© INRA, EDP Sciences, 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted use, distribution, and reproduction in any noncommercial medium, provided the original work is properly cited.

1. INTRODUCTION

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded enveloped RNA virus which is assigned to the family Arteriviridae [11, 26, 37], together with lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV). The Arteriviridae as well as the Coronaviridae and Roniviridae belong to the order Nidovirales [6, 12, 26].

PRRS is worldwide recognized as the economic most important viral pig disease [44]. Infection of pregnant sows may result in mummified and weak-born piglets, elevated pre-weaning mortality and abortion [9, 35, 59]. Infected boars may show a temporary decrease of sperm quality and virus shedding via sperm [51]. This virus shedding may lead to virus transmission to sows. Besides its effect on reproduction, PRRSV is also involved in the multifactorial respiratory disease complex in pigs, where the virus facilitates secondary bacterial infections and respiratory problems in pigs of all ages [55, 59].

Two main types of vaccines are currently used to prevent PRRSV infection, modified live virus (MLV) vaccines and killed virus (KV) vaccines [34, 68]. Commercial MLV and KV vaccines exist, but both have some disadvantages. MLV vaccines induce an immune response that can protect pigs against PRRSV infection, however only when the virus is not too distant from the vaccine strain [28, 29]. Attenuated viruses may cause safety problems. Some MLV vaccines may spread transplacentally [18, 36, 46], be shed via semen and reduce semen quality [10, 47], and may even revert to virulence [45]. KV vaccines are safe to use, but currently used KV vaccines insufficiently protect pigs against viremia upon challenge, since both magnitude and duration of viremia were not different between vaccinated and control animals [68]. A comparative study of MLV and KV vaccines in boars showed that while vaccination with an MLV vaccine decreased viremia and virus shedding in semen, vaccination with a KV vaccine did not change onset, duration or level of viremia, or virus shedding in semen [47]. Preliminary experiments in our lab showed that while an experimental inactivated PRRSV vaccine was able to induce neutralizing antibodies, it could only partly block viremia after challenge [41]. The current incomplete protection of KV vaccines against PRRSV infection might be caused by an over-inactivation of the virus, resulting in destruction of neutralizing viral epitopes. Neutralizing viral epitopes induce the production of neutralizing antibodies by the host, which are necessary for neutralization of the virus and reduction of overall infection. As a result, destruction of the neutralizing epitopes by over-inactivation will reduce the number of neutralizing antibodies raised by the host, which will ultimately lead to less efficient protection against a viral challenge.

To our knowledge, currently used KV vaccines against PRRSV have been evaluated for the quantity of viral antigens, but not the quality. For other viruses, the quality has been examined. For example, the effect of inactivation of influenza virus is investigated by measuring the hemagglutinating activity before and after inactivation [19]. For HIV, the attachment of neutralizing antibodies to viral epitopes is determined after inactivation by an enzyme-linked immuno sorbent assay (ELISA) [25, 50]. ELISA as a tool for quality control of the antigen is also used for rabies virus [21, 53] and poliovirus vaccines [43]. Quality control for PRRSV cannot be performed using the same methods as for HIV and influenza, because of the limited knowledge of PRRSV neutralizing epitopes. There are some neutralizing epitopes known on GP5 and GP4 [39, 48, 66] and there are possibly also neutralizing epitopes on GP3 [5], but it is not known if other important epitopes exist and which neutralizing epitopes are most important. Previous results from our lab showed that PRRSV neutralizing antibodies block infection by preventing the interaction of PRRSV with the internalization receptor sialoadhesin (Sn) on the target cells, macrophages [15, 16, 60, 61]. This indicates that neutralizing antibodies are directed to the viral epitopes that are involved in the attachment to the PRRSV receptors and internalization into the macrophage [15]. Based on this observation, a quality control of the viral antigen of a PRRSV KV vaccine was developed, by monitoring PRRSV attachment to and internalization into macrophages before and after inactivation. An ideal killed PRRSV vaccine should be able to attach to and internalize into macrophages during a quality-control assay, but it should be disabled to replicate in order to avoid viremia.

The aim of this study is to investigate the effect of different PRRSV inactivation methods on the viral entry-associated domains. To this end, inactivated PRRSV attachment to and internalization into macrophages were monitored.

