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ABSTRACT: Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR).
The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters.
The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl.
The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases.
Pathologie Biologie 06/2009; 59(3):142-5. · 1.53 Impact Factor
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ABSTRACT: Hepatitis A virus (HAV) is responsible for around half of the total number of hepatitis infections diagnosed worldwide. HAV infection is mainly propagated via the faecal-oral route and as a consequence of globalisation, transnational outbreaks of foodborne infections are reported with increasing frequency. Molecular procedures are now available and should be employed for the direct surveillance of HAV in food and environmental samples.
Letters in Applied Microbiology 08/2007; 45(1):1-5. · 1.62 Impact Factor
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ABSTRACT: Molecular methods for the detection and typing of hepatitis A virus (HAV) strains in sewage were applied to determine its distribution in Cairo and Barcelona. The study revealed the occurrence of different patterns of hepatitis A endemicity in each city. The circulating strains characterized, whether in Cairo or Barcelona, were genotype IB. The effects of a child vaccination programme and the increase in the immigrant population on the overall hepatitis A occurrence in Barcelona were evaluated. While vaccination contributed to a significant decrease in the number of clinical cases, the huge recent immigration flow has probably been responsible for the re-emergence of the disease in the last year of study, in the form of small outbreaks among the non-vaccinated population.
Epidemiology and Infection 03/2007; 135(2):270-3. · 2.84 Impact Factor
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Journal of Fish Diseases 04/2006; 12(2):185 - 191. · 2.00 Impact Factor
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ABSTRACT: Human astroviruses (HAstV) are important human pathogens causing gastroenteritis worldwide. The increased recognition of astroviruses as the cause of sporadic outbreaks of disease is due to the recent availability of improved diagnostic methods. During the last decade, most epidemiological surveys have chosen astrovirus-specific RT-PCR as screening methods. In addition to serotyping by molecular techniques, new typing methods are being developed that may also identify other viral properties related to virulence. The information provided by different typing assays is required for a better understanding of both the antigenic diversity and the molecular mechanisms of pathogenicity.
Letters in Applied Microbiology 02/2005; 41(2):103-5. · 1.62 Impact Factor
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M Divizia,
R Gabrieli,
D Donia,
A Macaluso, A Bosch,
S Guix,
G Sánchez,
C Villena,
R M Pintó,
L Palombi,
E Buonuomo,
F Cenko,
L Leno,
D Bebeci,
S Bino
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ABSTRACT: Three different studies are reported concerning the environmental pollution caused by viruses in Albania. The first study describes an outbreak of gastroenteritis in the capital city, involving 2,722 children attending the Paediatric Unit of Tirana Hospital. The age group with the highest morbidity was 0-5 years, with 89.5%; no fatalities were recorded during the outbreak. Rotavirus was detected in 26/28 faecal samples by RT-PCR, although astrovirus, adenovirus and calicivirus were also present. The second study describes an outbreak of hepatitis A virus involving the city of Lac. Two hundred cases were recorded, with the highest incidence in the age-group 5-9 years. Phylogenetic analysis of the VP1/2A region showed the presence of a unique sequence: genotype IA. Rotavirus was identified in drinking-water samples collected during the outbreak. The third study describes the prevalence of HAV and HEV in 202 sera randomly collected from 12 different cities in Albania. HAV showed a high incidence (66.2%), whereas none was positive for HEV. The genomic analysis of the VP1/2A junction revealed the presence of only one genotype (IA) with few point mutations and just two amino acid substitutions at codons 22 and 34. Additionally, two potential antigenic variants were detected, the first at position 46 of VP3 and the second at position 23 of VP1.
Water Science & Technology 02/2004; 50(1):57-61. · 1.12 Impact Factor
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C Villena,
R Gabrieli,
R M Pintó,
S Guix,
D Donia,
E Buonomo,
L Palombi,
F Cenko,
S Bino, A Bosch,
M Divizia
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ABSTRACT: By the end of December 2000, the epidemiological system 'Alert' of the Public Health Institute in Tirane reported an outbreak of acute gastroenteritis. The outbreak involved children in Tirane and in the rural area. In total, 2722 children were seen in Tirane Hospital and 982 (56.4%) were treated for acute gastroenteritis. The age group with the highest morbidity was 0-5 years (89.7%), followed by the 6-9 (6.2%) and 10-15 years age groups (4.1%). The distribution of acute gastroenteritis cases, which occurred along the same water distribution system, suggests a waterborne origin. The nucleic acid amplification confirmed the co-circulation of different genotypes of rotavirus, mainly P[8]G9 and P[8]G3, responsible for the outbreak. Other enteric viruses such as astrovirus serotype 1, adenovirus and Norovirus, genogroups I and II were detected. Co-infections with different rotavirus genotypes and even with different enteric viruses were detected in several samples.
