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ABSTRACT: Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the ''high risk'' oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies.
PLoS ONE 01/2013; · 4.09 Impact Factor
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ABSTRACT: Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the "high risk" oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies.
PLoS ONE 01/2013; 8(1):e52391. · 4.09 Impact Factor
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ABSTRACT: Noroviruses (NoVs) are a major cause of acute non-bacterial gastroenteritis in all age groups. They efficiently circulate
in both clinical and environmental contexts. In this study, real-time RT-PCR methods based on TaqMan probe technology were
used to enumerate human noroviruses (genogroups I and II) in clinical samples, and in estuarine, seawater and sewage water
samples, with the aim of obtaining quantitative information on the level of viral contamination. This was achieved through
a quantitative analysis of the highly conserved region between ORF1 and ORF2, using genogroup-specific assays. RNA standards
used to construct calibration curves for the two genogroups were generated by in vitro transcription from recombinant pCR4TOPO
vectors containing a partial sequence coding for RdRp polymerase. Sewages were found to contain from 6.8×102 to 6.7×104 genome copies (GC) per millilitre; seawater samples contained from 7.6×101 to 2.4×103 per 10l. As for clinical samples, the concentrations of NoVs per gram of stool varied, ranging from 6.1×103 to 1.4×108. Real-time PCR is an easy to use, sensitive and specific tool able to generate quantitative data, and could prove useful
in both environmental and clinical settings.
Food and Environmental Virology 04/2012; 1(1):15-22. · 1.56 Impact Factor
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ABSTRACT: We evaluated the presence of the enteric viruses: norovirus, adenovirus, enterovirus, astrovirus, hepatitis A virus, and hepatitis E virus in bivalves using nested PCR methods and cell culture assays. Noroviruses GII.4 and GIV.1, adenoviruses types 1 and 2, hepatitis A, and echovirus type 7 were detected in the shellfish tested, which were often co-infected. This is the first study to detect such a high level of viral contamination in Italian mussels (up to four different viral groups in a single sample), and the first to document the presence of GIV NoV in shellfish.
The new microbiologica: official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC) 01/2012; 35(1):27-34. · 1.00 Impact Factor
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ABSTRACT: Among microorganisms, viruses are best fit to become emerging pathogens since they are able to adapt not only by mutation but also through recombination and reassortment and can thus become able to infect new hosts and to adjust to new environments. Enteric viruses are among the commonest and most hazardous waterborne pathogens, causing both sporadic and outbreak-related illness. The main health effect associated with enteric viruses is gastrointestinal illness, but they can also cause respiratory symptoms, conjunctivitis, hepatitis, central nervous system infections, and chronic diseases. Non-enteric viruses, such as respiratory and epitheliotrophic viruses are not considered waterborne, as they are not readily transmitted to water sources from infected individuals. The present review will focus on viral pathogens shown to be transmitted through water. It will also provide an overview of viruses that had not been a concern for waterborne transmission in the past, but that may represent potentially emerging waterborne pathogens due to their occurrence and persistence in water environments.
Annali dell'Istituto superiore di sanita 01/2012; 48(4):397-406. · 0.94 Impact Factor
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ABSTRACT: Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The aim of the present study was to contribute to the body of knowledge available on the molecular epidemiology of acute hepatitis E in Italy. Three sets of HEV-specific primers targeting the ORF1 and ORF2 were used to examine serum samples collected from acute hepatitis patients positive for anti-HEV IgG and/or IgM, between 2007 and 2010. Seventeen patients (39.5%) tested HEV RNA-positive: 12 infections, due to genotype 1, were associated with travel to endemic areas (Bangladesh, India and Pakistan), while five infections, due to genotype 3, were presumably autochthonous. Risk factors identified in this group included exposure to raw seafood, pork liver sausages and wild boar. Results from the present study confirm that human HEV infection in Italy is caused by different genotypes, depending on whether the infection is travel-related or autochthonous.
Journal of General Virology 04/2011; 92(Pt 7):1617-26. · 3.36 Impact Factor
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ABSTRACT: The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.
