Rodrigo Gajardo

Viruses in Mussels: Public Health Implications and Depuration

Studies were conducted in the common mussel ( Mytilus spp .) to evaluate the public health implications derived from shellfish contamination with human pathogenic enteric viruses. In bioaccumulation experiments, we could verify that after 6 h of immersion of mussels in marine water contaminated with high levels of clay-associated enteric adenovirus (type 40) and human rotavirus (type 3), between 4 to 56% of the seeded viruses were adsorbed to shellfish tissues, mainly in the gills and digestive tract. We investigated the occurrence of wild-type enteric viruses in mussels from sites with different levels of fecal pollution. Pathogenic viruses could be detected in mussels from areas that, following current standards based on bacteriological quality, should be regarded as unpolluted, safe for swimming, and suitable for harvesting shellfish. Cooking experiments performed with contaminated mussels revealed that 5 min after the opening of the mussel valves, rotaviruses and hepatitis A virus could still be recovered in steamed shellfish. Under commercial depuration conditions, health-significant enteric viruses, such as rotavirus and hepatitis A virus, could be recovered from bivalves after 96 h of immersion in a continuous flow of ozonated marine water. Routine screening of bivalves for the presence of health-significant enteric viruses before public consumption may help in the prevention of outbreaks among shellfish consumers.

A new continuous epitope of hepatitis A virus

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 aminoacids 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 μg/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. J. Med. Virol. 54:95–102, 1998.

Adsorption-elution with negatively and positively-charged glass powder for the concentration of hepatitis A virus from water

Two methods based on virus adsorption and elution from glass powder were developed for the concentration of hepatitis A virus (HAV) from large volumes of water. The cytopathogenic pHM-175 strain of HAV was used to test these procedures in tap water, fresh water, sea water and raw sewage. HAV was quantitated by a plaque assay in the FRhK-4 cell line. HAV was concentrated by glass powder adsorption-elution from 20-liter samples with satisfactory efficiencies in all types of water: 100% for tap water, 80% for freshwater, 75% for sea water and 61% for sewage. The charge of glass powder was modified by polyethylenimine treatment to avoid the need to pretreat the sample. Concentration efficiencies of HAV in 20-1 samples through adsorption to and elution from positively-charged glass powder were 100% for tap water, 94% for sea water, and 61% for fresh water and sewage. Both methods were used for the detection of wild-type HAV in raw sewage. Wild-type HAV in concentrated sewage samples was detected by molecular hybridization with a digoxigenin-labelled cDNA probe.

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.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools

IntroductionFetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity.MethodsSCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation.ResultsSCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons.ConclusionsThe tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Prion removal capacity of plasma protein manufacturing processes

The variant Creutzfeldt‐Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large‐scale manufacturing of human plasma‐derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal.

Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction

BackgroundMesenchymal stem cells (MSCs) show promising characteristics for their use in advanced therapy medicinal products. However, there are some unresolved concerns, such as the use of animal components for their expansion.In this study we assessed the suitability of a xeno-free supplement for cell culture (SCC) derived from human plasma, to culture and expand human MSCs (hMSCs) from different origins. Characteristics of viable cultured hMSCs such as genetic stability, phenotype and multipotentiality were qualitatively evaluated.MethodshMSCs from adipose tissue (AT), bone marrow (BM) and umbilical cord (UC) and supplier sources (commercial/non-commercial) were used. After hMSCs expansion in a xeno-free medium, classical hMSCs markers were studied by immunocytochemistry, and genetic stability was tested by classic karyotyping. The capacity of hMSCs to differentiate into adipogenic, osteogenic, and chondrogenic cells in differentiation media was assessed using different staining. Different lots of SCC were used to assure consistency between batches.ResultsAll hMSCs tested maintained their morphology and adherence to plastic during their expansion, and preserved their genetic stability, phenotype and differentiation potential. No differences were observed when using different lots of SCC. Moreover, the proliferation rate, evaluated as population doubling time (PDT) of commercial BM and AT hMSCs, was higher in the xeno-free medium than in the control media provided by the suppliers of the cells (PDT of 4.6 for BM-hMSC and 6.4 for AT-hMSC in xeno-free medium, and 7.0 and 14.7 respectively in the commercial media). UC-hMSCs PDT was similar in all the media tested. When using non-commercial BM-hMSCs, PDT was lower in the xeno-free medium, but reverted to the control level with the addition of growth factors.ConclusionsSCC-containing medium can be a feasible xeno-free alternative to expand hMSCs for advanced therapies.

Absence of evidence of parvovirus B19 transmission by plasma-derived clotting concentrates derived from B19V nucleic acid technology-tested plasma and including effective steps for the inactivation or removal of nonenveloped viruses.

prolonged course of itraconazole therapy. Environmental stimuli may contribute to the production of alloantibodies. Tyler and colleagues report an infant with systemic-onset juvenile arthritis treated with infliximab (a tumor necrosis factor [TNF]-a inhibitor) who developed anti-E, anti-Fy, and anti-Jk alloantibodies and severe hemolysis in response to a single blood transfusion. TNF-a inhibitors alter the balance between Type 1 and 2 helper T-cells, favoring antibody formation. Our patient’s significantly elevated IL-2R level, indicating a high background inflammatory state, suggests an additional possible mechanism: synergy between Histoplasma infection and alloimmunization. Microbial sequencing data show striking homology of selected bacterial species with epitopes of Kell, Duffy, and Kidd blood group antigens. Prior case studies of neonatal alloimmunization were associated with concomitant, severe infection. It is possible the microbial infections reported in these studies stimulated an increased inflammatory state, with altered Th1/Th2 ratios, allowing for a more robust response to foreign RBC antigens. While the exact physiologic mechanism of neonatal alloimmunization is unclear, severe inflammation and infection appear to potentially induce alloantibody formation in certain patients. Conservative transfusion practices, including offering phenotypically matched RBCs to patients with clinical or laboratory evidence of a high inflammatory state, may be prudent.