Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper

Vaccine 27 (2009) 5178–5183

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Short communication

Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper Trine Hammer Jensen, Line Nielsen, Bent Aasted, Merete Blixenkrone-Møller ∗ Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark

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Article history: Received 21 March 2009 Received in revised form 9 June 2009 Accepted 22 June 2009 Available online 9 July 2009 Keywords: Canine distemper virus DNA vaccination Early life vaccination

a b s t r a c t Young mink kits (n = 8) were vaccinated with DNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodies were induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres. The VN antibody titres remained high for more than 4 months and the mink were protected against viraemia, lymphopenia, clinical disease and changes in the percentage of IFN-␥ producing peripheral blood leucocytes after challenge inoculation with a recent wild type strain of CDV. Essentially, these results demonstrate that early life DNA vaccination with the H gene of a CDV vaccine strain induced robust protective immunity against a recent wild type CDV. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Canine distemper virus (CDV) a member of the genus Morbillivirus causes a highly contagious and fatal multi-systemic infection of a broad range of domestic and wild carnivores globally [1,2]. Attenuated live vaccines have effectively reduced the number of CDV infections [3]. However, the attenuated live vaccines suffer some limitations. Most important is the interference between attenuated live vaccines and maternal antibodies resulting in insufficient protection of offspring from vaccinated females [4]. Additionally, the attenuated live vaccines are associated with a risk of reversion to virulence and various wildlife species have suffered from fatal infections caused by live vaccines [5]. DNA vaccines are being investigated as an alternative vaccination strategy against canine distemper and measles to overcome some of the limitations of attenuated live vaccines [6–8]. We have previously demonstrated that DNA vaccination with the H and nucleoprotein (N) genes of CDV can induce solid protection against virulent challenge inoculation of adult mink [8]. Others reported that DNA vaccination of dogs with the fusion (F), H and N gene of CDV induced suppression of severe clinical distemper disease [7]. Early life DNA vaccination against the closely related measles virus (MeV) in newborn macaques without maternal antibodies protected against viraemia after challenge inoculation with virulent MeV [9]. Also a lipid-based adjuvant DNA MeV vaccine induced

a humoral and T-cell immune response and protected macaques against disease after challenge inoculation [10]. Limited information exists about early life vaccination with DNA plasmid genes of CDV. A combination of DNA vaccines encoding the H, F and N proteins of CDV followed by vaccination with an attenuated live vaccine induced a humoral immune response in 2 weeks old dogs [11]. Others demonstrated that a lipid-formulated DNA vaccine with the H and F gene conferred partial protection against distemper after challenge with virulent CDV [12]. The immune response of a natural host of CDV to early life DNA immunisation with the H gene of CDV followed by challenge inoculation with virulent wild type CDV has so far not been described. In this study the H gene of CDV alone was chosen for DNA immunisation of young mink because the immune response to this viral protein is known to be of essential importance for protective immunity against morbillivirus disease [8,13,14]. Within different strains of CDV the H gene are the most heterogenic and antigenic variable of the CDV genes, these differences could result in induction of antibodies with different neutralisation capacity [15–17]. The genetic and antigenic variations of new wild type strains [17–19], encouraged us to evaluate the protective effect of immunisation with the H gene of an attenuated live vaccine strain of CDV against a recent Danish wild type strain of CDV. 2. Materials and methods 2.1. Experimental animals

∗ Corresponding author. Tel.: +45 35 33 27 40; fax: +45 35 33 27 42. E-mail address: [email protected] (M. Blixenkrone-Møller). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.06.077

Four pregnant mink (Mustela vison) were purchased from a Danish Mink farm (GT Mink, Denmark). The mink were free of Aleutian

