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1 Institute for Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany
2 Impfstoffwerke Dessau-Tornau GmbH, PF 400214, 06855 Rosslau, Germany
Correspondence
U. Truyen
truyen{at}vmf.uni-leipzig.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Interestingly, the homologous neutralizing antibody titres of the sera of all three pigs and both rabbits inoculated or immunized with PPV-27a were 100- to 1000-fold lower than the heterologous titres against any of the other viruses. The low homologous neutralizing antibody titres suggest a possible, yet undefined, immune escape mechanism of this PPV isolate. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). PPV is widespread in swine herds, despite vaccination (Siegl, 1976; Gillick, 1977; Wattanavijarn et al., 1985). The virulent strains cause reproductive failures in swine, represented by stillbirth, mummification, embryonic death, infertility (SMEDI–syndrome) and delayed return to oestrus (Redman et al., 1974; Mengeling & Cutlip, 1975; Joo et al., 1976a, b, c; Cutler et al., 1983). The manifestation of clinical disease depends on the pathogenicity of the virus and on the stage of gestation. Fetuses infected before day 70 of gestation usually die, whereas fetuses infected at a later time point develop antibodies against PPV, eliminate the virus and survive the infection (Johnson & Collings, 1971).
PPV strains can be distinguished by their different pathogenicity (Tijssen et al.,1995). Substitution of only a few residues in the VP2 capsid protein is thought to be responsible for distinct biological properties between PPV-NADL-2 and PPV-Kresse in vitro (Bergeron et al.,1996; Simpson et al., 2002). The non-pathogenic PPV-NADL-2 strain is widely used for the production of inactivated vaccines and causes a limited viraemia, but does not cross the placenta barrier (Mengeling et al., 1980, 1984; Paul et al., 1980). In contrast, the highly pathogenic isolate PPV-Kresse is able to kill even immunocompetent fetuses and has been associated with dermatitis in juvenile pigs (Kresse et al., 1985; Whitaker et al., 1990).
Phylogenetic analysis of the VP1/VP2 protein genes revealed that there is a relatively weak sequence similarity between PPV-NADL-2 and recent field isolates from Germany (Zimmermann et al., 2006). Two clusters could be defined: cluster 1 is formed by four German isolates [including PPV-143a and PPV-IDT (MSV)], as well as the English isolate PPV-Challenge (Engl.), and Asian and American isolates (PPV-NADL-2 and PPV-Kresse); cluster 2 is represented by three German isolates, including PPV-27a (Zimmermann et al., 2006). The aim of this study was to examine two of these recent field isolates, one from each cluster, under experimental conditions for their pathogenicity (in vivo) and antigenicity (in vitro), particularly in comparison to the vaccine viruses PPV-NADL-2 and PPV-IDT (MSV).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Virus.
Two German field isolates and two vaccine viruses were used for inoculation of pregnant gilts and for immunization of rabbits: PPV-143a, PPV-27a, PPV-IDT (MSV) and PPV-NADL-2. PPV-143a and PPV-27a were isolated from the livers of aborted fetuses in Germany in 2002 (Zimmermann et al., 2006). PPV-143a in the fifth serial passage onto an SPEV cell culture was used as inoculum with an infectivity titre of 106.5 TCID50 ml–1. PPV-27a was used in the third serial passage with a titre 105.1 TCID50 ml–1. PPV-IDT (MSV) (Mayr & Mahnel, 1964) was used in the 24th serial passage with a titre of 106.5 TCID50 ml–1 and PPV-NADL-2, a tissue culture-adapted virus (Mengeling, 1975), was used with a titre of 104.9 TCID50 ml–1. PPV-Challenge (Engl.) was used in the eighth serial passage for immunization of rabbits.
Cells were seeded at 1.0x105 cells ml–1 and after 1 h incubation they were inoculated with PPV at an m.o.i. of 0.5–2.0. Cells were grown in DMEM supplemented with penicillin (100 U ml–1) and streptomycin (100 µg ml–1), and incubated at 37 °C in a humid environment containing 5 % CO2. Cells with a marked cytopathic effect (CPE) were harvested 5–6 days post-inoculation by three freeze–thaw cycles. Cellular debris was removed by centrifugation at 5000 g for 20 min at 4 °C before further purification.
Virus purification.
