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The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 °C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 °C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure-inactivated IBDV could be obtained under proper conditions. The pressure-inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.
Hydrostatic pressure can be regarded as a thermodynamic parameter. Its effect is governed by Le Chatelier's principle, which predicts that the application of pressure shifts equilibrium towards the state that occupies a smaller volume. In the past years, high hydrostatic pressure has been used as a method both to initiate changes in the higher-order structures of macromolecules and to modulate enzyme activity . The perturbation of macromolecular structures by hydrostatic pressure depends solely on the volume change of the process, in contrast with the perturbations of either high temperature or denaturing agents where the denaturing effects depend on multiple factors. Recent studies on the effect of pressure on macromolecules have provided a great deal of information on macromolecular structures, protein–protein and protein–nucleic acid interactions (reviewed in [1–5]).
Because pressure in a moderate range (less that 300 MPa) does not have a significant effect on the structures of single-chain proteins or nucleic acids, pressure provides a powerful method to study protein–protein or protein–nucleic acid interactions [1,2]. The dissociation of oligomeric proteins and the capsids of icosahedral viruses caused by pressure is a very general phenomenon [4,6–8] which was studied in the mid-1950s to prepare a vaccine against poliomyelitis . By studying the process of pressure dissociation of large aggregates, important thermodynamic data and information may be obtained. Many studies on the dissociation of oligomeric proteins under pressure have been reported [10,11]. One of the interesting findings is that the separation of the protein subunits is followed by conspicuous conformation changes called conformational drift that may lead to reversible or irreversible changes that can sometimes be characterized using spectroscopy and biological activity [12–14].
Virus is complex of proteins and nucleic acids, as well as lipids in some cases. The effects of high hydrostatic pressure on several viruses have been studied . For instance, simian immunodeficiency virus (SIV)  and some strains of human immunodeficiency virus (HIV) [16,17] were inactivated by high hydrostatic pressure. For some other viruses, such as vesicular stomatitis virus (VSV) , bovine rotavirus and simian rotavirus , high pressure causes virus inactivation, but does not affect the original immunogenic properties. These interesting findings have engendered the idea of applying high hydrostatic pressure to prepare virus vaccines with high efficiency and safety , as well as to sterilize biological preparations . Consequently, some important rules about virus structure have been outlined through the studies on assembly of bacteriophages, plant viruses and animal viruses [4,8].
Chicken infectious bursal disease virus (IBDV) is a member of the Birnavirus group that specifies two genomic double-stranded RNAs [21,22]. IBDV is responsible for a highly immunosuppressive disease in young chickens and causes significant economic losses to the poultry industry worldwide. Significant antigenic differences were detected among serotype I IBDV . Some virulent strains with very high mortality were reported in the last decade [24,25]. IBDV is a nonenveloped isometric particle with a diameter of about 60–65 nm . Electron cryomicroscopy maps show that the structure of the virus is based on a T = 13 icosahedral lattice and that the subunits are predominantly trimmer clustered in the two layers of virus coat . Segment A (≈3.4 kb) of genomes encodes a polyprotein, which is processed into three viral proteins VP2 (≈ 40 kDa, 51%), VP3 (≈ 32 kDa, 40%), VP4 (≈ 28 kDa, 6%) . VP2 and VP3 are major structural proteins of the virus capsid. VP2 carries major neutralizing epitopes [29,30], suggesting that it is at least partly exposed to the outer surface of the capsid. VP3 contains a very basic C-terminal region, which is likely to interact with the packaged RNA and therefore inside the capsid . Segment B of (≈ 2.9 kb) encodes the viral RNA polymerase VP1 (≈ 90 kDa, 3%), which exists as a genome-linked protein.
Here, we describe the effects of moderate hydrostatic pressure (from 0.1 to 250 MPa) on the structure and the biological activity of IBDV. Fluorescence spectroscopy, light scattering, electron microscopy and trypsin digestion were used to explore the changes in the virus structure upon pressure application.
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- Materials and methods
The aim of this work was to study the effects of high hydrostatic pressure on the structure and activity of IBDV, a rather complicated virus, whose capsid is based on the architecture of a T = 13 lattice and made up of three different types of polypeptides (up to 1440 subunits) . We have shown that the protein capsid of IBDV was dissociated by pressure as demonstrated by fluorescence spectroscopy and light scattering measurement (Figs 1 and 3). Although the final state of the dissociated IBDV was not known exactly, the average ΔV for subunits from dissociation could be estimated to be about −29 mL·mol−1 by making reasonable assumptions. The change in the center of the spectral mass of IBDV at 240 MPa and 0 °C was not as much as that induced by 8 m urea, suggesting that high pressure does not affect the conformation of viral proteins as strongly as a high concentration of urea. However, we do not have evidence that IBDV is completely dissociated into monomers under high pressure. We assumed that VP2 and VP3 trimers may be yielded in the IBDV pressure dissociation because they are possible intermediate states.
