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Keywords:

  • infectious bursal disease virus;
  • hydrostatic pressure;
  • dissociation;
  • inactivation;
  • vaccine

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations
IBDV

infectious bursal disease virus

VSV

vesicular stomatitis virus

HIV

human immunodeficiency virus

SIV

simian immunodeficiency virus

IBD

infectious bursal disease

TCID50

tissue culture infectious dose50

bis-ANS

bis (8-anilinonaphthalene-1-sulfonate)

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 [1]. 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 [9]. 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 [8]. For instance, simian immunodeficiency virus (SIV) [15] 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) [18], bovine rotavirus and simian rotavirus [19], 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 [9], as well as to sterilize biological preparations [20]. 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 [23]. 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 [26]. 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 [27]. 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%) [28]. 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 [28]. 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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

All reagents were of analytical grade. The experiments were performed in the standard buffer: 50 mm Tris, 150 mm NaCl, pH 7.5. Bis (8-anilinonaphthalene-1-sulfonate) (bis-ANS) was from Molecular Probes (Eugene, OR, USA). Trypsin was purchased from Sigma (St Louis, MO, USA). All other reagents were A.R. grade. Distilled water was purified by a Millipore system.

Virus preparation

An IBDV virus strain LH, purified in China, was utilized in this study. Purified IBDV was prepared as previously described [31] from the Bursa of Fabricius of chickens (BF) that had died from infectious bursal disease (IBD). The concentration of viral protein was determined according to previous report [32].

Nonpurified virus were either propagated on chicken embryo fibroblasts (CEF) in MEM medium, or prepared from tissue homogenate of BF of chickens that had died from the disease [31]. The supernatant of the infected CEF was collected when a cytopathic effect was clearly visible. Infected bursae were homogenized and diluted as a 10% suspension in 0.9% NaCl solution. All cellular debris in samples were removed by a centrifugation of 3000 g for 10 min.

Fluorescence measurements under pressure

The high-pressure bomb was made by K.-C. Ruan's laboratory according to the description of Paladini & Weber [33]. Fluorescence spectra were recorded on an SLM 48000 spectrofluorometer (SLM Instruments, Inc). The preparation was kept for ≈ 15 min under pressure before the fluorescence spectrum was recorded. Fluorescence spectra were analyzed by the center of spectral mass 〈ν〉 [33,34]:

  • image(1)

where Fi stands for the fluorescence emitted at wavenumber νi and ΣFi is the spectral integral. The degree of dissociation at pressure Pp) is related to 〈ν〉 by the expression:

  • image(2)

where Q is the ratio of the quantum yields of dissociated and associated forms; 〈ν〉 is the center of spectral mass at pressure p, and 〈νD〉 and 〈νA〉 are the corresponding 〈ν〉 for dissociated and associated forms [35].

The thermodynamic dissociation constant K of the equilibrium between an aggregate and its n equal constitutive subunits is given by the relation:

  • image(3)

where α is the degree of dissociation into subunits and C the molar concentration of protein as aggregate, and C1/2 the protein concentration at which α = 1/2.

The free energy change of the dissociation process is:

  • image(4)

If we introduce the degree of dissociation at pressure pp) into Eqn (4), then:

  • image(5)

Kd0 being the dissociation constant at atmospheric pressure.

Eqn (5) permits the calculation of ΔV, the standard volume change upon dissociation at a certain protein concentration from pressure dissociation data. It is acceptable to treat ΔV as a pressure-independent constant due to low compressibility of proteins [36,37]. In this study, the final state of dissociated subunits of viral capsid was unknown, so in order to estimate the average ΔV for dissociation, the value of n was assumed.

Light scattering measurements were made in a SLM 48000 spectrofluometer by selecting the same wavelength for both excitation and emission monochromators with the smallest slit. Scattered light (360 nm) was collected at an angle of 90° of the incident light.