For immunization of rabbits, supernatants were purified by precipitation and CsCl density-gradient centrifugation as described by Molitor et al. (1983). Purified PPV antigen was analysed for purity by SDS-PAGE containing 10 % polyacrylamide. The protein concentration was determined using the Micro BCA Protein Assay Reagent Kit (Pierce) with serum albumin as the standard (Smith et al., 1985).
Animal experiments.
Animal experiments were approved by the Regierungspräsidium Leipzig (AZ 24-9168.11TVV11/05 and AZ 24-9168.22-02-V4/04). Twelve specific-pathogen-free PiétranxLarge White primiparous sows, 11 months of age, were randomly assigned to four groups of three gilts each and were kept separately throughout the experiment. Before infection they all tested negative for antibodies to PPV (see this study), porcine influenza virus [in-house test using RESPIPORC FLU3 viruses – H1N2, H1N1 and H3N2 (Impfstoffwerke Dessau-Tornau) – as antigens], Erysipelothrix rhusiopathiae (ELISA; Cypress Diagnostics) and porcine respiratory and reproductive syndrome virus [in-house test using INGELVAC PRRS MLV (Ingelheim Vetmedica) as antigen]. At day 40 of gestation the sows were inoculated with the respective viruses (field isolates and vaccine strains) by both the intranasal (i.n.) and intramuscular (i.m.) route. Group 1 pigs were inoculated with PPV-143a (106.9 TCID50 ml–1), group 2 pigs with PPV-27a (105.1 TCID50 ml–1), group 3 pigs with PPV-IDT (MSV) (106.5 TCID50 ml–1) and group 4 pigs with PPV-NADL-2 (104.9 TCID50 ml–1). Each gilt received 2.0 ml virus i.m. and 2.0 ml i.n. Clinical signs (general performance, respiratory activity, food and water intake, and rectal temperatures) were recorded daily for 50 days post-inoculation. Blood samples were taken at intervals as specified in Table 1 and Fig. 1. The EDTA-blood samples were analysed in the hospital of the Medical Animal Clinics, Veterinary Faculty, University of Leipzig, Germany, for differential cell counts, which included numbers of leukocytes, monocytes, lymphocytes, neutrophils, eosinophils, basophils and thrombocytes, as well as for determination of haemoglobin and haematocrit. Blood samples were also analysed for virus-specific parameters, such as virus isolation, antibody detection by haemagglutination inhibition (HI) testing, and by PCR. At day 90, about 3 weeks before term, all gilts were euthanized and the fetuses were aseptically delivered via Caesarean and euthanized by intravenous barbiturate injection. Their size, weight and position in the uterus, as well their general condition, were recorded. Blood and tissue samples (lung and kidney) were collected from all fetuses. Umbilical cord blood samples from non-mummified fetuses were examined for antibodies against PPV by HI testing; lungs and kidneys were tested for infectious PPV by cell culture isolation and subsequent direct immunofluorescence (IF) testing. PPV protein was detected by using a haemagglutination (HA) test.
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To prepare virus-specific sera for cross-neutralization tests with the selected field isolates (PPV-143a, PPV-27a), vaccine viruses and PPV-Challenge (Engl.), 4-month-old New Zealand rabbits were immunized with CsCl-density-gradient-purified virus. Animals were injected with 148 µg virus protein emulsified in 0.6 ml Freund's incomplete adjuvant (Sigma). Each animal received two booster injections at 2 week intervals with an increasing amount of virus protein (222 and 370 µg). The resulting sera were heat-inactivated at 56 °C for 30 min and stored frozen at –20 °C.
Virus isolation.
PPV isolation was performed as described by Soares et al. (2003) with minor modifications. Briefly, lungs and kidneys were ground using a pestle and mortar with sterile sea sand, and then resuspended in DMEM supplemented with antibiotics (200 U and 100 µg streptomycin ml–1, respectively) to give 20 % organ suspensions. After repeated freezing and thawing for three cycles and centrifugation for 20 min at 2000 g at 4 °C, the supernatant was passed through a 20 µm filter. The supernatant was tested for HA activity using 0.5 % human erythrocytes and then added onto SPEV cells for virus isolation. Cultures were observed for 5–6 days post-infection (p.i.) for a CPE. The supernatant was then titrated in SPEV cells and seeded in 96-well microtitre plates. The cells were fixed after 5 days with acetone/methanol (1 : 1) and stained immunofluorescently by using FITC-conjugated PPV antibody (VMRD).
