Patrizia Orsini

Molecular techniques for the identification of enteric viruses in marine waters

Abstract The RNAs extracted from 28 high spin pellets of clarified lysates from BGM cells, previously infected by concentrated Adriatic Sea water samples, were analyzed by a dot-blot test. We used a 32 P-labeled cDNA probe ( BamH I 220–670 ) from the 5′ non-coding end of the cloned poliovirus I (Mahoney) genome. The probe was selected for its broad range of genetic specificities. Based on the density of the hybridization signals on the autoradiograph, our dot-blot results showed a high degree of reactivity of the probe to homologous (poliovirus 1) and heterologous (echovirus 6 and coxsackievirus B3) reference RNA, as predicted by a computer analysis of their sequences, and a low reactivity to that of the samples leading us to exclude the presence of enteroviruses like the reference strains. In order to understand if there were other enteroviral serotypes or not, dot-blot tests were supplemented with Northern-blot hybridization assays. Results from the Northern-blot showed a series of fragments ranging from 4.3 to 1.2 kb but no signal corresponding to 7.5 kb (as did the positive control RNAs). These features suggested that the tested RNAs might derive from viruses of the Reoviridae family, which includes members with segmented genome. Traditional PAGE analysis showed clearly the 10-fragments pattern characteristic of reoviruses. Twenty-three out of the twenty-eight samples tested showed the presence of viruses, and this confirms the previously noted cytopathic effect (CPE) of the samples on the BGM cells.

Enteric viruses in a wastewater treatment plant in Rome

Over the course of a year a series of samples were taken from a wastewater treatment plant handling domestic sewage at each stages of the process, to detect the presence of enteric viral and classical bacterial indicators, and physicochemical parameters.The viruses were isolated on BGM cell cultures and counted according to the Most Probable Number method.The values of enteric viruses varied from 102 to 104/L in raw sewage and from 100 to 103 in final effluent.The efficiency of the plant at each stages during processing was evaluated.The parameters analysed show a systematic reduction of values between input and output, with average bacteriological reductions of 88% (fecal streptococci), 93% (fecal coliforms) and 94% (total coliforms), viral load reduced by 0–99%. COD and suspended solids showed a reduction of 61 % and 71% respectively.The 40% of isolated viruses were submitted to identification procedures using molecular techniques and pools of antisera. The viral types identified were enteroviruses (poliovirus and coxsackievirus B) and reoviruses.Viruses appear less easily removed than classical bacterial indicators. Reoviruses were removed less efficiently than enteroviruses.

Bacteriological and virological quality of seawater bathing areas along the Tyrrhenian coast

Monitoring was carried out during summer 1997 along a selected area of the Tyrrhenian coast near the Tiber river mouth. Fifty-eight seawater samples, collected from 19 stations, were examined for coliforms, streptococci, Enteroviruses, Salmonellae, coliphages, Bacteroides fragilis phages, Pseudomonas, alophilic Vibrios, Aeromonas and yeasts. Salmonellae and coliphages were isolated in 3 and 12 out of 58 samples, respectively. Enteroviruses and Bacteroides fragilis phages were not isolated. Reoviruses were isolated only from 2 out of 58 samples. A limited number of samples of the northern stations located near the Tiber and other river mouths exceeded the guide values for bathing water by the EU Directive. All the southern stations, located near canals, were of very good microbiological quality. Pseudomonas, Vibrio, Aeromonas and yeasts were isolated from all stations and their values in 100 ml of seawater were 10-10 6 , 10-10 6 , 0-10 6 and 1-10 3 , respectively. An extensive disinfection practice carried out on domestic wastes, which are discharged in rivers and canals, probably brought pollution levels of most stations to values within the bacterial standards. The spread of Pseudomonas, Aeromonas, etc. showed that all the coastal area studied was characterized by the presence of organic matter coming from land that can support the presence of opportunistic pathogens and other microbial flora.

Comparison of cDNA probe hybridizations and RT-PCR detection methods for the identification and differentiation of enteroviruses isolated from sea water samples

Abstract Fifteen enteroviruses (EVs) previously isolated from Tyrrhenian sea water samples were used. They were first identified by traditional dot-blot and Northern-blot hybridizations with a group of cDNA probes from cloned Poliovirus 1 and Coxsackievirus B4 and oligodeoxynucleotides complementary to echovirus 6 and 9 sequences. Using both wild viruses and known enteroviruses a reverse-PCR protocol was then set up followed by cDNA sequencing of the fragments generated. The sequences of primers were selected from a consensus of several 5′ non-coding ends of enterovirus genomes, representing highly conserved regions. The downstream (region 577–603) and the upstream (region 436–465) oligonucleotide primers carried an extra sequence in order to generate BamH I and a Hind III restriction sites at the 5′ and 3′ end respectively of the amplified cDNA fragments for directional cloning in a plasmid. The downstream 5′NC primer was 5′-biotinylated in order to allow direct sequencing of the amplicon, when possible, after strand separations on streptavidin coated magnetic beads. The PCR of reverse transcribed viral RNAs resulted in a 167–170 b.p. cDNA product on ethidium bromide-stained 2% agarose gels in all the samples and reference viruses. The test is negative on reoviruses, hepatitis A and uninfected BGM cells and detects 50 viral particles. Sequences of cloned fragments were compared with sequences of cloned enteroviruses stored in commercial data banks. The 5′NC region of a reference echovirus 5 was also cloned and sequenced to improve the comparison. On the basis of deduced genetic distances, three poliovirus 1, eight coxsakievirus B5, four coxsakievirus B1 were diagnosed. One poliovirus Sabin 2 was isolated together with a coxsackievirus-related strain in the same lysate sample. The reliability and sensitivity of this RT-PCR method makes it an attractive approach to virus detection in environmental samples.

Microbiological and chemical quality of sludges from domestic wastewater plants

Digested sludge samples from domestic wastewater treatment plants located in Northern Italy were tested as far as the presence of viruses (enteric viruses and coliphages), bacteria (faecal coliforms, salmonella) and helminth eggs is concerned. Heavy metals were also analysed. Escherichia coli bacteriophages and faecal coliforms were isolated from all samples, while salmonellae and helminth eggs were isolated only from four and three out of 27 total samples, respectively. The 66% of sludge samples, 46 and 82% of aerobic and anaerobic digested sludges respectively, showed the presence of enteric viruses (enteroviruses and reoviruses). The virus concentrations ranged from 0.6 to 123 MPNCU/g. Results of this study suggest that significant concentrations of pathogens such as enteric viruses can be present in digested sludge, which showed compliance with Italian legislation. As suggested by other authors, there is the need for surveillance and reference indications both in EU (European Union) and Italian regula...