Just like many other oligomeric proteins and several viruses, IBDV showed a significant departure in concentration dependence upon pressure-induced dissociation. A large discrepancy existed between the experimental (10.5 MPa) and the expected value (178 MPa) of half dissociation pressure, Δp1/2, for the two IBDV samples with a 10-fold difference in the virus concentration (Fig. 2). Ruan & Weber  suggested that the concentration independence resulted from a broad distribution of free energy of association in the native oligomer population. Thus the viral population behaves as individual units that undergo independent dissociation. A comparison between the observed and expected values of Δp1/2 for different oligomeric protein and viruses [44,45] showed that the heterogeneity of aggregates population increases as the order (n) of association increases. From dimers to tetramers, to larger oligomeric proteins, to viruses, the discrepancy between experimental and expected Δp1/2 increases. IBDV has the largest discrepancy in experimetnal and expected Δp1/2 (about 167.5 MPa) among viruses that have been studied by pressure, which correlates well with its more complicated structure.
The assembly of oligomeric proteins and viruses is usually an entropy-driven process, and low temperature could enhance the dissociation of aggregates, as has been shown in many cases [4,49,50]. Our results showed that low temperature enhanced the dissociation of IBDV and increased the sensitivity of the virus to high pressure, resulting in higher irreversibility in its spectral characteristics (Fig. 3), which also suggests an entropic contribution to the stability of this virus. Thus, the effect of high pressure on the activity of IBDV was mostly investigated at low temperature (0 °C). The infectivity of IBDV was completely lost under certain conditions, for instance, 230 MPa for 2 h or 140 MPa for 3 h; meanwhile high immunogenicity was retained. The same phenomenon was also observed in some other viruses such as VSV , bovine rotavirus and simian rotavirus . Several other viruses, such as SIV , HIV [16,17], and adenovirus  were inactivated by hydrostatic pressure without assay of their immunogenic properties. Our results and previous studies demonstrate the two potential applications of high hydrostatic pressure: preparation of whole virus vaccines and virus sterilization.
Up to now, the loss in activity of oligomeric proteins and viruses by high hydrostatic pressure has usually been ascribed to pressure-induced dissociation of these aggregates and subsequent conformational drift. The differences observed in the reversibility of the dissociation and in regaining the native properties are dependent on the number of subunits, and the complexity of their interactions in the original aggregates. The more complex the aggregate is, the slower it is in regaining the native properties [6,49]. The rapid reassociation and recovery of activity and spectral properties were observed in many dimers [7,14,33,43]. In contrast, the reassociation upon decompression for oligomeric proteins composed of many subunits were very slow [13,14]. The different viruses presented distinct stability to pressure; the level of inactivation also depended on the pressure and the incubation time under pressure. As described above, IBDV has a rather complicated capsid, which should greatly increase its complexity and difficulty in regaining its native state. The IBDV treated by pressure at 0 °C was different from the native virus, as revealed by intrinsic fluorescence spectra, bis-ANS binding, electron microscopy and trypsin digestion. Although the coat proteins reassociated after releasing pressure (Fig. 6B), the reassembled virus changed considerably in morphology and size. The surface of particles seemed to become uneven and more hydrophobic (Figs 4 and 6B). These structural changes of viruses may be the cause of the inactivation. In the case of other pressure-inactivated viruses, such as VSV or rotavirus, constant changes in the shape of viral particles and the integrity of membrane or the coat proteins were also observed [18,19]. Additionally, Ruan and Weber found that a pressure-induced conformational-drifted form of glyceraldehyde 3-phosphate dehydrogenase from yeast remained stable at low temperature. As for IBDV, low temperature might also be an important factor to maintain the conformational drift in viral proteins as well as changes in infectivity caused by high pressure.
The outermost coat protein, VP2, of IBDV carries major neutralizing epitopes that determine the immunogenic properties of the virus. Usually, pressure lower than 300 MPa does not have a great influence on the structure of single-chain proteins . It is unlikely that the structure of VP2 and VP3 undergo significant changes under our experimental conditions, and thus it is not surprising that there is little loss in the immunogenic properties of IBDV. Da Poian et al. found that pressure-inactivated VSV attached to the cellular membrane, but could not be internalized by endocytosis , suggesting that the structure of the binding site on viral surface was not destroyed by pressure treatment. This may be crucial to evoke immune response.
The present results give more evidence for the possibility of high hydrostatic pressure as a new approach for antiviral vaccine preparations . As a physical approach, high pressure has many advantages over other methods of preparing vaccines. The application of pressure does not introduce exogenous substances into vaccines as chemical methods usually do. The vaccines prepared by pressure would contain whole virus, which usually results in more efficient immunization than isolated subunits. In addition, it is also a simple and inexpensive means to prepare effective vaccines.