Haemagglutination inhibition.
Heat-inactivated sera were first absorbed with 25 % kaolin in borate/saline solution (0.5 M H3BO4, 1.5 M NaCl, pH 9.0) for 20 min at 25 °C and centrifuged to remove natural heat-stable non-specific inhibitors. Sera were then absorbed with 50 % human erythrocytes (PBS, pH 7.0). The now 1 : 4 diluted sera were titrated in 2-log steps in PBS (pH 7.0) in V-type microplates and the HI test was performed as previously described using 8 HA units of parvovirus [PPV-IDT (MSV)] and 0.5 % human erythrocytes (O, rhesus-negative) (Joo et al. 1976b). HI titres of lower than 8 were considered as a negative result.
Serum neutralization.
All sera were heat-treated at 56 °C for 30 min and stored at –20 °C. The sera were diluted in 5-log steps, and 200 TCID50 per 0.1 ml was mixed with an equal volume of diluted sera. After 2 h incubation at 37 °C, 100 µl of the serum/virus mixtures was added onto SPEV cells seeded in 96-well microtitre plates. Plates were incubated for 5–6 days at 37 °C. After this period, the cells were fixed and stained with FITC-labelled antibodies for the presence of virus antigen. The neutralizing antibody titres were calculated by the formula of Kaerber (1931).
Real-time PCR.
All mummified and non-mummified fetuses were examined by real-time PCR in an Mx3000P cycler with SYBR Green as described by Wilhelm et al. (2005).
Also, post-infection sera of sows from infection group 27a, as well as the sera prepared in rabbits by immunization against PPV-27a which were used in the neutralizing tests, were tested for the presence of PPV DNA as an indication of viraemia. DNA was purified using the QIAmp DNA Mini Kit (Qiagen).
Statistical analysis.
The mortality rates of the fetuses and antibody titres were analysed by using the Mann–Whitney U-test. Results were considered significant at P values of <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Fetal mummification was significantly higher (P<0.05) in the gilts infected with PPV-27a as compared to the other groups (85 % vs 5–18 %) (Fig. 2, Table 2). Almost all fetuses of the group 2 gilts infected with PPV-27a showed various ranges of fetal mummification. In contrast, only single mummified fetuses were found in litters of group 1 (PPV-143a), group 3 [PPV-IDT (MSV)] or group 4 gilts (PPV-NADL-2). The crown–rump lengths (data not shown) of these fetuses indicated that they died before PPV infection. No differences in length or weight were observed in the non-mummified fetuses of all four infection groups.
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). At the time of the Caesarean the antibody titres as determined by HI were between 64 and 4096.
Fetuses.
Umbilical cord blood of the non-mummified fetuses from all groups revealed HI antibody titres (Table 2), indicating transplacental infection of all PPV isolates examined. Pre-colostral HI antibodies were found in fetuses exposed to PPV-143a (88 %), PPV-27a (100 %), PPV-IDT (MSV) (100 %) and PPV-NADL-2 (86.0 %). The titres ranged from 80 to 1024.
Virus detection in fetuses
The supernatants from lung and kidney tissues were inoculated onto SPEV cells in an attempt to reisolate PPV. After two passages, no evidence for virus replication was observed in the fetuses of group 1 (PPV-143a), group 3 [PPV-IDT (MSV)] and group 4 (PPV-NADL-2). No HA activity could be demonstrated in any organ suspensions of any of the fetuses. In contrast, virus could be readily isolated from fetuses of group 2 (PPV-27a) (Table 3). From these fetuses, two non-mummified fetuses (HI titre of 80–128) showed neither HA activity nor infectious PPV. One non-mummified fetus (HI titre of 640) showed no HA activity, but virus could be isolated from the lungs and kidney. High concentrations of infectious PPV virus could also be detected in all eight mummified fetuses by virus isolation in cell culture. These tissues also had high HA titres of 800–3200.