Infectivity assays

Infectivity of IBDV was studied by determination of its tissue culture infectious dose50 (TCID50) on CEF. Confluent monolayers of CEF cells were infected with serial dilutions of IBDV for 1 h at 37 °C. After aspiration of the virus solution, medium was added and cells were kept at 37 °C for 24 h. Then the cells were examined microscopically and scored as infected or not. TCID50 was calculated according to the Reed & Muench method [38].

Immunogenicity and antibody assays

The immunogenic properties of IBDV exposed to 230 MPa and 0 °C for 2 h were determined. Different doses of pressure-treated IBDV were administered intramuscularly in the legs of 17-day-old SPF chickens. At periodical intervals during the vaccination, blood was collected and antibody responses to IBDV were evaluated by agarose diffusion assays. All the chickens were challenged with native IBDV 40 days after vaccination. Protection in terms of death-rate was determined 4 days later, and gross lesions typical of infectious bursal disease were examined.

Electron microscopy

The IBDV samples were observed in a JEOL JEM-1200EX electron microscope. Negative staining was performed with 2.0% uranyl acetate.

Trypsin digestion of IBDV coat proteins

IBDV samples of 102 µg·mL−1 untreated or treated by hydrostatic pressure at 0 °C were incubated with trypsin in a final concentration of 0.2 µg·mL−1 at 37 °C for 60 min. The reaction was stopped by freezing the samples at −20 °C. Then, the samples were treated with 1% SDS and 5% 2-mercaptoethanol, and boiled for two minutes before electrophoresis on 12% SDS-polyacrylamide gel. The gel was stained by the silver method.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dissociation of IBDV by pressure

The effects of pressure on IBDV were followed by measuring its intrinsic fluorescence spectra coming from the tryptophan residues [39] in the two major structural proteins VP2 (51%) and VP3 (40%), and a minor structural protein VP4 (6%) [23,27]. Figure 1A shows the pressure dependence of the center of mass of the intrinsic fluorescence of IBDV at 0 °C. As pressure was elevated, the center of spectral mass decreased and reached a plateau at about 240 MPa, resulting in a total spectral red shift of about 690 cm−1 (Fig. 1A). The fluorescence efficiency decreased at the same time, corresponding to a loss of 40% efficiency at 240 MPa, in good agreement with the red shift. These phenomena indicate that the tryptophan residues in the viral coat proteins are exposed to an environment of increased polarity, which was ascribed to the dissociation of virus capsid, as occurs with many other oligomeric proteins and several viruses [11,14,35,40,41]. The presence of 8 m urea also resulted in a red shift in the center of spectral mass of intrinsic fluorescence spectra (with a total shift of about 850 cm−1), larger than that seen at 240 MPa (690 cm−1) (Fig. 1B). The maximum wavelength of the IBDV fluorescence spectrum at 240 MPa (about 340 nm) was smaller that in the presence of 8 m urea (about 345 nm), suggesting that the dissociated IBDV subunit under pressure was significantly different from that in 8 m urea. The light scattering of IBDV was also lowered by elevating pressure (see Fig. 3B), indicates directly a decreasing of the size of viral particles. This is in agreement with the pressure-induced dissociation of IBDV.

image

Figure 1. Pressure dissociation of IBDV. (A) Effect of hydrostatic pressure on the center of spectral mass of IBDV: (●) for increase pressure and (○) for decrease pressure. (B) Normalized intrinsic fluorescence emission spectra of IBDV at atmospheric pressure (1), 240 MPa (3), after return to atmospheric pressure (2), and treated with 8 m urea (4). The samples were excited at 280 nm and the emissions were measured from 300 to 400 nm. Protein concentration: 51 µg·mL−1. Temperature: 0 °C.

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image

Figure 3. Pressure dissociation of IBDV at different temperatures. (A) Pressure-induced changes in the center of mass of intrinsic fluorescence. (B) Pressure induced changes in light scattering of IBDV. Symbols: ○ and ●, 20 °C; ▵ and ▴, 0 °C; the open symbols represents the values after pressure release. Other conditions were as for Fig. 1.

