A. Bosch

Propagation of adenovirus types 40 and 41 in the PLC/PRF/5 primary liver carcinoma cell line.

The sensitivity of cell cultures to adenovirus types 40 and 41 (Ad40/41) was compared by means of cell culture infectious dose (ID50) assays using monolayer cultures in microtitre plates. The PLC/PRF/5 cell line derived from a primary human hepatocellular carcinoma was 100 times more sensitive to a laboratory strain of Ad41, and 10 times more sensitive to a laboratory strain of Ad40 and two Ad41 stool isolates, than Graham 293 and Chang conjunctival cells commonly used for the propagation of these viruses. In microtitre plate titration assays PLC/PRF/5 cells retained an optimal condition for longer and displayed cytopathogenic effects earlier and more clearly than the other cell lines. In contrast to previously used cells, PLC/PRF/5 cells also proved successful for the quantitation of Ad41, but not Ad40, by conventional plaque assays. The reason for the exceptional susceptibility of PLC/PRF/5 cells has not been elucidated, but the findings open attractive new doors for research on Ad40/41.

Detection of fastidious infectious enteric viruses in water.

A procedure based on the infection of CaCo-2 cells and molecular hybridization with specific cDNA probes has been developed forthe detection of infectious fastidious enteric viruses in environmental samples. CaCo-2 cells, derived from a human colon adenocarcinoma, showed an increased sensitivity when compared to the usual routine host cell line to laboratory strains of rotavirus 3, reovirus 3, astrovirus 1, poliovirus 1, coxsackievirus A 24, enterovirus 70, and adenovirus 5, 40, and 41. Using this methodology, wild-type rotaviruses, enteric adenoviruses, enteroviruses, and for the first time, astroviruses have been detected in freshwater samples. Direct dot-blot hybridization alone was not sufficient for virus detection from environmental samples. CaCo-2 cells may be used as a universal in vivo amplification system for human enteric viruses, enabling the specific monitoring of infectious viral agents in the environment.

Identification of viruses isolated from sewage, riverwater and coastal seawater in Barcelona

Abstract In a virological survey carried out in Barcelona, enteric viruses were isolated in samples from a sewage outlet (90% positive isolations), from two rivers (92 and 80% positive isolations) and from three beaches (17, 17 and 8% positive isolations). Viral identifications revealed that Poliovirus was detected in all kinds of water under study, the prevalent strain being vaccine type 3.

Direct ultrafiltration performance and membrane integrity monitoring by microbiological analysis

The feasibility of substituting a conventional pre-treatment, consisting of dioxi-chlorination, coagulation/flocculation, settling and sand filtration, of a drinking water treatment plant (DWTP) by direct ultrafiltration (UF) has been assessed from a microbiological standpoint. Bacterial indicators, viral indicators and human viruses have been monitored in raw river, ultrafiltered and conventionally pre-treated water samples during two years. Direct UF has proven to remove bacterial indicators quite efficiently and to a greater extent than the conventional process does. Nevertheless, the removal of small viruses such as some small bacteriophages and human viruses (e.g. enteroviruses and noroviruses) is lower than the current conventional pre-treatment. Membrane integrity has been assessed during two years by means of tailored tests based on bacteriophages with different properties (MS-2, GA and PDR-1) and bacterial spores (Bacillus spores). Membrane integrity has not been compromised despite the challenging conditions faced by directly treating raw river water. Bacteriophage PDR-1 appears as a suitable microbe to test membrane integrity, as its size is slightly larger than the considered membrane pore size. However, its implementation at full scale plant is still challenging due to difficulties in obtaining enough phages for its seeding.

Fecal pollution in llobregat river: Interrelationships of viral, bacterial, and physico-chemical parameters

A 19-mo survey of enterovirus in the Llobregat river, located south of Barcelona was conducted. Total coliforms, fecal coliforms, and fecal streptococci counts, as well as several physico-chemical parameters were also measured at two sampling sites. The data show a very high level of fecal and chemical pollution caused by untreated wastewater effluents from several suburbs of Barcelona.