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mink disease virus and Canine distemper virus and had no records of Mink enteritis virus. The breeding cages were checked for new births every morning and the day of birth was recorded as the morning the litter was found. The 18 mink kits were kept with their dams until weaning according to standard procedures for mink farming. The care and use of the animals was in accordance with the guidelines of the Danish Animal Care and Ethics Committee. 2.2. Preparation of expression plasmids for vaccination Immunisations were performed with the plasmid vector pV1J containing an insert of the H gene (1815 base pairs, named pCDVH) of the Onderstepoort strain of CDV [8,20]. The expression of the plasmid was confirmed by transfection of Vero cell as described by Sixt et al. [20] and visualised by indirect immunofluorescence assay (IFA) as described below [8]. Consistent with previous studies by Sixt et al. [20] we detected a high extent of H antigen expression in Vero cells 48 h after transfection. Control animals were given the empty plasmid vector pV1J (pEmpty). The expression plasmids were amplified in the TOP 10 strain of Eschericia coli and purified by use of Endofree Giga plasmid preparation kit according to the manufacturers protocols (Qiagen, Germany) with the following modifications. The isopropanol precipitate was resuspended in 1/4× TE buffer (1× TE:2.5 mM Tris–HCl, 0.25 mM EDTA, pH 8.0) over night and 2 additional precipitations were done [8]. The purified plasmids were suspended in phosphate buffered saline to a concentration of 1 mg DNA/ml. 2.3. Administration of DNA vaccines to young mink kits The mink kits were vaccinated 4 times. The first vaccination was administered to 5 days old mink kits, the initial dose was 200 ␮g of the pCDV-H, and divided between 70 ␮g injected intradermally and 130 ␮g injected intramuscularly with 27 G 0.5 ml hypodermic needles (Terumo, Sweden). The intradermal injections were distributed on the posterior part of the back. The intramuscular injections were distributed in the quadriceps muscle of each thigh and in the tibialis muscles of each leg. The combination of intradermal and intramuscular injections was chosen because this combination induced solid protective immunity in adult mink [8]. The second vaccination was applied at 3 weeks of age under anaesthesia (1–15 mg/kg each xylazine and ketamine). Anaesthesia was applied for animal welfare reasons and to ascertain correct injections since mink are difficult to restrain. The second vaccination consisted of 400 ␮g with 140 ␮g distributed intradermally and 260 ␮g distributed intramuscularly. The third and fourth vaccinations each of 400 ␮g were given at 6 and 9 weeks of age following the same procedure as the second vaccination. 2.4. Challenge inoculation with a recent Danish wild type strain of CDV The wild type virus used for the experimental challenge inoculation was isolated from a distemper outbreak in farmed mink in Denmark in 2004. This virus was named Mink/DK2004. The H gene of Mink/DK2004 showed 90% identity at the amino acid sequence level to the corresponding sequences of the Onderstepoort vaccine strain (data not shown). Prior to the challenge experiment the Mink/DK2004, originating from naturally infected mink, was passaged twice in mink (data not shown). Spleen and lymph nodes from the mink used for passage of the virus were homogenised and suspended in 20% RPMI 1640 containing penicillin and streptomycin. A challenge dose of 3 × 105 TCID50 was administered partly intraperitoneally and intramuscularly (20% suspension) and to the conjunctival and nasal mucosa (10% suspension) to anaesthetised mink. The mink were examined daily for clinical symptoms and

