Infrared spectroscopy tracing of sediment sources in a small rural watershed (French Alps).

The present article describes a first attempt to use infrared spectroscopy to trace the origin of suspended river sediments. Fifty samples of the main potential sediment sources within a small catchment area (990 ha) in the French Alps were collected and compared with samples of suspended sediment from the river, collected on various dates during 2006 and 2007 using sediment traps. Two major categories of sediment source were identified: topsoils and river channel sediments. For the qualitative part of the study, each of these two main categories was divided into two sub-categories, that is to say, cultivated and pastureland topsoils, and riverbed and riverbank sediments. Discriminant analysis on the source samples showed that Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy can be used to differentiate between the four potential source materials. To determine whether or not immersion in the river altered the infrared spectra of these source materials, we measured the infrared spectra of samples that had been immersed in the river, in litter bags, for periods of up to 24 days. Immersion did not cause any major changes in the infrared spectra. The contribution of each type of source material to the suspended sediment in the river was quantified using partial least squares (PLS) analyses of DRIFT spectra to compare actual river sediment samples with an experimental model. This model was produced from the DRIFT spectra of a range of calibration samples produced by mixing source material samples in different ratios. The predictions of the model were valid and fell within the confidence interval calculated for the calibration set. Comparisons between suspended sediment samples and the model indicate that the predominant source of the sediment is riverbank erosion, which, in this case, is probably due to trampling by cattle.

Reduction of chloroform formation potential of humic acid by sonolysis and ultraviolet irradiation.

This study is concerned with the changes of chloroform formation potential during the reaction of humic acid (HA) and sodium hypochlorite caused by different oxidative pretreatments: ultraviolet (UV) irradiation, ultrasonic (US) irradiation or combined UV-US irradiations. The UV and US decomposition of a reagent HA in water was investigated. The characterization of the oxidized HA sample by UV absorptiometry, synchronous fluorescence spectroscopy and size exclusion chromatography points a synergetic effect of the combined process. The values of the chlorine demand and chloroform formation potential were conventionally determined after a 96 h reaction at neutral pH. It was found that all applied processes decreased the concentration of chloroform but the highest decrease was observed for the UV-US treatment.

Selection of receptor-binding variants of human influenza A and B viruses in baby hamster kidney cells.

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D --> G (H1N1 virus), 128T --> A and 226I --> V (H3N2), and 187N --> D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L --> H, 208R --> H, and 225D --> G (H1N1), 194L --> I (H3N2), and 137G --> R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3'-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6'-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6'-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.

Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.

Vero cells, MDCK cells and embryonated chicken eggs (eggs) were used to evaluate influenza virus growth characteristics and immunogenicity induced by inactivated influenza B vaccines. Both cell lines produced comparable quantities of total viral and haemagglutinin (HA) proteins. Sequence analysis indicated genetic identity of the HA of Vero- and MDCK-grown virus counterparts with maintenance of antigenic characteristics of viruses derived from humans. The egg-grown influenza B/Memphis/1/93 variant differed from cell-grown counterparts at amino acid position 198 (Pro-Thr) and lost a glycosylation site. The level of neuraminidase (NA) activity was the highest in egg-grown virus, while MDCK- and Vero cell-grown viruses possessed 70% and 90% less NA activity respectively when fetuin was used as a substrate. Although each of the vaccines induced high and comparable levels of serum antibodies, mammalian cell-derived vaccines induced antibodies that were more cross reactive, and those antibodies induced by egg-derived vaccine were more specific to the homologous antigen. ELISPOT analysis indicated that the mammalian cell-grown vaccines induced high frequencies of IgG-producing cells directed against both cell- and egg-grown antigens, while egg-grown vaccine induced high frequencies of IgG and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown virus antigen. Taken together, our results suggest that mammalian cells are a viable option for the production of influenza virus vaccines.

Immunogenicity and protective efficacy in mice of influenza B virus vaccines grown in mammalian cells or embryonated chicken eggs.

The immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated chicken eggs (hereafter referred to as eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.

A varicella vaccine stable at 5 degrees C.

Varicella-Zoster virus (VZV), which causes Chicken Pox and Zoster, belongs to the Herpes viridae family [1, 2]. The virus is strongly cell dependent and its in vitro stability is very low. Following Takahashi's work, we have developed and prepared a vaccine with the OKA strain virus in the Japanese stabilizer. To improve the stability of the virus in a freeze-dried form, we have finalized a new formulation of stabilizer, called V 15-1. This stabilizer is a protein-free solution with defined contents of amino acids, salts and sugars. Comparative stability studies between the Japanese stabilizer and V 15-1 have been performed at different temperatures. We have demonstrated good stability for two years at + 5 degrees C and at + 25 degrees C and + 37 degrees C for the vaccine in freeze-dried form. We have also found a good stability of this vaccine at + 5 degrees C and + 25 degrees C after reconstitution. The use of V 15-1 thus allows us to prepare a Varicella vaccine with the OKA strain (> 2000 PFU per dose) which has good stability at 5 degrees C. A Varicella vaccine using a live attenuated virus strain OKA was developed in 1974 by Takahashi in Japan [3, 4]. The virus was isolated from vesicular fluid collected from a child with chicken pox. After propagation in human embryonic lung cells (HEC), guinea pigs embryonic cells (GPE) and human diploid cells (WI 38), the virus was attenuated. All the licensed vaccines are prepared with the OKA strain [5].

Good immunogenicity of GBM strain inactivated hepatitis A vaccine in healthy male adults.