Compensation of Cyclophosphamide Immunosuppression by a Bacterial Immunostimulant (Broncho-Vaxom) in Mice

The compensatory effect of a bacterial lysate, Broncho-Vaxom (BV) on the immunosuppressive action of cyclophosphamide (CY) was investigated. In CY immunosuppressed mice, BV treated animals recovered to normal levels of IgM and IgG in serum as well of IgA and IgG in gut secretions significantly earlier than controls. Furthermore, normal cell proliferation in thymus, as estimated by measuring the relative size of this organ was achieved earlier in BV treated mice than in control mice. Oral treatment with BV restores the number of IgM anti SRBC producing cells in spleen, in CY immunosuppressed mice. Since immunosuppression induced by CY increases the susceptibility to various infections, we tested in immunosuppressed animals the protective effect of BV towards IP challenge infections with Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae var ozaenae, Pseudomonas aeruginosa and Candida albicans. BV led to an enhanced resistance towards both pneumococci and staphylococci challenge infections but not to the other challenge microorganisms.

Viral contaminants of molluscan shellfish: detection and characterisation

Abstract: Environmental virology started with the detection of poliovirus in water. Since then other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection. Most shellfish-borne viral outbreaks are restricted to norovirus and hepatitis A virus, making them the main targets for bivalve virological analysis. The inclusion of virus analysis in regulatory standards for viruses in molluscan bivalve samples must overcome several shortcomings such as the technical difficulties and high costs of virus monitoring, the lack of harmonised and standardised assays and the challenge posed by the ever-changing nature of viruses. Nowadays methods are available to detect, quantify and characterise viral pathogens in molluscan shellfish to reduce the risks of shellfish-borne virus diseases.

Retroviral properties inherent to viral erythrocytic infection in sea bass

SummaryThe characterization of the aetiological agent of viral erythrocytic infection (VEI) of sea bass suggests a retroviral origin of the disease. RNA from viral erythrocytic infection virus (VEIV) and DNA from blood and organs of VEI-affected fish hybridized to a specific retrovirus cDNA probe. Sequences homologous to retrovirus genome were also detected in non-infected SBL cells (a sea bass cell line), however, Southern blot analysis showed that the DNA restriction patterns in VEI-affected erythrocytes differed from those of SBL cells. RNA-dependent DNA polymerase activity was detected in VEI-affected sea bass blood. This reverse transcription was strongly Mn2+-dependent and is the first report of its occurrence in a marine fish and in fish blood samples. Nucleic acid sequences homologous to retrovirus RNA were detected in chromatographic fractions exhibiting reverse transcriptase activity and the presence of virus-like particles, 125–150 mm in diameter. The density of VEIV in sucrose was 1.17–1.18 g/cm3. The symptomatology of VEI is not far from those described for some retroviral diseases.

Concentration of fish enveloped viruses from large volumes of water

The validity of several concentration procedures for the detection of fish enveloped viruses present in large volumes of water was determined. Viral haemorrhagic septicaemia virus (VHSV) was used to evaluate adsorption/elution to positively-charged MK filter cartridges for the concentration of enveloped viruses. For fresh water, the efficiency of the procedure ranged from 12 to 100%, with a mean recovery of 57%. In seawater samples, the recoveries varied from 15 to 100%, with a mean recovery of 59%. The same virus was used in methods such as organic flocculation and ammonium sulphate flocculation with very poor recoveries of infectious virus, caused by the inactivation of VHSV in both procedures. Concentration of seawater samples from tanks housing sea bass or gilthead affected by viral erythrocytic infection and lymphocystis, respectively, were carried out. In both cases, the viruses responsible for the outbreaks were detected by electron microscopy in the concentrated water samples.

Foodborne viruses: understanding the risks and developing rapid surveillance and control measures

Abstract: Viruses are increasingly recognized as pathogens involved in foodborne infections. The viruses most adapted and likely to be carried in this way are those transmitted by the faecal-oral route. These include viral agents causing gastro-intestinal disease in humans such as noroviruses (NoV), but also agents such as hepatitis A virus (HAV), which, although being transmitted by the faecal-oral route, exhibit their classical clinical symptoms elsewhere in the body. Data from a Europe-wide study of NoV outbreaks illustrate that outbreaks are commonly detected in all countries that have sufficient diagnostic capacity and undertake systematic evaluation of outbreaks of gastro-enteritis other than for bacterial infections. Experience with other pathogen/food matrix combinations has demonstrated the necessity to establish sampling strategies in order to undertake effective monitoring of foodstuffs for risk management and control purposes. The ultimate goal in source tracing is the possibility to rapidly type the isolates. This is particularly relevant for the highly variable NoV, which together with HAV, is the main target for detection in food.