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According to Bottcher et al. [27], the capsid of IBDV is composed of 780 VP2 and 600 VP3 as well as 60 VP4. Both VP2 and VP3 exist as trimers in capsid. It is likely that trimers of VP2 and VP3 are intermediate states in the dissociation process of IBDV. This assumption is reasonable, although there is not evidence for this in our present experiments. As the real final size of the pressure-dissociated capsid protein is unknown, we simply assumed that the capsid of IBDV is dissociated by pressure into VP2 and VP3 trimers and VP4 monomers to estimate the standard molar volume change of dissociation. Thus the number of the dissociated subunits is 520. According to Eqn (5), the average ΔV value for each subunit was estimated to be about −29 mL·mol−1, very similar to that of other large aggregates [42], indicating that our assumption is reasonable.

Concentration dependence of pressure dissociation

The change in the half-dissociation pressure (Δp1/2) corresponding to the change in concentration can be deduced from Eqn (5):

  • image(6)

The identity of experimental value and expected value of Δp1/2 calculated from Eqn (6) for two concentrations C1 and C2 means the concentration dependence characteristic of a stochastic process, as having been observed for many other dimers [33,35,43]. The dissociation curves of the two IBDV concentrations 51 µg·mL−1 and 510 µg·mL−1 vs. pressure are shown in Fig. 2. The experimental half-dissociation pressure Δ p1/2 is only about 10.5 MPa, much smaller than the expected value (about 178 MPa) giving a significantly abnormal concentration dependence, which is one of the characteristics of relatively large oligomeric proteins (n ≥ 4) and viruses [13,14,40,44,45].

image

Figure 2. Concentration dependence of the spectral changes of intrinsic fluorescence of IBDV induced by pressure. Plot of center of spectral mass vs. pressure at two concentrations: 51 µg·mL−1 (▵) and 510 µg·mL−1 (●). Other conditions are as for Fig. 1.

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Effects of temperature on the dissociation of IBDV

Figure 3A shows the dissociation curves of IBDV at 20 and 0 °C in terms of the center of spectral mass. It is clear that the dissociation curve shifted to higher pressure as temperature was increased from 0 to 20 °C. The two curves at two temperatures in Fig. 3A gave a difference of approximately 30 MPa between their half dissociation pressures, indicating that pressure-induced disassembly was facilitated by lower temperature, as seen in many other large aggregates. It has also been reported previously that the dissociation of oligomeric proteins and viruses by pressure is promoted by low temperatures [14,35,46,47]. It was noticed that for the dissociation of IBDV at 0 °C, the decompression curve showed an clear hysteresis to the compression curve. Upon pressure release to 0.1 MPa, the center of spectral mass only recovered 70% of that of the original value (Figs 1A and 3A). In contrast, pressure-induced dissociation of IBDV at 20 °C did not reach a plateau at 240 MPa, and showed much more recovery in the center of spectral mass upon pressure release (Fig. 3A). A similar result in the light scattering measurement was also obtained (Fig. 3B). Fluorescence and light scattering are two methods currently used to follow the protein structure changes. We have used these two methods to make sure that the effects of high-pressure on IBDV are different at 0 and 20 °C, respectively. However, it is not surprising that sometimes they do not give exactly the same results. In this study, it seems that fluorescence is better to follow the IBDV dissociation.

Bis-ANS Binding to IBDV

Bis-ANS is a hydrophobic probe and its fluorescence yield may increase greatly upon binding to hydrophobic sites of proteins [48]. It is currently used to probe the conformation change of proteins. Figure 4A shows that, at atmospheric pressure, bis-ANS can bind to native IBDV with a clear increase in its quantum yield, together with a blue shift of its maximum emission spectrum to 507 nm. This suggests that, at this pressure, there exist hydrophobic areas accessible to bis-ANS on the surface of IBDV coat. As the pressure was raised, the bis-ANS fluorescence yield was increased gradually, but without any shift in the maximum emission wavelength. This increase in intensity of bis-ANS indicates an appreciable enlargement of the hydrophobic sites on the surface of IBDV coat proteins, which may be ascribed either to the exposure of hydrophobic interface of subunits or to a conformational change of coat proteins at high pressures. It is striking that at two different temperatures the pressure-induced changes in hydrophobic properties of the surface of coat proteins are different (Fig. 4B). At 20 °C, a small increase in fluorescence intensity with elevated pressure was observed (Fig. 4B), and the intensity almost returned to the original value after pressure was released. Meanwhile, at 0 °C, there was a sharp and partially reversible increase in fluorescence yield with rising pressure (Fig. 4B). This result correlates well with the pressure induced intrinsic fluorescence changes described above.

