of their transmission, to evaluate their endemicity and to explain the extent of epidemics when they occur. Beyond type identification, sequencing of the VP1 region has also proved to be a reliable method for such molecular epidemiological studies.Echovirus 6 (E6) and echovirus 11 (E11) are among the most commonly isolated HEVs worldwide, and are frequently associated with outbreaks and sporadic cases of aseptic meningitis, as well as with several diseases ranging from mild non-specific illness to encephalitis, paralysis, myocarditis and severe systemic infections in neonates (Abe et al., 2000;Ashwell et al., 1996;Atkinson et al., 1998;Bahri et al., 2005;Belguith et al., 2007a;Boyd et al., 1987;Cabrerizo et al., 2008;Chomel et al., 2003;Druyts-Voets, 1997;El-Sageyer et al., 1998;Joo et al., 2005;Khetsuriani et al., 2006;Mao et al., 2010;Mirand et al., 2008;Miwa & Sawatari, 1994;Somekh et al., 2001;Ventura et al., 2001). However, despite their important impact on human health, studies of the molecular epidemiology of E6 and E11 remain li...
Genetic variability of hepatitis B virus (HBV) in the C gene and its association with the different stages of chronic liver disease has been studied inadequately with controversial results. The objectives of the current study were to determine the frequency of core promoter and precore mutations in chronic hepatitis B in Tunisia and to evaluate their impact on viral replication and disease progression. Sequencing was performed in upstream regulatory sequence (URS), pre-core (PreC) and basal core promoter (BCP) regions for 123 chronic infected patients by HBV genotype D at different status of disease. Mutations were detected in 98.4% of cases, affecting URS, BCP and Pre-C in 95.1%, 95.9% and 87.8% respectively. Multi-mutations increased significantly from asymptomatic carrier to advanced liver disease status. G1896A (74.8%), G1764A/T/C (71.5%), G1899A (54.4%) and T1678C (52%) were the most common. Special attention should be paid to A1703T, T1678C/G-A1703T, and A1652G-A1679G mutations probably specific of Tunisians sequences; they were observed in 40.6%, 41.5% and 30.1% respectively. A1679G/C, T1753C/G/A, A1762T/G and A1762T-G1764A were more prevalent in older patients. High DNA levels were associated with G1899A or G1764T/C-C1766G-C1799G and advanced liver disease with mutations at positions 1762, 1764 and/or 1899 alone or in double or triple mutations. It was also shown that substitutions at nucleotides 1762, 1764 and 1899 have an impact on the disease progression. It is the first report for specific mutations in the URS region for genotype D. It should be completed by studying eventual correlation with clinical progression and the response to treatment.
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