2. MATERIALS AND METHODS

2.1. Cells and virus

The European PRRSV strain, Lelystad virus (LV) [63], grown on Marc-145 cells (4th passage) was used for inactivation. Marc-145 cells cultivated in minimum Eagle’s medium (MEM) with 5% fetal bovine serum (FBS), 0.3 mg/mL glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.1 mg/mL kanamycin were used for LV production and titration. Virus attachment and internalization were investigated in macrophages cultivated in RPMI 1640 with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 0.1 mg/mL kanamycin, 0.1 mg/mL gentamycin, 0.01 mg/mL tylosin, 0.3 mg/mL glutamine, 1 mM nonessential amino acids and 1% sodium pyruvate (100×) for 24 h.

2.2. PRRSV concentration and purification

The European PRRSV strain, LV, was grown on Marc-145 cells. The medium containing the virus was purified as described by Delputte et al. [15], this with some modifications to allow purification of larger quantities of virus. Virus supernatant was first filtrated through a 0.45 μm filter and then ultra-centrifuged for 2 h at 31 000 rpm with a rotor type 35 at 4 °C (Beckmann Coulter, Analis, Ghent, Belgium) to pellet the virus. The resuspended virus pellet was centrifuged 10 min at 13 000 rpm (Heraeus fresco, Thermo Fisher Scientific, Zellik, Belgium) to remove cell debris and large aggregates and the supernatant was ultra-centrifuged through a 30% sucrose cushion for 3 h at 30 000 rpm with a SW 41 Ti rotor at 4 °C (Beckmann Coulter). Finally, the virus pellet was resuspended in 1 mL PBS by incubating it for 1 h on ice.

2.3. Inactivation methods

An overview of the inactivation methods used and their mode of action is shown in Table I. Purified virus (10⁷ TCID₅₀/mL) was used for inactivation. For inactivation with formaldehyde, glutaraldehyde or 2,2-dithiodipyridine (AT-2), virus was incubated for 4 h at 37 °C with different concentrations formaldehyde (Sigma, Bornem, Belgium) [14, 54], glutaraldehyde (Sigma) [14] or AT-2 (Aldrich, Bornem, Belgium) [7, 54]. Formaldehyde, glutaraldehyde or AT-2 was afterwards removed by ultracentrifugation. For inactivation by changing the pH, the pH was adjusted with HCl and NaOH and virus was incubated for different times at a pH2 or at a pH12. After incubation, the pH was neutralized [14]. For temperature inactivation, virus was incubated for different times at 37 °C [14, 33]. Inactivation of PRRSV with gamma irradiation was performed using an electron accelerator (Prof. L. Van Hoorebeke, Ghent University, Faculty of Science, Department of Subatomic and Radiation Physics). For PRRSV inactivation with ultraviolet (UV) radiation, virus was radiated with UV light from a UV cross-linker (BRS, Drogenbos, Belgium) [14]. Inactivation with binary ethyleneimine (BEI) was performed by incubating virus with 1 mM BEI (Aldrich) for different times at 37 °C. The reaction was stopped with 0.1 mM sodium thiosulfate (Sigma) [2, 42].

2.4. Analysis of virus inactivation

Virus titration was performed on 3 days cultivated Marc-145 cells following the standard procedure [3]. After 5 days, occurrence of cytopathic effect (CPE) was investigated and the 50% tissue culture infective dose (TCID₅₀) was calculated.

To confirm that all virus was completely inactivated using selected inactivation procedures, either a complete dose (10⁷ TCID₅₀/mL virus) or 10× more virus was inactivated and used for inoculation of Marc-145 cells, followed by two blind passages. The Marc-145 cells were investigated every week for CPE and cells were stained by immunoperoxidase monolayer assay (IPMA) [30]. Additionally, a bioassay was also done for selected inactivation methods by injecting 10⁷ TCID₅₀/mL inactivated PRRSV in RPMI 1640 medium intramuscularly in a pig. Blood was taken every week and serum was checked for viremia by virus titration and PRRSV specific antibodies by IPMA up to 2 weeks post inoculation.

