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Aims: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification., Methods and Results: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = -0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively)., Conclusions: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water., Significance and Impact of the Study: This study gives valuable advance in the methods of concentration and diagnosis of virus in water., (Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.)

An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.

The L1 major capsid protein of human papillomavirus type 45 was expressed in insect cells using recombinant baculovirus technology. Human papillomavirus type 45 L1 major capsid protein self-assembled into empty virus-like particles (VLPs). These 50-60 nm diameter particles were present in the nuclei of recombinant baculovirus-infected insect cells and had a density of 1.29 - 1.30 g/cm3 in cesium chloride. The expressed human papillomavirus type 45 L1 protein sequence is identical to the reference human papillomavirus type 45 strain except for one amino acid located at position 49 of the human papillomavirus type 45 L1 protein. It must be noted that the quantity of purified human papillomavirus type 45 virus-like particles is at a lower level than that previously observed with human papillomavirus type 16. Nevertheless, the ability to generate preparative amounts of human papillomavirus type 45 virus-like particles is of great importance for the production of an anti-human papillomavirus vaccine.

The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty virions were identified by electron microscopy at densities of 1.29-1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.

Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84-88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.

The polymerase chain reaction was used to search for hepatitis B virus (HBV)-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer. Amplified HBV-DNA sequences were detected by hybridization with a digoxigenin-labelled HBV-DNA probe. HBV-DNA was detected in 17% of HBsAg negative Senegalese subjects from the general population and in 44% and 58% of the patients suffering from cirrhosis or primary hepatocellular carcinoma (PHCC) respectively. In the control group, amplified HBV-DNA was detected in 25% of the subjects without HBsAg and anti-HBs antibodies, and in 6% of subjects positive for anti-HBs antibodies. This study confirmed the hypothesis that there is an etiologic link between HBV and PHCC in HBsAg-negative patients.