47
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles

      case-report

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.

          Related collections

          Most cited references17

          • Record: found
          • Abstract: found
          • Article: not found

          Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.

          Norwalk virus capsid protein was produced by expression of the second and third open reading frames of the Norwalk virus genome, using a cell-free translation system and baculovirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty viruslike particles similar to native capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk virus challenge. These particles also induced high levels of Norwalk virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk virus infection as well as the implementation of structural studies.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant.

            Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. Three regions (England and Wales, Germany, and the Netherlands) had sustained epidemiological and viral characterisation data from 1995 to 2002. In all three, we noted a striking increase in norovirus outbreaks in 2002 that coincided with the detection and emergence of a new predominant norovirus variant of genogroup II4, which had a consistent mutation in the polymerase gene. Eight of nine regions had an annual peak in 2002 and the new genogroup II4 variant was detected in nine countries. Also, the detection of the new variant preceded an atypical spring and summer peak of outbreaks in three countries. Our data from ten European countries show a striking increase and unusual seasonal pattern of norovirus gastroenteritis in 2002 that occurred concurrently with the emergence of a novel genetic variant. In addition to showing the added value of an international network for viral gastroenteritis outbreaks, these observations raise questions about the biological properties of the variant and the mechanisms for its rapid dissemination.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature.

              Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 degrees C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 degrees C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner.
                Bookmark

                Author and article information

                Contributors
                +358-40-1901506 , +358-3-35518450 , lllete@uta.fi
                Journal
                Arch Virol
                Archives of Virology
                Springer Vienna (Vienna )
                0304-8608
                1432-8798
                19 August 2010
                19 August 2010
                November 2010
                : 155
                : 11
                : 1855-1858
                Affiliations
                [1 ]Vaccine Research Center, University of Tampere Medical School, Biokatu 10, 33520 Tampere, Finland
                [2 ]Institute of Medical Technology, Tampere University Hospital, University of Tampere, Tampere, Finland
                [3 ]Department of Pediatrics, Tampere University Hospital, Tampere, Finland
                Article
                768
                10.1007/s00705-010-0768-z
                2970802
                20721592
                c11e155d-00ac-4b47-9e67-d7f99aa3f399
                © The Author(s) 2010
                History
                : 4 June 2010
                : 22 July 2010
                Categories
                Brief Report
                Custom metadata
                © Springer-Verlag 2010

                Microbiology & Virology
                Microbiology & Virology

                Comments

                Comment on this article