2.5. Effect of PRRSV inactivation on virus attachment, internalization, disassembly and infection by immunofluorescence staining

For the detection of PRRSV during the course of PRRSV infection, macrophages were inoculated with untreated or treated PRRSV (moi 2) for 1 h at 4 °C or 1, 5 and 10 h at 37 °C, fixed with methanol at −20 °C and stained as described by Delputte et al. [15]. Briefly, the capsid protein was stained with a primary antibody P3/27 [64] and a secondary antibody fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse immunoglobulins (Molecular Probes, Invitrogen, Merelbeke, Belgium). A virus stock with a ratio of 1/100 of infectious virus/non-infectious virus was used for inoculation. This is determined by application of diluted samples of virus suspension to glass slides, fixed with methanol and stained against nucleocapsid as described above. This showed that the virus stock with 10⁶ TCID₅₀/mL contained approximately 10⁸ particles/mL. Confocal analysis was performed using a TCS SP2 laser scanning spectrum confocal system (Leica Microsystems GmbH, Heidelberg, Germany) to determine the amount of internalized PRRSV. The numbers of bound and internalized PRRSV were counted from the acquired images. The amount of internalized PRRSV of both treated and untreated virus in a macrophage were determined by counting the small fluorescently labeled dots and set relatively to internalized untreated virus.

2.6. Inhibition of phagocytosis

Macrophages were pre-treated for 30 min with 0.1 μM wortmannin to block phagocytosis. Afterwards the macrophages were inoculated with untreated or treated virus for 1 h at 37 °C in the presence of 0.1 μM wortmannin. Then the cells were fixed with methanol at −20 °C and stained as described by Delputte et al. [15]. Briefly, the capsid protein was stained with a primary antibody P3/27 [64] and a secondary antibody FITC-conjugated goat polyclonal anti-mouse immunoglobulins (Molecular Probes, Invitrogen). Confocal analysis was performed to check for endocytosis.

2.7. Analysis of the viral proteins of inactivated PRRSV by SDS-PAGE and Western blotting

SDS-PAGE, Western Blotting and ECL of viral proteins were performed essentially as described by Delputte et al. [17]. The membranes were stained for 1 h with a primary mouse monoclonal antibody against one of the proteins of LV, M (126.3) [38], N (P3/27) [64], GP5 (4BE12) [52], GP4 (122.29) [39], GP3 (P9A3-20) (Intervet, Boxmeer, the Netherlands) or pig polyclonal antibody.

3. RESULTS

3.1. Effect of different treatments on PRRSV infectivity

To test the potential of the different PRRSV inactivation treatments, purified virus (10⁷ TCID₅₀/mL) was treated with formaldehyde, glutaraldehyde, AT-2, pH, 37 °C, gamma irradiation, UV light or BEI, using different concentrations, time periods and/or doses. Treated PRRSV was titrated to determine an inactivation curve (Fig. 1). There was no infectious virus detected when PRRSV was treated with the lowest concentration of formaldehyde (0.1 μg/mL) (Fig. 1A), the lowest gamma irradiation dose (0.25 kGy) (Fig. 1F), the lowest UV irradiation dose (100 mJ/cm²) (Fig. 1G), or at the first time point investigated for inactivation with pH2 (1 h) (Fig. 1D), pH12 (data not shown), and BEI (6 h) (Fig. 1H). For glutaraldehyde, the amount of infectious virus decreased in function of the concentration and no infectious virus could be detected upon incubation with a concentration of 0.5 μg/mL glutaraldehyde or more (Fig. 1B). PRRSV was still infectious after 4 h incubation at 37 °C with the highest concentration of 2 mM AT-2 (Fig. 1C). For 37 °C treatment, the amount of infectious virus decreased in a time dependent way and there was no infectious virus detected after 48 h incubation at 37 °C (Fig. 1E). For formaldehyde, glutaraldehyde and AT-2, the titer at time point 0 was lower than 10⁷ TCID₅₀/mL, because of loss of some virus during ultracentrifugation. These results showed that for all inactivation methods, except for AT-2, a minimal treatment that allowed complete virus inactivation could be determined.

Figure 1. Effect of different inactivation methods on PRRSV infectivity. Untreated LV or inactivated LV was titrated on 3 days cultured Marc-145 cells to determine presence of infectious virus in the samples. The dotted line resembles the detection limit of the assay.