A formalin-inactivated aluminium hydroxide adsorbed hepatitis A vaccine was evaluated in a dose-response study on 195 healthy male adults (age range: 18-31 years) in two French hospitals (Lyon, Rouen). Four doses (20, 40, 80, 160 RIA antigen units) were administered intramuscularly (i.m.) in two injections over a 6-month period. At the time of the first vaccine injection, 32 subjects (16.4%) were found positive (> 20 mIU ml-1) for HAV antibody (total Ig RIA HAVAB assay, Abbott Laboratories) and were excluded from the analysis of immunogenicity criteria. Fourteen days after the first vaccine injection, 78.1% (95% confidence interval (CI): 62-90) of seronegative subjects who received the 160 RIA antigen unit dose seroconverted with a geometric mean titre (GMT) of 43 mIU ml-1 (95% CI: 33-56). Seroconversion was 100% (95% CI: 91-100) at 1 month with a GMT of 95 mIU ml-1 (95% CI: 79-112). Statistical analysis revealed a significant dose-related effect (p < 0.0001) on GMT by multivariate regression analysis of the results after the first injection. Biological safety was evaluated and alanine aminotransferase and aspartate aminotransferase levels were similar prior to and 14 days after the first injection in the four groups. Reactions after injection were similar in the four dosage groups: 6.2% of subjects reported immediate reactions after first vaccination (feeling sick, spontaneous pain, headache), 8.9% reported local reactions at the site of injection (spontaneous pain, haematoma, local adenopathy) and 13.5% reported general reactions ('flu-like' syndrome, gastrointestinal tract disorders, fatigue, headache).(ABSTRACT TRUNCATED AT 250 WORDS)

Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier.

In 1980, the authors reported preliminary results of large-scale production of inactivated poliovirus vaccine in which virus was produced in Vero cell culture on a microcarrier. For this first stage of development, 150-liter tanks were used. The virus is now produced in 1,000-liter tanks. The main point concerning the quality of Vero cells, namely the absence of tumorigenicity, has been demonstrated, qualifying them for use in the Institut M erieux cell bank. The purity of the cell line has also been determined by checking for the absence of bacteria, fungi, mycoplasmas, and viruses. The search for oncornavirus and for reverse transcriptase activity was carried out, and the results were negative but are not described in this paper. The quality of the purification process was checked by a search for residual cellular DNA in concentrated, purified, and inactivated vaccine. With use of a molecular hybridization procedure, a specific probe was prepared to detect approximately 50 pg of DNA per filter. The preliminary results show that the purification procedure fulfills the World Health Organization's requirements. T1 oligonucleotide mapping has also shown the identity of poliovirus RNA extracted from virus grown on Vero cells and that from primary monkey kidney cells. These data have led to the awarding of a license by the French government to the Institut M erieux for production of this new, reassessed, inactivated poliovirus vaccine.

Thousand litre scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results.

Through the progress of scientific knowledge the Vero cell line was considered to be a suitable alternative cell substrate for the industrial production of Polio Virus. Using microcarrier culture, more than 10(12) cells could be obtained weekly for virus inoculation. The virus yield is around 60 D units/ml for type I; 20 D units/ml for type II, and 50 D units/ml for type III.

The large-scale cultivation of VERO cells in micro-carrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine.

As the increasing shortage of monkeys is a reality, the application of an alternative cell substrate for large-scale production of Killed Poliomyelitis Vaccine (KPV) was studied. Through progress of scientific knowledge the non-tumorigenic VERO cell line was considered to be a suitable alternative cell substrate for this purpose. The Master-Cell-Bank and Working-Cell-Banks prepared by us are giving a practically inexhaustible cell source. Using micro-carrier culture, weekly more than 400 billions of cells at a concentration of 10(6) cells per ml could be obtained for virus inoculation. The virus yield per cell was at least as high as for primary monkey kidney cells. Processing of virus harvests could be performed according to the methods used at the production on primary monkey kidney cells. From a technological view-point large-scale production of KPV on VERO cells appears to be possible economically. More research on the safety control might be necessary.

Comparison of sensitivity of VERO cell line versus primary monkey kidney cells in the detection of residual live polio virus during and after inactivation.

Substitution of Primary Monkey Kidney (PMK) Cells by Monkey Cell Lines (MCL) for detection of residual live virus in poliovaccine control has been considered for various reasons. The sensitivity of VERO Cells versus PMK cells has been investigated. This study was done with poliovirus during and just after formalin inactivation. The amount of residual live virus was tested by TCID50 end-point titrations in roller-tubes and small bottles at different stages in the inactivation process. Just before or at the end of inactivation, a 1 000 doses vaccine equivalent volume was tested on Roux bottles. Thus far the results show that VERO cells seem to be less sensitive than PMK cells, especially for type 3 virus. Additional studies will be needed to investigate whether the sensitivity of the VERO cells can be improved to detect residual live poliovirus during and after inactivation with the same sensitivity as PMK cells.

Direct enzyme linked immunosorbent assay (ELISA) for quantification of poliomyelitis virus D-antigen.

The proposed micro-ELISA assay by means of the double antibody method involves three steps: adsorption of type specific antiserum on micro-wells; simultaneous incubation of antigen and Horse Radish Peroxydase (HRPO) conjugated antiserum; substrate incubation followed by photometric measurement of absorbance at 403 nm. Preliminary results seem generally in good agreement with those obtained by other tests such as gel diffusion and indirect ELISA.