University of Leipzig, An den Tierkliniken 1, Leipzig, Germany

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1 Journal of General Virology (2009), 90, DOI /vir Short Communication Correspondence U. Truyen Vaccination against porcine parvovirus protects against disease, but does not prevent infection and virus shedding after challenge infection with a heterologous virus strain A. Jóźwik, 1 J. Manteufel, 1 H.-J. Selbitz 2 and U. Truyen 1 1 Institute for Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, Leipzig, Germany 2 IDT Biologika GmbH, PF , Rosslau, Germany Received 20 March 2009 Accepted 10 June 2009 The demonstration of field isolates of porcine parvovirus (PPV) that differ genetically and antigenically from vaccine strains of PPV raises the question of whether the broadly used inactivated vaccines can still protect sows against the novel viruses. Ten specific-pathogen-free primiparous sows were assigned to three groups and were vaccinated with one of two vaccines based on the old vaccine strains, or served as non-vaccinated controls. After insemination, all sows were challenged with the prototype genotype 2 virus, PPV-27a, on gestation day 41; fetuses were delivered on gestation day 90 and examined for virus infection. The fetuses of the vaccinated sows were protected against disease, but both the vaccinated and the non-vaccinated sows showed a marked increase in antibody titres after challenge infection, indicating replication of the challenge virus. All sows (vaccinated and non-vaccinated) shed the challenge virus for at least 10 days after infection, with no difference in the pattern or duration of virus shedding. Porcine parvovirus (PPV) belongs to the family Parvoviridae, which comprises small and highly stable viruses (Zee & MacLachlan, 2004). Infection of swine with PPV occurs worldwide and may lead to reproductive failure in swine or cutaneous lesions in piglets. Transplacental infection of fetuses occurs readily and may lead to death and mummification of the fetus. The reproductive disorder in swine is known as stillbirth, mummification, embryonic death and infertility (SMEDI) syndrome (Soares et al., 1999; Zee & McLachlan, 2004; Cao et al., 2005; Wilhelm et al., 2006; Wolf et al., 2008). The establishment of clinical disease and severity of symptoms depend on the virulence of the virus strain and the stage of gestation at the time point of infection. 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 (Nielsen et al., 1991). Zimmermann et al. (2006) showed that PPV is more diverse than thought previously and that some recent German field isolates differ genetically from the reference and vaccine strains. By phylogenetic analyses based on VP1 sequences, two genotypes could be defined. Zeeuw et al. (2007) showed that these genotypes also differ antigenically when tested in serum neutralization tests, showing fold titre differences. The prototype genotype 2 virus, PPV-27a, is highly virulent in pregnant gilts after experimental infection, as demonstrated by the high mortality among the fetuses of sows infected with PPV- 27a (85 %) compared with sows infected with the other strains of PPV, e.g. PPV-NADL-2, PPV-IDT or PPV-143a (mortality 5 18 %) (Zeeuw et al., 2007). The currently available vaccines against PPV are based on inactivated whole-virus preparations of PPV genotype 1 strains isolated some 30 years ago. Therefore, the aim of this study was to examine whether vaccinations with genotype 1 viruses (one commercially available vaccine and one experimental vaccine) can protect pregnant sows against disease or infection after challenge with the highly virulent genotype 2 strain PPV-27a. Two PPV vaccines containing inactivated whole virus were used: Porciparvac (IDT Biologika GmbH), which contains the reference strain IDT [master seed virus (MSV)], and an experimental vaccine containing the strain Stendal, which except for the vaccine virus was an identical preparation to Porciparvac. The Stendal virus is related closely to the IDT (MSV) virus and clusters with the genotype 1 viruses (not shown). The challenge virus PPV-27a (Zimmermann et al., 2006) was propagated in SPEV (porcine kidney) cells, obtained from the Friedrich Loeffler Institute (Insel Riems, Germany). The animal experiment was approved by the Landesdirektion Leipzig (TVV 25/07). Ten specific-patho G 2009 SGM Printed in Great Britain 2437