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Serum neutralization
Neutralizing antibody titres were determined in the post-infection sera of the sows and rabbit sera raised against the various PPV-isolates. The neutralizing antibody titre in sera raised against PPV-143a, PPV-IDT (MSV), PPV-NADL-2 and PPV-Challenge (Engl.) against the PPV isolate 27a were generally very low, with SN titres ranging from 0.5 to 0.69, but were high against PPV-143a, PPV-IDT (MSV), PPV-NADL-2 and PPV-Challenge (Engl.). Sera raised against PPV-27a neutralized all heterologous PPV isolates with high titres ranging from 2.99 to 3.99 (overall geometric mean titre). The homologous virus, however, was less efficiently neutralized (titre 0.69–1.19, see Table 4).
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, column 4 and Table 4, column 5).
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and Table 5).
Real-time PCR
Parvovirus DNA was only detected in the post-infection sera of sows of day 8 p.i. However, neither the day 42 and 49 p.i. samples from the sows nor the sera of the rabbits were positive for PPV-DNA, indicating that the low homologous antibody titres of the PPV sera were not due to inhibition by virus present in these sera.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, the fact that in this study antibodies could be detected in fetuses of all four groups provides indirect evidence for transplacental infection of both the PPV field isolates and the vaccine viruses PPV-IDT (MSV) and PPV-NADL-2. This is in contrast to previous reports where it was postulated that PPV-NADL-2, which is widely used as a vaccine strain, is not able to cross the placental barrier (Mengeling & Cutlip, 1976; Mengeling et al., 1980; Paul et al., 1980). It must be stressed, however, that no virus could be isolated from any fetus, and only viral DNA was amplified by PCR from single fetuses from all groups. Furthermore, the amount of viral DNA differed greatly between the fetuses of the PPV-27a-inoculated sows and all others. The amount of virus in the piglets of the sows inoculated with PPV-NADL-2, PPV-IDT or PPV-143 was 109-fold lower than that of the PPV-27a sows, and close to the detection limit of the PCR method used. Although this suggests transplacental transmission, direct evidence, i.e. the isolation of infectious virus, is still missing. Positive PCR results may represent the transplacental transfer of non-infectious virus or virus DNA. The seroconversion of all piglets indicates virus transmission only if a transfer of maternal IgG through the placenta can be completely excluded. Further studies are needed to clarify this.
Transplacental infection is believed to be dose-dependent (Paul & Mengeling, 1984). In our experiments, the inocula had a different virus titre, with PPV-27a and PPV-NADL-2 having the lowest titres (105.1 and 104.9 TCID50 ml–1, respectively) and PPV-143 and PPV-IDT having the highest titres (106.9 and 106.5 TCID50 ml–1, respectively). A dose-dependent influence on transplacental transmission in this study cannot be deduced.
A difference in the virulence of PPV-27a to members of the other cluster [PPV-143a, PPV-IDT (MSV), PPV-NADL-2] was indicated by the high mortality of the fetuses. PPV spreads inside the uterus from fetus to fetus. Virus spread was probably slower between fetuses of the PPV-143a, PPV-IDT (MSV) and PPV-NADL-2 groups than between those of the PPV-27a group. Considering the time required for the virus to cross the placenta, infection most probably occurred after day 70 of gestation (Joo et al., 1976a, b, c). This would explain the detection of antibodies in the fetuses, which were then immunocompetent (Bachmann et al., 1975; Mengeling & Cutlip, 1975; Redman et al., 1974). Considering the crown–rump lengths of the single mummified fetuses where no PPV could be detected, their death most probably occurred before PPV infection.
The failure to detect PPV-infection in fetuses of groups 1, 3 and 4 [PPV-143a, PPV-IDT (MSV) and PPV-NADL-2] could be interpreted as false-negative results due to autolysis or mummification, the presence of virus-neutralizing antibodies, or low virus titres in fetuses (Belak et al., 1998; Joo et al., 1976a, b, c; Soares et al., 1999), and some studies suggest that some strains may require adaptation to cell culture (Choi et al., 1989; Mayr & Mahnel, 1964). By examining some fetuses by real-time PCR with SYBR Green (Wilhelm et al., 2005), low amounts of PPV DNA could be detected in non-mummified fetuses of groups inoculated with PPV-143a, PPV-IDT (MSV) and PPV-NADL-2, and in one mummified fetus of the group infected with PPV-NADL-2. However, high amounts of PPV-DNA were only observed in both mummified and non-mummified fetuses of the group of sows infected with PPV-27a.