3.2. Effect of different inactivation procedures on virus internalization into macrophages

To investigate the preservation of the entry-associated domains of the treated PRRSV, an immunofluorescence staining was performed to determine whether internalization of inactivated PRRSV into macrophages was still possible. The results of these internalization experiments are shown in Figures 2 and 3. PRRSV inactivated with 1 μg/mL formaldehyde (Figs. 2A and 3), 50 μg/mL glutaraldehyde (Figs. 2B and 3), pH2 (Figs. 2D and 3) or pH12 (data not shown) for 1 h was no longer able to internalize into macrophages. The internalization experiments with AT-2 inactivated PRRSV (Figs. 2C and 3) showed that PRRSV treated with all concentrations of AT-2 tested, could still internalize into macrophages, but this was to be expected since the virus was still infectious even at the highest concentration of AT-2. The internalization experiments for 37 °C and BEI inactivation (Figs. 2E, 2H and 3) showed that PRRSV incubated at 37 °C or treated with 1 mM BEI could still internalize into macrophages for all time points investigated. PRRSV treated with all doses of gamma irradiation or UV examined could still internalize into macrophages, but the internalization diminished in a dose dependent way for gamma irradiation inactivated PRRSV (Figs. 2F, 2G and 3).

Figure 2. Effect of different inactivation methods on PRRSV internalization. Macrophages were incubated with untreated LV or inactivated LV for 1 h. After 1 hpi, the virus was stained by immunofluorescence and internalization of PRRSV particles was determined by confocal microscopy. The amount of internalized PRRSV particles of both treated and untreated virus in a macrophage were counted by counting the small fluorescently labeled dots and set relatively to internalized untreated virus.

Figure 3. Effect of different inactivation methods on different stages of the viral replication cycle. Macrophages were incubated with inactivated LV or untreated LV for 1 h at 4 °C and 1, 5 or 10 h at 37 °C. After fixing and immunofluorescence staining of the cells, attachment (1 hpi at 4 °C), internalization (1 hpi at 37 °C), disassembly (5 hpi at 37 °C) and replication (10 hpi at 37 °C) was analyzed by confocal microscopy.

Together, these data show that the entry-associated domains were not preserved when PRRSV is inactivated with formaldehyde, glutaraldehyde or pH changes, while they were preserved using AT-2, 37 °C, gamma irradiation, UV or BEI. About 20% of the cells inoculated with PRRSV inactivated with methods that still allowed internalization, showed internalization, because untreated PRRSV infects only about 20% of the macrophages in vitro. From the cells inoculated with PRRSV inactivated with methods that did not allow internalization anymore, there were no cells that showed internalization at all.

3.3. Effect of the different inactivation methods on different stages in the virus replication cycle in macrophages

PRRSV inactivated with formaldehyde, glutaraldehyde and pH changes was not able to internalize into macrophages, which is a crucial step in the viral replication cycle. Because internalization did not occur, the uncoating, virus replication, assembly and release did also not occur (Fig. 3).

PRRSV inactivated by 37 °C, gamma irradiation, UV and BEI could still internalize into macrophages, thus it was able to perform this step of the viral replication cycle. After 5 h, the uncoating occurred, thus also the second step of the viral replication cycle could be performed. The inactivated virus was however not capable of replicating and this thus confirmed the completely inactivated status of the virus (Fig. 3).

3.4. Inhibition of phagocytosis to confirm receptor-mediated endocytosis after inactivation

To investigate if entry of the inactivated PRRSV in macrophages occurred via Sn and CD163 as described for infectious PRRSV [60, 61], and not simply taken up by phagocytosis, entry was also determined using a phagocytosis inhibitor wortmannin. After treatment of the macrophages with the phagocytosis inhibitor wortmannin, untreated PRRSV still attached to and internalized into macrophages. PRRSV inactivated with UV and BEI also attached to and internalized into macrophages, whereas formaldehyde and glutaraldehyde inactivated PRRSV could not attach and internalize.