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mink showing clinical symptoms were euthanised with barbiturate under anaesthesia as described above. The experiment was completed 35 days after the challenge inoculation by euthanasia of the remaining animals. 2.5. Mink specimens Blood samples were taken for serum collection from the tail vein at 3 and 6 weeks of age and from vena cephalica at 9 weeks of age. Blood were sampled for serum collection, cell counts, isolation of peripheral blood mononuclear cells (PBMCs) and percentage of IFN␥ producing peripheral blood leucocytes (PBLs) before challenge inoculation and at days 2, 6, 10, 14, 21 and 28 after challenge inoculation from vena cephalica. At the end of the experiment blood was sampled by cardiac puncture under anaesthesia and immediately followed by euthanasia. Lung, trachea and brain samples were transported on dry ice and stored at −80 ◦ C until RT-PCR was performed. A mixed conjunctival and nasal swab was taken 14 days after challenge inoculation. The swabs were transported in RPMI 1640 including 200 units/ml penicillin and 200 ␮g/ml streptomycin on ice and stored at −80 ◦ C before virus isolation and titration was attempted. 2.6. Virus neutralising antibody assay Virus specific neutralising antibody titres were determined in Vero cells using a TCID50 format microtitre assay as described by [21] with few modifications. Duplicate serial 2-fold serum dilutions starting at 1:10 through 1:10,240 were added to a standard virus inoculum (approximately 50 TCID50 /well) of the Onderstepoort strain of CDV. Serum from a mink vaccinated with attenuated live CDV vaccine was used as positive control and virus dilution without serum was used as negative control. The titres were calculated using the Reed and Munch method [22]. 2.7. CDV antigen staining of peripheral blood mononuclear cells PBMCs were isolated by centrifugation of heparinised blood on a Ficoll density gradient (Ficoll-Paque, Amersham Pharmacia Biotech, Sweden) [23]. The PBMCs were fixed on glass slides and stained for intracellular CDV antigen as previously described [15]. Monoclonal antibody against the CDV N protein (no. 4.272) was used as primary antibody [24]. Fluorescein isothiocyanate (FITC)labelled rabbit F(ab’)2 antibody to mouse immunoglobulins (No. F0313, DakoCytomation, Denmark) was used as secondary antibody. Parallel preparations incubated with normal mouse serum (No. X0910, DakoCytomation) instead of monoclonal antibody were used as negative controls. 2.8. Flow cytometry analysis of lymphocyte counts and IFN- producing peripheral blood leucocytes Heparin stabilised blood were lysed with ammonium chloride lysing buffer (0.16 M NH4 Cl, 0.01 M KHCO3 , 0.1 mM EDTA, pH 7.3) as described previously [25]. Subsequently, 5 × 103 PBLs were analysed in a FACS Calibur flow cytometer (Becton Dickinson, CA, USA). Populations of lymphocytes, monocytes and granulocytes were gated based on their light scatter characteristics in a forward-scatter-versus-side-scatter diagram [26]. For determination of absolute counts of leucocytes in blood, the instrument particle (cell and latex bead) registration efficiency was measured using BD TruCount tubes (Becton Dickinson) [27]. The percentage of IFN-␥ producing PBLs was measured essentially as described previously [28]. In short, PBLs were cultured for 4 h at 37 ◦ C and 5% CO2 with 1 ␮g/ml ionomycin (Sigma, MO, USA),

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10 ␮g/ml brefeldin (Sigma) and 20 ng/ml phorbol-12myristate-13acetate (PMA, Sigma) in RPMI 1640 containing 10% fetal bovine serum (FCS), 200 units/ml penicillin and 200 ␮g/ml streptomycin. After the PBLs were fixed with 4% paraformaldehyde and treated with 0.1% saponin, the PBLs were incubated with mouse anti-bovine IFN-␥ (MCA 1783; Serotec, United Kingdom) followed by a second incubation with F0313 (DakoCytomation) and finally analysed by flow cytometry. The percentage of cytokine positive cells was calculated after subtraction of eventual positive signals from an isotype matched immunoglobulin control preparation (No. X0931 DakoCytomation). The values for all animals in a group were plotted and were found to be normally distributed. Student’s t-test was used for statistical evaluations. 2.9. Detection of viral RNA in tissue samples from the respiratory tract and brain by RT-PCR Lung and brain tissue (the cranial part of cerebrum) were homogenised in RNA Now (Ozyme, France). Total RNA was isolated following the RNA Now protocol (Ozyme). Random hexamer primers were used for cDNA synthesis essentially as described previously [8]. Amplification of cDNA was done with primers specific for the gene coding for the morbillivirus phosphoprotein resulting in a 430 base pair product [8,29]. A primer set for the cellular glyceraldehyde-3-phosphate dehydrogenase gene resulting in 568 base pair sequence was included in the PCR amplification as positive control for the RNA isolations [8]. 2.10. Virus titration from conjunctival/nasal swabs Virus titration was carried out by use of Vero cells expressing canine signalling lymphocyte activation molecule (Vero-DST) donated by Yanagi and co-workers [30]. First, SLAM expression by the Vero-DST cells were verified by staining the Vero-DST cells with a mouse anti-influenza virus H monoclonal antibody 12CA5 (Roche Diagnostics, Denmark) followed by FACS analysis [30]. The swab sample which was a pooled sample from the conjunctival and nasal mucosa was diluted 2-fold in 96-well plates in triplicate. Subsequently, 2 × 104 Vero-DST cells in MEM including 5% FCS, 400 units/ml penicillin, 400 ␮g/ml streptomycin and 2.5 ␮g/ml fungizone (Gibco, United Kingdom) were added each well. Then the suspensions were incubated for 1.5 h at 37 ◦ C and 5% CO2 followed by medium change. In the next 7 days the cells were examined daily for cytopathic effects. Virus titres were calculated by use of the Reed and Munch method [22].