PLoS ONE 01/2011; 6(10):e24073. · 4.09 Impact Factor
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ABSTRACT: Human adenoviruses (HAdVs) are common pathogens associated with a variety of clinical manifestations. Although most infections are self-limiting, HAdVs can cause severe or lethal infections in immunocompromised as well as in healthy individuals. Several HAdVs have recently been characterized as emerging pathogens. In Italy, epidemiological, and especially molecular epidemiological, information on this pathogen is scarce. This study describes the characterization by cell culture, PCR and phylogenetic analysis of HAdV strains originating from a small collection of clinical samples gathered between 2008 and 2009. The distribution of different HAdV species was studied and the possible presence of newly emerging types was ascertained. A broad-range primer pair was used, targeting a portion of the hexon gene, in combination with species-specific primer pairs targeting a portion of the fiber gene. Human and animal reference AdV strains were included in the study. The broad-range assay identified all HAdV strains (study and reference samples), as well as three out of four animal AdV reference strains. Seven different types belonging to three HAdV species (B, C and F) were identified in the study samples. Species C was by far the most frequent. Two co-infections were detected, each with two serotypes within species C (types 1/2 and 2/6). The combined use of these two PCR assays--allowing not only the identification of known types but also, potentially, the discovery of newly emerging ones--can provide valuable epidemiological information on the spread of HAdVs.
Journal of General Virology 10/2010; 92(Pt 2):412-20. · 3.36 Impact Factor
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ABSTRACT: Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. In Italy, the true burden of HEV infection is unknown. Molecular HEV screening of raw sewage samples from 11 wastewater treatment plants yielded 19 positives (16%; 18 genotype I, 1 genotype III) evenly distributed throughout Italy. Evidence that HEV could be establishing itself in our region is accumulating and may justify more active surveillance to monitor its spread.
Applied and environmental microbiology 09/2010; 76(17):5870-3. · 3.69 Impact Factor
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ABSTRACT: Noroviruses cause acute viral gastroenteritis worldwide. They are classified in five genogroups, of which GI, GII, and GIV infect humans. Little information is available on the prevalence and clinical effects of GIV noroviruses. We conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 11 wastewater treatment plants throughout Italy, with the aim of studying the circulation of GIV NoV, as well as its genetic diversity. Eight percent of samples tested positive, and sequence analysis showed a considerable degree of genetic variability. These results imply the need for further studies to elucidate the role of this virus as a gastroenteritis-causing pathogen.
Archives of Virology 03/2010; 155(4):589-93. · 2.11 Impact Factor
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ABSTRACT: The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.
Annali dell'Istituto superiore di sanita 01/2010; 46(3):266-73. · 0.94 Impact Factor
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ABSTRACT: Microbiological indicators are commonly used in the assessment of public health risks associated with fecal contamination of freshwater ecosystems. Sediments are a reservoir of microorganisms, and can thus provide information on past pollution events, not obtainable through the testing of surface water. Moreover, pathogens present in sediment may represent future threats to human health. Clostridium perfringens, a typical colonizer of sediments, has been suggested as an alternative indicator of fecal pollution. In order to be suitable for such purpose, the microorganism should be widely distributed in contaminated environments. The objective of this study was thus to determine the composition of the anaerobic community in sediment samples of the lower Tiber basin, in central Italy, through a combined approach involving granulometric analysis of sediment samples, as well as a microbiological and molecular (16S rRNA) analysis of strains.
Granulometry showed a similar, clayey sediment composition, in most sampling sites. The microbiological method, employing, an adaptation of the standard method, proved to be effective in isolating anaerobic bacteria from the environmental matrix for the purpose of genetic analysis. Eighty-three strains of bacteria were isolated and the partial 16S rRNA gene sequenced. While biochemical analysis detected only C. perfringens strains, phylogenetic analysis indicated the presence of three clusters: C. perfringens, C. bifermentans and B. cereus, comprising eight taxa. C. perfringens, the commonest in almost all sediment sampling sites, was present in all sites, and in both seasons (seasonal sampling was carried out only along the Tiber and Aniene rivers). None of the described genetic profiles showed complete similarity with GenBank sequences.
The study underlines the value of C. perfringens as an alternative microbial indicator of fecal contamination in river sediments. This is supported by the bacterium's presence in all sampling sites, and in both seasons, coupled with its detectability using commercial diagnostic kits. The study also illustrates the presence of an anaerobic community of considerable biodiversity in the lower Tiber basin, with C. perfringens as its main component. The 16S rRNA analysis, while confirming the phylogenetic relationships among isolated species, also showed haplotype patterns different from those present in the NCBI database.