2 A. Jóźwik and others gen-free primiparous sows (mixed breed Deutsche Landrasse/Pietrain ) were synchronized by using routine protocols of gestagen and subsequent human and equine choriogonadotropin treatment, and inseminated at the age of 7 months. They tested negative for antibodies to PPV [by haemagglutination-inhibition (HI) test], for antibodies to Salmonella enterica (by ELISA) and for the presence of porcine respiratory and reproductive syndrome virus (by RT-PCR) prior to the experiment. The animals were divided into three groups. The first group contained four sows that were vaccinated with the experimental vaccine containing strain Stendal, the second group contained two sows vaccinated with Porciparvac and the third group served as the control group (four sows that were not vaccinated). The sows were vaccinated twice at days 235 and 221 before insemination by subcutaneous inoculation of 3 ml per dose (Porciparvac) or by intramuscular inoculation of 2 ml per dose (Stendal). For challenge infection, pregnant sows were inoculated at day 41 of gestation with 4 ml PPV- 27a (titre, 10 6 TCID 50 ) by the intranasal route. Two millilitres were applied into each nostril. Clinical signs (general condition, food and water intake, consistency of faeces, rectal temperature and external appearance of the genital organs) were recorded daily for 30 days post-infection (p.i.). Blood samples were taken on the day of infection, at 3 weeks p.i. and at 7 weeks p.i. for antibody testing. Rectal swabs were taken from sows six times during the first 3 weeks p.i. and on day 50 p.i. Except for those taken on days 21 and 50 p.i., swabs were analysed by PCR, virus isolation was performed on cell cultures and PCR from cell-culture supernatant was used to detect possible virus shedding. On day 90 of gestation, about 3 weeks before term, all sows were euthanized and the fetuses were delivered aseptically via Caesarean and euthanized by intracardial barbiturate injection. Their size, weight and position in the uterus, as well their general condition, were recorded. Tissue samples (lung and kidney) were collected from all fetuses and tested for virus replication by direct PCR, for virus isolation by cell culture and for virus shedding by PCR from cellculture supernatant. Umbilical-cord blood samples from non-mummified fetuses were examined for antibodies against PPV by HI test. PPV isolation from swabs or fetuses was performed in SPEV cells as described previously (Soares et al., 1999; Zeeuw et al., 2007). The amount of material that was tested in one reaction of the real-time PCR represented onethousandth of the swab material. DNA from the tissue probes of the fetuses (lungs and kidneys) and from the rectal swabs of the sows was extracted by using a QIAamp DNA Mini kit (Qiagen) according to the manufacturer s instructions. PCR using the primer pair PPVs (59- GGGGGAGGGCTTGGTTAGAATCAC-39) and PPVas (59-ACCACACTCCCCATGCGTTAGC-39) was performed as described previously (Wilhelm et al., 2006). The HI test was performed as described previously (Joo et al., 1976; Zeeuw et al., 2007) with 0.5 % human erythrocytes (group O, Rh 2 ) and 8 haemagglutinating units of PPV-NADL-2. HI titres,8 were considered negative. The serum neutralization test was performed as described previously (Zeeuw et al., 2007) using the two vaccine viruses, PPV-IDT (MSV) and PPV-Stendal, or PPV-27a as test viruses. The end point was detected by an indirect immunofluorescence test using porcine hyperimmune sera and fluorescein isothiocyanate-labelled antipig conjugate (Dianova). The significance of the differences in mortality rate of the fetuses and antibody titres was analysed by using the Mann Whitney and x 2 tests. Results were considered significant at P-values of,0.05. All sows remained clinically healthy throughout the experiment. The mortality rate among the fetuses of the vaccinated and non-vaccinated sows differed significantly: whilst in both vaccine groups, nearly 100 % (66 of 67) of the fetuses were alive and unaffected, 78 % (39 of 50) of the fetuses of the control group were dead and showed various degrees of fetal mummification (Table 1). Infectious virus and