Fig. 1. Serum CDV neutralising antibody titres induced after neonatal vaccination and subsequent challenge inoculation. Mink were vaccinated with pCDV-H (n = 8) or pEmpty (n = 5) 4 times (at 5 days of age and at 3, 6 and 9 weeks of age). The mink were challenge inoculated with the Mink/DK2004 strain of CDV at 27 weeks of age (shown as 0 d). Values shown are geometric means ± standard deviation log10 values. The horizontal dotted line indicates log10 that equals a neutralising titre of 100.

3.2. Virus neutralising antibody response after early life DNA vaccination with pCDV-H After 2 immunisations with pCDV-H at the age of 5 days and 3 weeks, 6 out of 8 vaccinated kits developed detectable serum VN antibodies. After the third immunisation the average VN antibody titre of all vaccinated mink was above 1:100 (Fig. 1). Importantly, the VN antibody titres remained above 1:100 without any significant variations between 9 and 27 weeks of age (Fig. 1). The pCDV-H vaccinated mink were boosted (more than a 4-fold increase in titre) after challenge inoculation with wild type CDV. 3.3. Virus neutralising antibody response in non-vaccinated mink after inoculation with the Mink/DK2004 strain The 5 kits vaccinated with pEmpty all had VN antibodies 10 days after challenge inoculation, of these kits 2 developed detectable VN antibody titres 6 days after challenge inoculation (Fig. 1). The levels of VN antibodies of 2 fatally infected mink remained below 1:100 and also below the levels of VN antibodies of the 3 clinical unaffected mink during the 35 days of observation (data not shown).

3. Results 3.4. Virulence of the Mink/DK2004 strain 3.1. Experimental design Eight mink kits were vaccinated with the pCDV-H, 5 mink kits were vaccinated with pEmpty and 5 mink kits served as negative controls; they were non-vaccinated and mock inoculated. The first of 4 vaccinations was given at 5 days of age followed by revaccinations at 3, 6 and 9 weeks of age. When the mink were 6.5 months old they were challenge inoculated with the wild type strain Mink/DK2004. From the day of challenge inoculation the 13 vaccinated mink were kept in strict isolation and the 5 mock inoculated negative control mink were housed separately. The 5 mock inoculated mink did not experience any sign of disease and we did not detect lymphopenia, CDV antigen, RNA or virus excretion nor did they develop detectable CDV neutralising antibodies. These results proved these mock inoculated mink to be negative controls for the IFN-␥ measurements throughout the experiment.

The mink vaccinated with pEmpty developed viraemia 6 days after challenge inoculation and they had viraemia throughout the observation period of 35 days. Lymphopenia was defined as less than 500 lymphocytes/␮l blood based on values for normal lymphocyte counts of mink obtained in this work and by Chen and Aasted [31]. Lymphopenia was initially demonstrated 6 days after challenge inoculation and was most severe 14 days after the challenge inoculation (Table 1). Lymphopenia persisted until euthanasia in the fatally infected mink vaccinated with pEmpty (Table 1). In all but 1 mink vaccinated with pEmpty (n = 5), viral RNA was found in lung tissue and cerebrum and the 4 mink excreted virus from conjunctival and nasal mucosa (Table 1). Two mink vaccinated with pEmpty developed lethargy, conjunctivitis, diarrhoea, respiratory distress and dyskeratosis of the footpads 3 and 4 weeks after challenge inoculation, respectively. These 2 mink were also

T.H. Jensen et al. / Vaccine 27 (2009) 5178–5183 Table 1 Protective effect of early life vaccination with the H gene of CDV.