3.5. Safety test to confirm complete inactivation

Gamma irradiation, UV and BEI seemed to be useful methods for killed PRRSV vaccine development, since the inactivated virus attaches and enters macrophages in a similar way as the virulent virus. Therefore, safety tests were done to confirm that the inactivated virus was completely inactivated and safe for use in pigs. First, 10⁷ TCID₅₀/mL virus was inactivated and Marc-145 cells were inoculated for two passages with the inactivated virus. This test indicated that virus inactivated with 0.25 kGy gamma irradiation, 100 mJ/cm² UV or 6 h incubation with BEI were completely inactivated (data not shown). To be sure the virus was inactivated, 1.5 kGy gamma irradiation, 1 000 mJ/cm² UV or 24 h incubation with BEI were selected as a safe method to inactivate PRRSV. A second test was performed by inactivating a higher concentrated virus suspension under the same conditions (10⁸ TCID₅₀/mL virus). The results demonstrated that 1 000 mJ/cm² UV or 24 h incubation with BEI could still completely inactivate 10 times more virus (data not shown). Virus (10⁸ TCID₅₀/mL virus) inactivated with gamma irradiation was not completely inactivated with a dose of 1.5 kGy and starting from a dose of 2 kGy, the preservation of entry-associated domains decreases. Therefore, inactivation with gamma irradiation is not useful for further vaccine development. Finally, to confirm complete inactivation, a bioassay was performed. Therefore, 10⁷ TCID₅₀/mL virus was inactivated with 1 000 mJ/cm² UV or 24 h incubation with BEI and intramuscularly injected in pigs. Viremia was not detected up to 2 weeks after injection, while virus specific antibodies were induced, which suggests that the virus was completely inactivated and safe to use in pigs (data not shown).

3.6. Western blot analysis of the viral proteins of inactivated PRRSV

The effect of different inactivation methods on viral proteins was further investigated by analyzing the protein pattern of the virus before and after inactivation by Western blotting (Fig. 4, Tab. II). Under reducing as well as under non-reducing circumstances, all the viral proteins were present after inactivation with formaldehyde, AT-2 and gamma irradiation, but to a lesser extent as untreated PRRSV. For glutaraldehyde, under reducing as well as under non-reducing conditions not all the viral proteins could be detected after inactivation. The protein pattern of the virus before and after inactivation by UV irradiation was not identical, which suggests that UV irradiation resulted in the degradation of the viral proteins. For pH changes, 37 °C and BEI all the viral proteins were present after inactivation. Complexes like N-dimer and M-GP5 however were more degraded in comparison to the untreated virus when treated with pH changes.

Figure 4. Effect of different inactivation methods on PRRSV proteins. Western blot analysis of untreated LV or inactivated LV in reducing (A) and non-reducing (B) conditions.

The formaldehyde, glutaraldehyde, pH, AT-2, gamma irradiation and UV inactivated PRRSV did not have the same protein pattern as the untreated virus. These results indicated that with these inactivation procedures inactivated PRRSV did not resemble infectious PRRSV. The 37 °C and BEI inactivated PRRSV showed the same protein pattern as the untreated virus. These results suggest that PRRSV inactivated at 37 °C or by BEI resembles infectious PRRSV.

4. DISCUSSION

Virus inactivation procedures with the aim to develop inactivated virus vaccines should have two major goals: complete inactivation of infectious virus (safety), while conserving epitopes of the inactivated virus that are important for the induction of protective immunity (antigen quality). This study aimed to test different inactivation procedures for PRRSV to evaluate the effect on both reduction of infectivity and conservation of viral entry-associated domains, the latter being a measure for the quality of the antigen used.