Epidemiology and Infection 01/2004; 131(3):1105-10. · 2.84 Impact Factor
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J Buesa,
B Collado,
P López-Andújar,
R Abu-Mallouh,
J Rodríguez Díaz,
A García Díaz,
J Prat,
S Guix,
T Llovet,
G Prats, A Bosch
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ABSTRACT: The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a "new variant" designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.
Journal of Clinical Microbiology 09/2002; 40(8):2854-9. · 4.15 Impact Factor
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ABSTRACT: A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.
International Journal of Food Microbiology 03/2002; 74(1-2):119-30. · 3.33 Impact Factor
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ABSTRACT: Procedures for the detection of astroviruses in wastewater samples have been developed and evaluated. Following these methodologies, we investigated the occurrence of astroviruses in wastewater samples from three different sewage treatments plants located in Southern France and two in the Barcelona area. Some positive samples were genotyped by analysis of a fragment of the ORF1a by restriction fragment length polymorphism (RFLP) with endonuclease DdeI. The amplimers generated contain several sites for the DdeI restriction endonuclease, being the number and location of sites different between strains.
Water Science & Technology 02/2001; 43(12):73-6. · 1.12 Impact Factor
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ABSTRACT: An outbreak of hepatitis A, affecting 183 people, occurred in Valencia (Spain). Epidemiological evidence pointed to an association of the outbreak with consumption of Coquina clams (Donax sp), imported frozen from Peru. Shellfish were analysed for the presence of hepatitis A virus (HAV), enteroviruses, rotaviruses, astroviruses, caliciviruses and hepatitis E virus. HAV was detected in 75% of assayed shellfish samples. Other enteric viruses were occasionally found in the same samples. Molecular epidemiological analysis of fragments of the VP1/2A and the 5' end of the genome from shellfish and sera isolates, revealed the presence of six variants belonging to a single genotype.
Water Science & Technology 02/2001; 43(12):61-5. · 1.12 Impact Factor
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ABSTRACT: The immune responses elicited in mice by different forms of the VP3(110-121) B-epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T-epitope, and a third containing a putative T-epitope of HAV (VP3(102-121)) were assayed. While this latter T-epitope induced an enhancement of the response against the VP3 B-epitope, the artificially coupled T-epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110-121) and VP1(11-25) HAV sequences and the second only the VP1(11-25) sequence, also suggested the presence of a T-epitope, since the response against the VP1 peptide was increased in the first construct.
FEBS Letters 11/1998; 438(1-2):106-10. · 3.54 Impact Factor
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A Bosch
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ABSTRACT: Water virology started around half a century ago, with scientists attempting to detect poliovirus in water samples. Since that time, other enteric viruses responsible for gastroenteritis and hepatitis, among a great variety of virus strains, have replaced enteroviruses as the main target for detection in the water environment. Technical molecular developments, polymerase-chain reaction (PCR) amplification being the method of choice, enable the detection of fastidious health-significant viruses. However, shortcomings of molecular procedures include their potential incompatibility with concentration methods, indispensable to reduce the water sample volume to assay for viruses, the inability to discern between infectious and non infectious material. On the other hand, these procedures are restrained to sophisticated laboratories and detection of alternative indicator organisms has been proposed. Bacterial indicators fail to give a reliable clue of the virological quality of water. Selected bacteriophage groups appear as a better choice for their use as virus indicators.