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(Table 1). We did not detect any clinical signs of disease in any of the pCDV-H vaccinated mink.

Diagnostic features after challenge inoculation with wild type CDV

Mink vaccinated with pCDV-H

Mink vaccinated with pEmpty

Clinical symptoms of distemper Lymphopeniaa CDV antigen in PBMCsb

0/8 0/8 0/8

2/5 5/5 5/5

CDV viral RNA in Respiratory tract Cerebrum

0/8 0/8

4/5 4/5

Viral excretionc

0/8

4/5

a

Lymphocyte counts were performed by FACS analysis on days 0, 6, 10, 14, 21, 28 and 35 after challenge inoculation. Day 14 after challenge inoculation all the pEmpty vaccinated mink suffered lymphopenia determined by FACS analyses. For this reason day 14 is represented in the table. b Detected by IFA days 0, 6, 10, 14, 21, 28 and 35 after challenge inoculation. The PBMCs from mink vaccinated with pEmpty were positive at all time points checked after challenge inoculation. c Viral excretion was tested by titration of conjunctival/nasal swabs on Vero-DST cells 14 and 21 days after challenge inoculation.

excreting high titres of virus (780 TCID50 /ml and 5500 TCID50 /ml, respectively). In contrast, the mink in which no viral RNA was detected did not excrete virus and the 2 infected but not diseased mink excreted low levels of virus (<5 TCID50 /ml). The mink were euthanised shortly after the onset of clinical symptoms due to a fast progression of the symptoms. 3.5. Early life DNA vaccination induce solid protection against inoculation with Mink/DK2004 The 8 kits vaccinated with the pCDV-H were protected against viraemia and lymphopenia and we did not detect systemic spread of the virus or virus excretion from conjunctival and nasal mucosa

3.6. T-cell immunity The pCDV-H vaccinated mink experienced no statistical significant difference in IFN-␥ producing PBLs compared to the mock inoculated mink indicating the vaccine had induced protective Tcell immunity against distemper (Fig. 2). The average percentage of IFN-␥ producing PBLs of the mink vaccinated with the pEmpty peaked 28 days after challenge inoculation and the percentage of IFN-␥ producing PBLs stayed significantly higher than in the pCDVH vaccinated until the end of the experiment (Fig. 2). 4. Discussion We have established an infection model in mink with a currently circulating wild type strain of CDV, Mink/DK2004. Mink are highly susceptible to CDV [23,32] which is a recurrent problem for animals kept for fur production [2,33]. The infection model was used to evaluate the protective capacity of early life DNA vaccination with the H gene of the Onderstepoort vaccine strain of CDV against challenge inoculation with a recent wild type Mink/DK2004. Vaccination early in life is important because many diseases including distemper are most critical for young animals [1,34,35]. However, early life vaccination is complicated by the period of susceptibility during which the maternal antibodies have declined below a protective titre but still can interfere with live vaccine. DNA vaccines against morbillivirus have the potential to overcome interference with maternal antibodies [6]. For this reason the present study was conducted to evaluate the capacity of neonatal mink to respond to DNA immunisation. An additional advantage of early life vaccination is that production animals are easiest to handle as infants.

Fig. 2. (A) Percentage of IFN-␥ producing PBLs. * illustrate a significant difference between the percentage of IFN-␥ producing PBLs of pCDV-H vaccinated mink (n = 8) and the mink vaccinated with pEmpty (n = 5) 6, 21, 28 and 35 days after challenge inoculation. (B) IFN-␥ antibody staining profile of PBLs of a mink immunised with the pCDV-H 6 days after challenge inoculation and the corresponding control profile using IgG1 iso-type control.