Currently, there are no data available in literature on inactivation procedures of PRRSV. Therefore, inactivation methods and conditions used in this study are based on studies where inactivation of other viruses was evaluated. Evaluation of the capacity of different inactivation procedures to completely inactivate PRRSV showed similarities and differences with that of other viruses. PRRSV could not be inactivated with AT-2, not even after treatment with 2 mM for 4 h at 37 °C, while HIV type 1 is already inactivated with 100 μM AT-2 after 1 h at 37 °C [54]. AT-2 modifies free thiol groups of internal viral proteins like the nucleocapsid of HIV-1, more specifically zinc-finger motifs important for HIV-1 infection, leaving disulfide bridges of glycoproteins in the virus envelope unaffected [7, 65], but for PRRSV the formation of homodimers of nucleocapsid proteins via disulfide bridges is important for virus infection [67]. Since PRRSV seems not to be sensitive for AT-2, this product cannot be used to develop an inactivated PRRSV vaccine. For PRRSV inactivation with formaldehyde and glutaraldehyde, respectively 0.1 and 0.5 μg/mL for 4 h at 37 °C was sufficient. In comparison, HIV-1 can be inactivated with 2 μg/mL formaldehyde after 24 h at 37 °C [54] and SARS-CoV with 90 μg/mL formaldehyde or 20 μg/mL glutaraldehyde after 24 h at 37 °C [14]. For PRRSV inactivation by changing the pH, incubation of the virus at a pH2 or pH12 for 1 h at 37 °C was effective. Also SARS-CoV could be inactivated after incubation of 1 h at 37 °C at a pH2 or pH12 [14]. Incubation of PRRSV for 48 h at 37 °C was efficient for its inactivation, while SARS-CoV was inactivated after incubation of 20 min at 56 °C [14] and adenovirus type 5 after 10 min at 50 °C [33]. For this study, inactivation of PRRSV at higher temperatures was not considered, since PRRSV proteins, similar to what is observed with other viruses [56], will most likely be denaturated, thereby destroying important epitopes. PRRSV inactivation with gamma irradiation or UV could be achieved with an irradiation dose of respectively 0.25 kGy or 100 mJ/cm². SARS-CoV could not be inactivated with gamma irradiation, even not after an irradiation dose of 15 000 rad. For inactivation with UV, an irradiation dose of 3614 mJ/cm² was needed [14]. BEI inactivation of PRRSV could be achieved with 1 mM BEI after 6 h incubation at 37 °C. Berhane et al. [2] inactivated Nipah virus with 3 mM BEI for 24 h at room temperature and Mondal et al. [42] used 1.6 mM BEI for 24 h at 37 °C to inactivate rabies virus. In conclusion, for all tested inactivation procedures, except for AT-2, conditions that allowed PRRSV inactivation could be determined.

Besides inactivation, conservation of viral entry-associated domains is equally important for the development of an inactivated virus vaccine. An in vitro assay to check the conservation of viral domains that are important for the induction of a protective immunity is preferred to avoid time consuming vaccination studies and to allow precise fine tuning of inactivation methods. For influenza virus and HIV for example, the major neutralizing epitopes are known and an in vitro assay to analyze the conservation of these domains after inactivation can be performed by measuring haemagglutination for influenza virus [19] or by ELISA for HIV [25]. However, for PRRSV there is currently no in vitro assay to evaluate this due to a limited knowledge on the PRRSV neutralizing epitopes. In our lab, it was shown that neutralizing antibodies were preventing infection by disturbing entry of the virus into macrophages [15]. Therefore, it was hypothesized that viral domains important for viral entry (entry-associated domains) are also important for the induction of viral neutralizing antibodies. In this study, different inactivation methods for PRRSV were analyzed for their effect on the viral domains important for entry into macrophages, which are most likely also important for the induction of neutralizing antibodies. Our experiments with formaldehyde, glutaraldehyde and pH inactivated PRRSV suggest that the viral entry-associated domains are modified, since the virus can no longer attach to and internalize into macrophages. Similarly, Western blotting showed that the viral proteins were not or to a lesser extent present, which is probably due to cross-linking of proteins by formaldehyde and glutaraldehyde or denaturation of viral proteins by pH changes. Formaldehyde and glutaraldehyde are known to have a similar effect on proteins, as they are able to induce protein cross-linking [1, 8, 22], while pH changes affect proteins by denaturation [62]. Cross-linking or denaturation of viral domains that are involved in attachment and internalization of PRRSV might interfere with the subsequent presentation of viral domains to cells of the adaptive immune system. As a result, inactivation with formaldehyde, glutaraldehyde and pH changes will probably give a poor preservation of viral immunogenicity [13, 54]. In conclusion, formaldehyde, glutaraldehyde and pH changes are not effective methods to inactivate PRRSV with preservation of the entry-associated domains and for future vaccine development.

Because gamma irradiation mainly has an effect at the genomic level, one could assume that this would be an effective method to inactivate PRRSV. However, the range between complete inactivation and preservation of the entry-associated domains was too small to use this method in a safe way for vaccine development. PRRSV inactivated with a dose of 1.5 kGy was still infectious, while PRRSV inactivated with a dose of 2 kGy was no longer infectious, but could not efficiently internalize into macrophages. At this dose viral entry-associated domains are most likely destroyed due to the formation of free radicals which damage proteins [24]. This viral protein degradation was also seen on Western blot. In conclusion, gamma irradiation is not suitable as a method to inactivate PRRSV.