International Microbiology 10/1998; 1(3):191-6. · 1.80 Impact Factor
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ABSTRACT: A new continuous epitope of hepatitis A virus (HAV) was defined in the VP3 protein. Convalescent sera recognised the synthetic peptide 3110-3121 (FWRGDLVFDFQV). The replacement of the arginine, glycine, or aspartic acid at positions 112, 113, or 114, respectively by other amino acids induced the loss of synthetic peptide recognition by human convalescent sera, thereby confirming the presence of an epitope in the original VP3(110-121) sequence. Shorter VP3 peptides such as VP3(110-119). VP3(110-117), and VP3(110-116) and a tandem repeat of VP3(111-116) failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. The maximum inhibition of human convalescent binding to HAV by the VP3(110-121) peptide was around 60%, and 50% inhibition was achieved at a peptide concentration of 2.3-2.4 micrograms/ml. Antibodies generated by this peptide bound to intact HAV and neutralised its infectivity. Antipeptide antibodies inhibited convalescent serum binding to HAV. Monoclonal antibodies H7C27 and MAK-4E7 inhibited completely binding of the antipeptide antibodies to HAV.
Journal of Medical Virology 03/1998; 54(2):95-102. · 2.82 Impact Factor
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ABSTRACT: The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus.
FEMS Microbiology Letters 12/1997; 156(1):107-11. · 2.04 Impact Factor
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ABSTRACT: A method based on infection of CaCo-2 cultured cell monolayers (CC) and reverse transcription-PCR (RT-PCR) was developed for the specific detection of infectious astrovirus. The procedure was validated by titrating poliovirus stocks in parallel in CaCo-2 cells by determining the most probable number of cytopathogenic units and by cell culture and subsequent RT-PCR (CC-RT-PCR). CC-RT-PCR was then employed to measure the persistence of astrovirus suspended in dechlorinated tap water. After 60 days, the decay of astrovirus infectivity was 2 log units at 4 +/- 1 degrees C and 3.2 log units at 20 +/- 1 degrees C, while after 90 days, the titer reduction was 3.3 and 5 log units at 4 +/- 1 degrees C and 20 +/- 1 degrees C, respectively. Astrovirus decay in the presence of free chlorine (FC) was monitored by CC-RT-PCR. Residual infectivity was found after 2 h in the presence of 1 mg of FC/liter. Under these conditions, astrovirus shows a log titer reduction (LTR) or 4, while 0.5 mg of FC/liter induced an LTR of 2.4. The possibility of acquiring data on the survival of fastidious viruses in the environment opens new perspectives on the epidemiology of some significant infections transmitted by the fecal-oral route.
Applied and Environmental Microbiology 09/1997; 63(8):3119-22. · 3.83 Impact Factor
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ABSTRACT: Volume 62, no. 5, p. 1811, column 2, line 2: Reference "(16)" should read "(15)." Page 1812, column 1, line 17: "Willcocks and Carter (15)" should read "Willcocks et al. (14)." [This corrects the article on p. 1811 in vol. 62.].
Applied and Environmental Microbiology 09/1996; 62(8):3073. · 3.83 Impact Factor
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ABSTRACT: Five microwell non isotopic hybridization assays, based on colorimetric immunoenzymatic reading, were developed and evaluated for the rapid and automatable detection of enteroviruses. Virus nucleic acids and/or capture probes were covalently bound to microtiter wells, and digoxigenin-11-dUTP was used as label for the detection of hybridized material. Among these procedures, a reverse transcriptase polymerase chain reaction (RT-PCR) hybridization assay was the most sensitive, enabling the detection of 10 MPNCU of poliovirus, and offering detection specificity for other enteroviruses, such as coxsackieviruses and echoviruses. The second most sensitive method was a complementary hybridization assay, simultaneously using three detection probes, one from the 5' end and two from the 3' end of poliovirus genome, offering a sensitivity for poliovirus detection of 5 x 10(3) MPNCU.
Molecular and Cellular Probes 05/1996; 10(2):81-9. · 2.08 Impact Factor
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ABSTRACT: Rotaviruses from environmental samples have been genotyped by a seminested reverse transcription PCR assay with serotype-specific primers derived from variable regions of gene 9, which produce different characteristic segment sizes for serotypes 1 to 4. The method enabled the detection and identification of type 1, 2, and 3 group A rotaviruses in sewage.
Applied and Environmental Microbiology 10/1995; 61(9):3460-2. · 3.83 Impact Factor
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Environmental Science and Technology 10/1995; 29(10):2636-8. · 5.23 Impact Factor