Antigenic virus variants characterized by single amino acid substitutions within the antibody-binding site, may be less efficiently neutralized by antibodies (Strassheim et al., 1994; Yuan & Parrish, 2000). In the present study we investigated post-infection sera of pigs and antisera of rabbits immunized with the respective viruses in two independent cross-neutralization tests. Cross-neutralization of the sera raised against the vaccine viruses PPV-NADL-2 and PPV-IDT (MSV) against the field isolates PPV-143a and PPV-27a as well as against PPV-Challenge (Engl.) revealed low neutralization activity (0.5–0.69) against PPV-27a, indicating incomplete protection. Neutralizing antibodies are known to play a prominent role in protection against parvovirus infection. Therefore, if PPV-27a is representative for current PPV-isolates in the population, this indicates that vaccines, which have been used for 30 years, may no longer be fully protective.
Beside the evidence for distinct antigenic types of PPV, another interesting phenomenon became obvious in this study. Interestingly, all sera raised against the field isolate PPV-27a neutralized all heterologous PPV isolates with high efficiency (2.99–3.99), but homologous neutralization was much less efficient (0.69–1.19). This was seen for all three pigs inoculated as well as with the sera of both rabbits immunized with PPV-27a. To test that this possible immune escape was not due to an inhibitory effect of virus present in the sera, sera from different time points post-infection were used in cross-neutralization tests and were tested for PPV DNA by PCR. Only early sera (8 days p.i.) where shown to be viraemic, and as the neutralization tests were done with later sera, the obtained SN titres against PPV-27a were therefore not an artefact due to the presence of virus in the test sera. The mechanisms behind this phenomenon are currently unknown and studies are underway to define the viral determinants responsible for this interesting feature.
The determinants of PPV virulence are unknown. Soares et al. (2003) and Simpson et al. (2002) discussed the importance of the 127 nt repeat for virulence, as that repeat is found in the VP1-gene of PPV-NADL-2, but not in any of the pathogenic viruses tested. The results reported by Zimmermann et al. (2006) that all recent field isolates from Germany [PPV-27a, PPV-143a and PPV-IDT (MSV)] lacked the 127 nt repeat confirm this hypotheses. However, based on the results of the study presented here, both PPV-IDT (MSV) and PPV-143a appeared non-pathogenic, even though they lack the repeat.
Comparison of the PPV-NADL-2 and PPV-Kresse genomes revealed three amino acid changes within the coding region of VP2 (D378
G, H383Q and S436P) which are considered to be responsible for the different tissue tropism in vitro (Bergeron et al., 1996). Whether these three amino acid differences also determine the pathogenicity of the viruses is unknown. All three amino acid differences are present in PPV-Challenge (Engl.), but not in PPV-IDT (MSV), PPV-143a and PPV-27a (Zimmermann et al., 2006). In PPV-143a and PPV-27a, the VP2 residue Pro-436 is changed to Thr. This indicates that it is unlikely that all three changes in the capsid protein reported by Bergeron et al. (1996) determine pathogenicity. Moreover, Vasudevacharya & Compans (1992) demonstrated with a PPV-mutant, P2, that only two changes were sufficient to alter an important biological property of the virus, its host range. One of these mutations occurred in the NS gene and the other in the capsid gene.
The phylogenetic cluster containing the German isolate PPV-27a is defined by three amino acid substitutions (Q228E, E419Q and S436T) in VP2 (Zimmermann et al., 2006). All three residues are located in accessible regions on the capsid surface, and position 228 has been identified as part of one of the nine known linear epitopes on VP2 (Kamstrup et al., 1998; Simpson et al., 2002). To what extent the capsid structure will be altered by changing amino acid 228 from Gln to Glu and amino acid 419 from Glu to Gln, and whether they are even involved in the apparent immune escape, needs further investigation.
In conclusion, our results indicate that possible antigenic variation represented by PPV-27a may influence effective vaccination against PPV. Further studies and animal inoculation experiments using PPV-27a mutants will be required to address this important issue.
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Received 16 June 2006;
accepted 14 October 2006.
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, PPV-143a; 
, PPV-27a; 
, PPV-NADL-2; 
, PPV-IDT (MSV)] in peripheral blood at different time points after infection. The transient relative lymphopenia at day 6 is indicated with an arrow.