image

Figure 4. Bis-ANS binding of IBDV coat proteins. (A) Fluorescence emission spectra of bis-ANS in the absence or presence of IBDV at 0 °C and various pressures represented by the numbers in the bar above or under the curves. (B) Effects of pressure on the fluorescence of bis-ANS bound to IBDV at different temperatures. Symbols: ○ and ●, 0 °C; ▵ and ▴, 20 °C; the open symbols refer to the values after pressure release. Concentration of IBDV: 51 µg·mL−1; concentration of bis-ANS: 20 µm. Excitation wavelength: 395 nm.

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Pressure inactivation of IBDV

Figure 5A shows the pressure effect (at 0 °C) on the infectivity of IBDV determined by measurement of the tissue culture infectious dose50 (TCID50) on chicken embryo fibroblast. As seen in Fig. 5A, the titers of the virus decreased dramatically with pressure, following a single exponential fitting. At 60 MPa, about half the infectivity of IBDV was lost, and at pressure higher than 140 MPa, no viral infectivity could be detected. The inactivation of IBDV induced by pressure was clearly dependent on the incubation time at the high pressure as shown in Fig. 5B, indicating that a complete loss of infectivity for IBDV could be reached in less than about 60 min (the half time of the exponential inactivation being about 4–5 min). Both curves in Fig. 5 indicate that it is possible to entirely inactivate IBDV by means of pressure under certain conditions. Experiments on SPF chickens provided further evidence for the inactivation of IBDV by high pressure. Chickens injected with the IBDV treated at 230 MPa and 0 °C for 2 h showed neither death nor typical signs of infectious bursal disease (IBD), suggesting a complete loss of infectivity. In contrast, a high mortality was observed in the control chickens exposed to the native virus (data not shown). It was also found that higher pressure could shorten the time needed for inactivation. For instance, at 230 MPa, only about 10 min was needed for a decrease of 4.5 TCID50 (Fig. 5B), whereas at 90 MPa, 3 h was required to obtain the same effect (Fig. 5A). It seems that IBDV is easier to inactivate than vesicular stomatitis virus [18], but similar to several other viruses such as bovine rotavirus [41] or SIV [15].

image

Figure 5. Effects of high hydrostatic pressure on the infectivity of IBDV. Unpurified IBDV was utilized. (A) Infectivity vs. pressure, all the treatments were performed at 0 °C for 3 h. (B) Infectivity vs. incubation time, all the treatments were performed at 230 MPa and 0 °C.

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Immunogenic properties of pressure-treated IBDV

Table 1 shows the result of immunity assay of the immunogenic properties of the inactivated IBDV at 230 MPa at 0 °C for 2 h. All the three groups of SPF chickens immunized with different doses of pressure-inactivated IBDV remained alive 4 days after challenge, and no typical lesions of IBD were observed. Meanwhile all the control chickens developed severe clinical IBD and a high death-rate was present 4 days after challenge. These results indicate that pressure-inactivated IBDV could confer to SPF chickens effective protection against the infection of the native IBDV. The serum antibody against the native IBDV could be detected 7 days after immunization with the pressure-treated IBDV for the groups of 0.5 mL and 1.0 mL dose (Table 2), and the titers of antibody increased with time, reaching 1 : 32 in all the three groups 3 weeks later (Table 2). These results suggest that hydrostatic pressure treatment does inactivate IBDV without affecting its ability to elicit neutralizing antibodies. The similar results have also been reported for several other animal viruses, i.e. VSV [18], bovine rotavirus and simian rotavirus [41].

