Antigenic, Immunologic and Genetic Characterization of Rough Strains B.abortus RB51, B.melitensis B115 and B.melitensis B18

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

Molecular and biological characterization of poliovirus 3 strains isolated in adriatic seawater samples

ÐIn a previous study [Muscillo, M., Carducci, A., La Rosa, G., Cantiani, L., Marianelli, C. (1997a) Enteric virus detection in adriatic seawater by cell culture, polymerase chain reaction and poly- acrylamide gel electrophoresis. Water Res. 31, 1980±1984] enterovirus strains were isolated from Adria- tic seawater and estuarine water from the Foglia River, by infecting susceptible cells with ultra®ltrated water samples. In the present work we have studied three of those samples, in which routine reverse transcriptase±polymerase chain reaction (RT±PCR) and sequencing analysis had identi®ed the presence of poliovirus type 3 (P3). In order to better estimate the risk to human health of such occurrence in bathing water (having bacteriological standards in line with the EEC directive 76/160), we set up a pro- tocol to distinguish wild from Sabin P3 strains. Three sets of RT±PCR primers were engineered and their predicted products were: 593 nucleotides (nt) in the 5' noncoding (5'NC) region (11±603), 350 nt at the Vp3±Vp1 junction (2438±2787) of the capside protein genes, and 420 nt in the 2C (4209±4628) region, which is regarded as the hotspot of recombinant polioviruses. Eight reference ATCC strains, whose sequences were known, were also tested under the same experimental conditions in order to ver- ify the accuracy of the RT±PCR reactions. The amplicons were directly sequenced by Big-dye2 termin- ator sequencing using a capillary automatic sequencer. The latter two regions found the same viral species Polio 3 in all the sample strains, with no meaningful distinction between P3/Leon/37 and P3/ Leon/12a1b, the vaccine strain. The analyses in the 5'NC region were more useful, where genetic re- lationships and the predicted secondary structure suggested that the viruses were of vaccinal sources. Molecular data were con®rmed by in vitro phenotypic marker tests rct/40, where all the examined samples displayed a temperature sensitive phenotype rct/40(ÿ). Our results suggest that the 472U4C transition alone, is not a predictive marker of reversion to neurovirulence. Finally, we conclude that the 220U constantly found in the consensus sequences of the samples can serve as a good predictor of rct/ 40(ÿ) phenotype

Enteric virus detection in Adriatic seawater by cell culture, Polymerase Chain Reaction and Polyacrilamide Gel Electrophoresis assays

Abstract--Forty samples of sea and estuary water were collected from a 40 km strip along the Adriatic coast of Italy between June 1994 and September 1995. Each sample consisted of t0 1 of water. Routine bacteriological analyses were carried out and viral particles concentrated on cross-flow membranes; the concentrated water samples, equally divided into two parts, were used to infect both BGM and Hep-2 cells. Lysates from all cell cultures were further tested for the presence of enteroviruses by reverse-transcribed polymerase chain reaction (RT-PCR) and reoviruses by polyacrylamide gel eleetrophoresis (PAGE). The results showed widespread viral contamination of the waters tested, particularly in late summer. Under our experimental conditions, BGM cells were more efficient than Hep-2 in recovering viruses. In fact, enteroviruses were detected in up to 33% and reoviruses in 80% of BGM infected with seawater, compared to 8% and 53%, respectively, for the Hep-2 cells. In estuarine samples, enteroviruses were detected in 30% and reovirus in 54% of BGM, compared to 23% and 30% of Hep-2. Twenty nine out of 40 samples showed the presence of infectious particles on the basis of the CPE appearance; after identification of the isolated viruses, only 13 turned out to be specifically contaminated by enteroviruses. Of the latter, five were below the bacteriological standards set by the Italian legislation in line with the EEC Directive 76/160 IEEC for bathing waters

Molecular analysis of poliovirus 3 isolated from an aerosol generated by a wastewater treatment plant

We examined three samples of lysates from cell cultures that had previously been infected by the aerosol generated by a waste water treatment plant near Pisa. We first attempted to confirm that we were dealing with one of the enterovirus family, using an inverse PCR analysis of the RNA extracted from the cell lysates. This identified a single genome sequences in all the samples, which corresponded to poliovirus 3. Sequence analyses revealed that the genotyping of poliovirus 3 was accurate for the species, irrespective of the genomic region sequenced. Subspecies genotyping is only possible for the translated region, and in this case, it identified the Poliovirus type 3 Leon 12alb strain as ancestor of the virus isolated. Molecular analyses were then carried out on the wild virus in the regions VP2 (9623 bp), VP3 (922bp) and VPI (l128bp) and found various nucleotide mutations (e.g. 472T~C; 970C~C.Cys,S,; ! 3196~r, Val.t,,u; 1743r~c. Va~A~a; 1817C~X. Hys~ryr). Thermosensitivity tests at 34°C and 44°C showed a slight reversion to the heat resistant phenotype. The need for adequate protective measures for plant staff, together with the frequent use of treated water for irrigation, means that effective methods of aerosol borne pathogen detection and risk determination must be adopted