BMC Microbiology 11/2008; 8:171. · 3.04 Impact Factor
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ABSTRACT: This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.
The new microbiologica: official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC) 11/2006; 29(4):307-10. · 1.00 Impact Factor
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ABSTRACT: There is little information on the epidemiology of Human Adenoviruses (HAdVs) in Italy. In this study, 103 HAdV isolates, collected by the A. Gemelli Hospital (Catholic University Medical School of Rome, Italy) between 1987 and 2005, were genotyped by sequencing and phylogenetic analysis on a partial hexon gene region. Nine different serotypes belonging to all six HAdV species were identified. Serotype 2 was the most frequent (53.4%), followed by serotype 1 (15.53%) and serotype 41 (9.7%). Partial-hexon-based identification was confirmed as an effective tool for studying the molecular epidemiology of HAdVs.
The new microbiologica: official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC) 08/2006; 29(3):177-84. · 1.00 Impact Factor
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ABSTRACT: To identify the risk factors of an outbreak of meningitis associated with echovirus 30-infection that occurred in Rome, Italy, in late 1997 among children from two different schools.
A case-control study was carried out. A case was defined as a child from either of the two schools, A or B, who presented meningitis-like (fever, headache and vomiting), diarrhea, or respiratory tract symptoms. All asymptomatic students were included in the analysis as controls.
Among 446 pupils (80%) who answered the questionnaire, 68 met the case definition. Twenty pupils developed a meningitis-like illness. Echovirus 30 was isolated from cerebrospinal fluid (CSF) in four and from stools in six. Forty-eight pupils reported other symptoms. The attack rate was 10.8% in school A and 0.8% in school B for meningitis-like illness; it was 12% and 10%, respectively, for other enterovirus-like illnesses. The risk of meningitis-like illness was higher among children attending school A (crude OR = 14.9; 95% CI = 4.3-52.1), among children using any public pool (OR = 3.8; 95% CI = 1.5-9.9) and those using an outside swimming pool X (OR=13.4; 95% CI=2.7-65.8 versus no swimming pool and OR = 8.3; 95% CI = 1.1-62.6 versus other pools). The epidemic curve appears to suggest a person-to-person transmission.
The epidemic occurred by person-to-person transmission in a number of classrooms and at swimming pool X.
International Journal of Infectious Diseases 08/2006; 10(4):291-7. · 1.94 Impact Factor
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ABSTRACT: The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.
Infection and Immunity 03/2006; 74(2):1161-70. · 4.16 Impact Factor
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ABSTRACT: In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml. The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures. In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs. Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain. No explanation could be found for the different susceptibility of the viruses to the polysaccharides. Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.
Antiviral Research 05/2003; 58(2):139-47. · 4.30 Impact Factor
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ABSTRACT: The availability of new polysaccharide-protein conjugate vaccines against Neisseria meningitidis serogroup C prompted European National Health authorities to carefully monitor isolate characteristics. In Italy, during 1999-2001, the average incidence was 0.4 cases per 100,000 inhabitants. Serogroup B was predominant and accounted for 75% of the isolates, followed by serogroup C with 24%. Serogroup C was isolated almost twice as frequently in cases of septicemia than in cases of meningitis, and the most common phenotypes were C:2a:P1.5 and C:2b:P1.5. Among serogroup B meningococci, the trend of predominant phenotypes has changed from year to year, with a recent increase in the frequency of B:15:P1.4. Only a few meningococci had decreased susceptibility to penicillin, and, in the penA gene, all of these strains had exogenous DNA blocks deriving from the DNA of commensal Neisseria flavescens, Neisseria cinerea, and Neisseria perflava/sicca. Fluorescent amplified fragment-length polymorphism analysis revealed the nonclonal nature of the strains with decreased susceptibility to penicillin.
Clinical Infectious Diseases 03/2003; 36(4):422-8. · 9.15 Impact Factor
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ABSTRACT: Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5' noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.
Journal of Clinical Microbiology 01/2003; 40(12):4554-60. · 4.15 Impact Factor
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ABSTRACT: The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 10 l were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3 : 3 : 4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.
Water Research.