Table 1. Immunity test of pressure-treated IBDV.
 ImmunizationDeath rate 4 daysSymptoms of IBD (% lesions)
Groupsdose (mL per chicken)postchallenge (%)BursaStomachMuscle
Immunized chickens0.2 0 0  0 0
0.5 0 0  0 0
1.0 0 0  0 0
Control chickens838310083
Table 2. Serum antibody levels in chickens immunized with pressure-treated IBDV.
Days afterTiters of antibody (mLa)
immunization0.20.51.0
  • a

    Dose of immunization;

  • b

    b antibody undetectable;

  • c

    c antibody detectable.

 7– bcc
141 : 41 : 81 : 8
211 : 321 : 321 : 32
281 : 641 : 641 : 16

Electron micrographs

Figure 6 shows the transmission electron micrographs of IBDV after incubation at atmospheric pressure or at 230 MPa (0 °C for 2 h). The native IBDV is an icosahedral particle as shown in Fig. 6A. The viruses treated by high pressure had a smaller size compared with the native viruses, and their shells were not as continuous or regular as those of the native viruses (Fig. 6B). Additionally, the viruses treated by high pressure had a tendency to aggregate, indicating the existence of more hydrophobic viral surfaces, in agreement with the results of bis-ANS binding experiment. It should be emphasized that the micrographs shown here were typical for the whole grid. These electron micrographs suggest that IBDV pressurized at 0 °C may lose some of their outer coat proteins. This alteration caused by high pressure and low temperature correlates well with the spectral results and accounts for the inactivation of IBDV described above.

image

Figure 6. Electron microscopy of IBDV subjected to high pressure. IBDV was negatively stained with uranyl acetate after 2 h incubation at 0 °C at (A) atmospheric pressure and (B) 230 MPa. Magnification: 100 000×. Concentration: 102 µg·mL−1.

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Trypsin digestion of IBDV coat proteins

Due to the protection of protein–protein and/or protein–nucleic acid interactions, capsid proteins in complete viruses often show higher stability than dissociated subunits or incomplete viruses. This was examined by trypsin digestion. Figure 7 shows the polyacrylamide gel electrophoresis of trypsin digested coat proteins of IBDV treated under different conditions. Four major bands, representing VP1 to VP4 of IBDV, can be seen for both the control viruses in the absence of trypsin (lane 1) and the viruses not subjected to high pressure but treated with trypsin (lane 2), indicating the insensitivity of proteins in capsid to trypsin digestion. The proteins in IBDV subjected to pressure at 20 °C were only slightly digested (lane 3), suggesting that reassociated viruses are very similar to the native ones in their resistance to proteolysis. In contrast, coat proteins in IBDV subjected to high pressure at 0 °C (lane 4) were greatly digested, as revealed by the weakening of the four major bands and the appearance of some other bands of smaller molecular mass. This result suggests that after pressure-treatment at 0 °C, the proteins of the capsid cannot return to the same state as that of the native IBDV, and are more easily digestible by trypsin. This may result either from the changes in protein conformation or from the weakened protein–protein or protein–RNA interactions caused by the dissociation–reassociation.

image

Figure 7. Polyacrylamide gel electrophoresis of trypsin-digested IBDV coat proteins. Lane 1, control native virus without trypsin; lane 2, native virus with trypsin; lane 3, virus subjected to 230 MPa at 20 °C with trypsin; lane 4, virus subjected to 230 MPa at 0 °C with trypsin.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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) [27]. 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 [14] 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 [18], bovine rotavirus and simian rotavirus [19]. Several other viruses, such as SIV [15], HIV [16,17], and adenovirus [19] 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[14]. 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 [6]. 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 [51], 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 [52]. 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by a grant from National Natural Science Foundation of China and a grant from INSERM/Academia China (K. R. and C. B.). C. B. thanks Prof. T. Randolph (University of Colorado, Boulder) for very stimulating discussions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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