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DNA plasmids encoding the H protein of CDV was used for immunisation because the H protein appears essential for inducing neutralising antibodies against CDV in adult mice and mink [8,20]. Similarly, DNA plasmids encoding the H gene of MeV have been shown to induce VN antibodies [10,13,36–38]. Vaccination with the H gene of MeV was demonstrated to be essential for protecting macaques against measles [10,13]. The present study demonstrated that 2 immunisations with pCDV-H given to kits 5 days and 3 weeks old induced detectable VN antibodies. Due to the fragility of the kits at 5 days of age we did not draw blood to test for VN antibodies. The VN antibody responses induced within the first month of life after only 2 immunisations indicate that the immune system of newborn mink is competent and able to respond to DNA vaccination early in life as reported for other animals [6,39–41]. At the age of 9 weeks all CDV DNA vaccinated mink (n = 8) had VN antibody titres of more than 1:100 which in standardised measurements of dogs vaccinated with attenuated live vaccines are believed to correlate with protection against distemper [42]. Previous studies showed that DNA vaccination of adult mink with both the H and N genes of CDV induced VN antibody titres of 1:100 which conferred with protective immunity [8]. The VN antibody titres of the mink in the present study vaccinated only with the H gene of CDV were boosted after challenge inoculation suggesting a limited local viral replication at the challenge inoculation sites. However, all the H gene vaccinated kits in this study were protected against disease and no signs of viraemia or systemic infection were found. Possibly, 2 or 3 vaccinations would have been sufficient to induce protective immunity because 3 vaccinations were enough to induced VN antibody titres of more than 1:100 (Fig. 1). Additionally, studies by Cherpillod et al. [7] indicated that DNA vaccination of dogs with the H, F and N genes of CDV induced protection against severe distemper [7]. In contrast, the 5 control mink vaccinated with the pEmpty developed viraemia 6 days after challenge inoculation and virus infection was found in the respiratory tract and the CNS of all but 1 mink. Two mink given pEmpty developed persistent lymphopenia and clinical symptoms typical for CDV infection. These 2 mink had persistently lymphopenia, they were excreting large amounts of infectious virus and had lower VN antibody titres compared to the other 3 mink given pEmpty. The differences in the outcome of the CDV infection between the mink might be attributed to individual differences in immune responses [43]. The results of the FACS analysis of the percentage of IFN-␥ producing PBLs indicate that the pCDV-H vaccinated mink was protected against CDV infection. Because no statistical significant differences were seen in the percentage of IFN-␥ producing PBLs of the pCDV-H vaccinated mink after challenge inoculation and the mock inoculated mink (Fig. 2). Investigated CDV wild types have been found to differ genetically and antigenically from the vaccine strains of CDV [18,19,44,45]. Thus, it is important to monitor the efficacy of the available live vaccine strains of CDV against currently circulating wild type CDVs. It has been suggested that previously described vaccine failures could be attributable to antigenic differences between currently circulating wild type CDVs and the strains of CDV employed in the commercially available live vaccines [46–48]. In conclusion, the results presented here suggest that DNA immunisation with the H gene of the Onderstepoort vaccine strain induced cross-protection against a current wild type CDV. Our findings of solid immune response following early life CDV DNA vaccinations encourage further studies on DNA immunisations in newborns specifically aiming at reducing the number of vaccinations and the dose of DNA.

Acknowledgements The Danish Fur Breeders Research Fund and Faculty of Life Sciences, University of Copenhagen are acknowledged for their financial support. We wish to thank Leon Langli, Jørgen Østergaard and Klaus Hartmann for help with handling and care of the mink and Tove Dannemann Jensen, Alma Orantes and Morten Lund for excellent technical assistance. References [1] Appel MJ. Pathogenesis of canine distemper. Am J Vet Res 1969;30(7):1167–82. [2] Appel MJ, Summers BA. Pathogenicity of morbilliviruses for terrestrial carnivores. Vet Microbiol 1995;44(2–4):187–91. [3] Carmichael LE. Canine viral vaccines at a turning point—a personal perspective. Adv Vet Med 1999;41:289–307. [4] Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB. Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines. J Virol 2000;74(14):6358–67. 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