virus DNA could be detected readily in fetuses of the control group, but in neither of the two vaccine groups (Table 1). This may indicate that the fetuses of the vaccinated sows were not infected. However, when umbilical blood of the fetuses from the vaccinated sows was examined, low antibody titres, ranging from 8 to 35.91, could be demonstrated in some piglets (Table 1). It is not clear whether this should be interpreted as virus replication in immune-competent fetuses or whether it represents a euthanization artefact, resulting from contamination of fetal blood with maternal blood. All sows seroconverted after vaccination with either of the two vaccines. The antibody titres after vaccination were rather low and ranged from 32 to 512, as determined by HI test. After challenge infection, both the vaccinated and the non-vaccinated sows showed a significant increase in antibody titres to levels between 2048 and (Table 2). There was no difference between the antibody titres of the vaccinated and non-vaccinated sows after challenge infection. After challenge infection, virus replication and shedding of the virus in the faeces was observed for all non-vaccinated and vaccinated sows. There was no difference in the pattern of virus shedding among the three groups. Virus could be isolated from sows of every group and virus DNA could be detected by PCR in most sows. The maximum of virus shedding lasted until day 7 (Table 3). The challenge experiments revealed that HI antibody titres ranging between 128 and 256 at the day of the infection were able to prevent mummification, and possibly even infection, of the fetuses. This is remarkable, as antibody titres were as low as 10 when the sera were tested in neutralization tests against the heterologous challenge virus (Table 2). The homologous titres against the genotype Journal of General Virology 90

3 Cross-protection against a heterologous PPV genotype Table 1. Number of mummified and non-affected fetuses in the groups and results of virological and serological analyses of tissues and umbilical blood from fetuses Group (vaccine); sow no. No. (%) fetuses PPV-PCR Antibody* GMTD Virusd Total Alive Mummified (100) 0 Neg Pos Neg (100) 0 Neg Pos Neg (100) 0 Neg Neg 0 Neg (90) 1 Neg Neg 0 Neg (100) 0 Neg Pos Neg (100) 0 Neg Pos Neg (53) 7 Pos Pos Pos (27) 8 Pos Pos 8.00 Pos (0) 17 Pos ND 0 Pos (0) 7 Pos ND 0 Pos *Antibody to PPV in umbilical cord blood. ND, Not done because of mummification of fetuses. DGeometric mean of antibody titre (HI test). dvirus isolation in cell culture (SPEV cells). vaccine viruses were much higher (up to 2560), confirming the results of a previous study (Zeeuw et al., 2007). The most remarkable result of this study was that vaccination of sows with an inactivated whole-virus vaccine based on a genotype 1 virus does not prevent infection and subsequent shedding of a genotype 2 challenge virus. Even more surprising was the result that vaccinated and non-vaccinated sows showed the same degree of serological response after infection and a virtually identical pattern of virus shedding. At the herd level, vaccination with these vaccines may not be a perfect tool to manage PPV infection. PPV is considered to represent a single serotype. Between the genotypes, an overall identity of about 95 % at the amino acid level (VP1) is observed. Amino acid residues 381, 596 and 586 are consistently different between the genotypes (Zimmermann et al., 2006); however, it has not yet been shown that these amino acids determine the Table 2. Antibody titres of the sows after vaccination and challenge infections as determined by HI and neutralization tests against the various test viruses Group (vaccine); sow no. HI antibody titre at day: Neutralizing antibody titre at day 0 against test virus: (vaccination 1) 40 (vaccination 2) 0 (infection) 19 p.i. 50 p.i. Stendal IDT (MSV) PPV-27a 414,8, ,8, ,8, ,8, ,8, ,8, ,8,8,8, ,20,20,20 417,8,8,8, ,20,20,20 419,8,8,8, ,20,20,20 420,8,8,8, ,20,20,