Temperature inactivation has been documented to inactivate viruses by denaturation [31, 56]. As with protein cross-linking, denaturation of viral proteins may also destroy the entry-associated domains of PRRSV. However, a modest increase of temperature (37 °C) did not prevent PRRSV to attach to and internalize into macrophages, while preventing viral replication. Incubating PRRSV at 37 °C would thus be an interesting option to generate a killed PRRSV vaccine. Clearly, inactivation at 37 °C does not result in denaturation of viral proteins. This is confirmed by Western blotting, since no difference was observed in the banding pattern between not inactivated control virus and inactivated virus. However, since the mechanism of inactivation is unknown, it might not be safe to use this method to inactivate PRRSV for vaccine development.

Of all methods tested, the most promising methods to inactivate PRRSV for KV vaccine development are UV and BEI, because they mainly have an effect on genomic level, preserving entry-associated viral domains. This was shown by internalization of the inactivated virus into macrophages. However, for UV the destruction of capsid proteins has been reported [40]. Western blot analysis in this study indeed showed degradation of viral proteins upon UV inactivation.

By investigating internalization into macrophages, the possibility exists that the inactivated virus is taken up by phagocytosis instead of using the PRRSV entry receptor Sn. However, PRRSV inactivated by UV and BEI did also attach to and internalized into macrophages after treatment with a phagocytosis inhibitor. These results confirm that inactivated PRRSV could still interact with PRRSV receptors and that thus internalization of BEI or UV inactivated PRRSV into macrophages did not occur via phagocytosis.

In summary, the results of this study show that inactivation methods that have a direct effect on viral proteins, like formaldehyde, glutaraldehyde and changing the pH are not good candidates for viral inactivation, as they do not conserve viral entry-associated domains. On the other hand, UV, BEI and gamma irradiation, which mainly have an effect on the genome, could be interesting methods to inactivate PRRSV for vaccine development, as inactivated virus is still able to internalize into macrophages, but no longer replicates. However, UV and gamma irradiation may give problems with photoproducts or free radicals formed during irradiation, which in turn could indirectly degrade viral proteins. This study also revealed that exposure of PRRSV to increased temperature was an efficient method of viral inactivation, but the mechanism of action still requires further investigation.

To investigate if the methods that seem the most suitable according to the in vitro screening assay described in this study are indeed good for preserving the immunogenicity of PRRSV inactivated vaccines, new experimental vaccines based on BEI and UV inactivation will be tested in animals.

Acknowledgments

The authors would like to thank C. Vanmaercke and C. Bracke for their highly appreciated and excellent technical assistance. We also want to thank J. Vandoorsselaere and D. Helderweirt (KATHO, Roeselare) for virus production and purification. L. Van Hoorebeke and P. Vanderniepen (Ghent University, Faculty of Science, Department of Subatomic and Radiation Physics) are acknowledged for advice and usage of their electron accelerator and technical assistance. This work was funded by the Agricultural Research Program from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-LO 040721). P.L. Delputte is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO).

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All Tables

All Figures

	Figure 1. Effect of different inactivation methods on PRRSV infectivity. Untreated LV or inactivated LV was titrated on 3 days cultured Marc-145 cells to determine presence of infectious virus in the samples. The dotted line resembles the detection limit of the assay.
In the text

Figure 2. Effect of different inactivation methods on PRRSV internalization. Macrophages were incubated with untreated LV or inactivated LV for 1 h. After 1 hpi, the virus was stained by immunofluorescence and internalization of PRRSV particles was determined by confocal microscopy. The amount of internalized PRRSV particles of both treated and untreated virus in a macrophage were counted by counting the small fluorescently labeled dots and set relatively to internalized untreated virus.

In the text

Figure 3. Effect of different inactivation methods on different stages of the viral replication cycle. Macrophages were incubated with inactivated LV or untreated LV for 1 h at 4 °C and 1, 5 or 10 h at 37 °C. After fixing and immunofluorescence staining of the cells, attachment (1 hpi at 4 °C), internalization (1 hpi at 37 °C), disassembly (5 hpi at 37 °C) and replication (10 hpi at 37 °C) was analyzed by confocal microscopy.

In the text

	Figure 4. Effect of different inactivation methods on PRRSV proteins. Western blot analysis of untreated LV or inactivated LV in reducing (A) and non-reducing (B) conditions.
In the text