4 A. Jóźwik and others Table 3. Shedding of PPV-27a in rectal swabs from the day of challenge infection until day 14 p.i. Values represent virus isolation/pcr detection directly from rectal swabs/pcr detection from first-passage tissue-culture supernatant. Numbers show the virus load as log 10 (TCID 50 per swab). Group (vaccine); sow no. Time post-challenge infection (days) /2/2 2/2/2 2/2/3 2/2/2 2/2/ /2/2 2/2/2 2/2/2 2/2/2 2/2/ /2/2 2/2/2 2/2/2 2/2/2 2/2/ /2/2 2/2/2 2/2/3 2/2/2 2/2/ /2/2 2/0/1 2/2/2 2/2/2 2/2/ /2/2 2/2/2 2/2/2 2/2/2 2/2/ /2/2 2/2/2 2/1/5 2/2/2 2/2/ /2/2 +/1/4 2/2/2 2/2/2 2/2/ /2/2 2/2/2 2/2/2 2/2/2 2/2/ /2/2 2/2/2 +/1/7 2/2/2 2/2/2 antigenic differences (Zeeuw et al., 2007). The viruses that are used in the two vaccines examined in this study are both genotype 1 viruses and differ in the VP1 gene by only 2%. Although low antibody titres apparently prevent disease, they will not stop the distribution of the virus in a vaccinated herd. This may explain the phenomenon of single clinical cases of PPV disease in regularly and wellmanaged pig-breeding herds. Some non-protected animals will be infected and may develop disease. As there is crossprotection between the genotypes, strain variation may not play an important role in the protection of the individual pig against disease. The replication of the heterologous virus in the face of substantial titres of neutralizing antibodies is remarkable. It is not clear whether this is due to the antigenic difference between the viruses, and it would be interesting to see whether, after homologous challenge, a complete block of virus infection can be observed. For successful herd management, vaccines are required that will stop virus spread at the herd level and will eventually even allow the eradication of the virus from a herd or population. This study revealed that vaccinated pigs will still shed virus after infection, although the virus titre observed appeared to be very low. However, as the titres of the non-vaccinated sows were of the same order of magnitude in our study, it is rather likely that they will be high enough to initiate infection or distribution in the affected herd. It will be necessary to define further the determinants of protection and other possible mechanisms of immunity besides neutralization by antibodies. The currently available vaccines still appear suited for protecting the individual pig against PPV disease. However, the necessity to revaccinate all breeding sows at regular intervals of 4 6 months, which has been shown to be necessary in the field, emphasizes the need for more efficient vaccines. Modified live-virus or vector vaccines that may induce some cellular immunity may be possible candidates. By using the genotype 2 virus PPV-27a, we will try to address some of these questions in future experiments. Acknowledgements A. J. was the recipient of a fellowship from the German Academic Exchange Service (DAAD). References Cao, S., Chen, H., Zhao, J., Lü, J., Xiao, S., Jin, M., Guo, A., Wu, B. & He, Q. (2005). Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR. Vet Res Commun 29, Joo, H. S., Donaldson-Wood, C. R. & Johnson, R. H. (1976). A standardised haemagglutination inhibition test for porcine parvovirus antibody. Aust Vet J 52, Nielsen, J., Rønsholt, L. & Sørensen, K. J. (1991). Experimental in utero infection of pig foetuses with porcine parvovirus (PPV). Vet Microbiol 28, Soares, R. M., Durigon, E. L., Bersano, J. G. & Richtzenhain, L. J. (1999). Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1. J Virol Methods 78, Wilhelm, S., Zeeuw, E. J. L., Selbitz, H.-J. & Truyen, U. (2006). Realtime PCR protocol for the detection of porcine parvovirus in field samples. J Virol Methods 134, Wolf, V. H. G., Menossi, M., Mourão, G. B. & Gatti, M. S. V. (2008). Molecular basis for porcine parvovirus detection in dead fetuses. Genet Mol Res 7, Journal of General Virology 90

5 Cross-protection against a heterologous PPV genotype Zee, Y. C. & MacLachlan, N. J. (2004). Parvoviridae and Circoviridae. In Veterinary Microbiology, 2nd edn, pp Edited by D. C. Hirsh, N. J. MacLachlan & R. L. Walker. Oxford: Blackwell. Zeeuw, E. J. L., Leinecker, N., Herwig, V., Selbitz, H.-J. & Truyen, U. (2007). Study of the virulence and cross-neutralization capability of recent porcine parvovirus field isolates and vaccine viruses in experimentally infected pregnant gilts. J Gen Virol 88, Zimmermann, P., Ritzmann, M., Selbitz, H.-J., Heinritzi, K. & Truyen, U. (2006). VP1-sequences of German porcine parvovirus isolates define two genetic lineages. J Gen Virol 87,