- 31 minutes, 7 seconds
- Expertise article
- P. Rensona,, C. Fabletb, M. Le Dimnaa, S. Mahéa, F. Touzainc, Y. Blanchardc, F. Paboeufd, N. Roseb, O. Bourrya et.al.
ABSTRACT
The porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses for the swine industry worldwide. In the past several years, highly pathogenic strains that lead to even greater losses have emerged. For the Western European swine industry, one threat is the possible introduction of Eastern European PRRSV strains (example Lena genotype 1.3) which were shown to be more virulent than common Western resident strains under experimental conditions. To prepare for the possible emergence of this strain in Western Europe, we immunized piglets with a Western European PRRSV field strain (Finistere: Fini, genotype 1.1), a new genotype 1 commercial modified live virus (MLV) vaccine (MLV1) or a genotype 2 commercial MLV vaccine (MLV2) to evaluate and compare the level of protection that these strains conferred upon challenge with the Lena strain 4 weeks later. Results show that immunization with Fini, MLV1 or MLV2 strains shortened the Lenainduced hyperthermia. In the Fini group, a positive effect was also demonstrated in growth performance. The level of Lena viremia was reduced for all immunized groups (significantly so for Fini and MLV2). This reduction in Lena viremia was correlated with the level of Lena-specific IFNγ-secreting cells. In conclusion, we showed that a commercial MLV vaccine of genotype 1 or 2, as well as a field strain of genotype 1.1 may provide partial clinical and virological protection upon challenge with the Lena strain. The cross-protection induced by these immunizing strains was not related with the level of genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is attributed to a better cell-mediated immune response.
1. Introduction
Since the emergence of the porcine reproductive and respiratory syndrome virus (PRRSV) almost 30 years ago, the disease has spread to most swine-producing countries around the world. PRRSV infection is mainly characterized by reproductive disorders in sows and respiratory syndrome and growth retardation in growing pigs (Albina, 1997). Due to the direct and indirect costs of the disease, PRRS is recognized as one of the most important economic diseases for the swine industry worldwide (Neumann et al., 2005). PRRS also must be considered in terms of public health, because studies have shown that PRRSV infection may facilitate the spread or the maintenance of zoonotic bacteria or viruses in pigs, such as Salmonella or the hepatitis E virus (Beloeil et al., 2004; Salines et al., 2015).
Historically, two different PRRS viruses emerged nearly at the same time in the late 1980s; genotype 1 in Europe and genotype 2 in North America and Asia. Additional genetic analyses can distinguish between different subtypes among genotype 1 strains (Stadejek et al., 2013). Subtype 1 strains — mainly circulating in Western Europe but also present in Asia and North America — are considered to be predominantly low pathogenic strains, whereas subtype 3 strains circulating in Eastern Europe are considered to be more virulent (Morgan et al., 2013). Lena strain is a prototype subtype 3 strain which has been extensively studied over the past few years (Karniychuk et al., 2010; Renson et al., 2017; Trus et al., 2016; Weesendorp et al., 2013). This strain was isolated in 2007 in a Belarusian farm where severe reproductive failure and high mortality rate in growing pigs were reported (Karniychuk et al., 2012). 
Due to the growing economic exchanges between Western and Eastern Europe, and in particular the flow of live pigs and trucks circulating between these two parts of Europe, the risk of spread of a subtype 3 PRRSV strain from Eastern to Western Europe is high. Considering (i) the virulence these strains had demonstrated under numerous experimental conditions (Karniychuk et al., 2010; Morgan et al., 2013; Renson et al., 2017; Weesendorp et al., 2014), and (ii) the naïve immune status of the Western Europe pig populations regarding subtypes 3 strains (Stadejek et al., 2008), their introduction in the Western European pig industry would certainly have a huge economic impact.
Preparation for the possible emergence of such strains in Western Europe calls for an investigation on the protection provided by previous immunization of pigs with field or vaccine strains. Regarding vaccines, two studies have already explored the protection afforded by a modified live virus (MLV) vaccine of genotype 1 upon challenge with the Lena strain in growing pigs. Both studies showed partial clinical and virological protection in vaccinated pigs (Bonckaert et al., 2016; Trus et al., 2014). Concerning PRRSV field strains, only one study has explored the heterologous protection provided by genotype 1.1 Belgian PRRSV strains (Trus et al., 2016). Here also, the obtained protection was partial. In contrast, a recent study demonstrated that immunization with the Lena strain provides a complete protection against a homologous challenge (Weesendorp et al., 2016).
Although these previous studies have provided very interesting data, they also have some caveats because they used MLV vaccines developed from old PRRSV strains and they did not evaluate MLV vaccines of genotype 2 which are now licensed for use in many Western European countries. Another limitation is that the protection provided by PRRSV vaccine and field strains was not investigated simultaneously, hindering comparison of the protection level conferred by attenuated or non-attenuated PRRSV strains.
Thus, considering these remaining questions, the objective of the present study was to evaluate and compare the protection provided by the immunization with a Western European field (genotype 1.1) PRRSV strain, a new genotype 1 MLV vaccine or a genotype 2 MLV vaccine upon challenge with the genotype 1.3 Lena strain in growing pigs.
2. Materials and methods
2.1. Vaccine and virus strains
The genotype 1 Ingelvac PRRSFLEX® EU vaccine (Boehringer Ingelheim France, Paris, France, 94881 strain, GenBank accession no. KT988004) and the genotype 2 Ingelvac® PRRS MLV vaccine (SCS Boehringer Ingelheim Comm, Brussels, Belgium, USA ATCC VR2332 strain, GenBank accession no. EF484033) were used in the in vivo experiment as MLV1 and MLV2 vaccines, respectively. For in vitro (enzyme-linked immunospot) ELISPOT analyses, MLV1 and MLV2 vaccine strains were obtained by suspending the respective lyophilized Ingelvac vaccines in Eagle's minimal essential medium (EMEM), propagating them once and titrating them on MARC145 cells.
The genotype 1.1 Finistere PRRSV strain (PRRS-FR-2005-29-24-1) was isolated in France in 2005 from a herd with reproductive failures in sows (abortions). In specific pathogen-free (SPF) pigs, Finistere infection induces a mild clinical expression (Rose et al., 2015). The genotype 1.3 Lena PRRSV strain (GenBank accession no. JF802085) was kindly provided by Dr. Hans Nauwynck (University of Ghent, Belgium). The Lena strain was isolated in Belarus in 2007 from a herd with mortality, reproductive failures and respiratory disorders (Karniychuk et al., 2010) . The Finistere and the Lena strains were propagated and titrated on pulmonary alveolar macrophages for 2 and 5 passages, respectively, for animal inoculations, and for 4 and 7 passages, respectively, for ELISPOT analyses. For virus neutralizing tests, a Lena strain adapted to MARC145 cell culture was kindly provided by Dr. Hans Nauwynck (University of Ghent, Belgium), then propagated for 5 passages and titrated on MARC145 cells.
2.2. Finistere strain full genome sequencing
The full genome sequence of the PRRSV Finistere strain was obtained using next-generation sequencing (NGS). Viral RNA purification, cDNA synthesis and library construction was prepared as described in (Brown et al., 2016). NGS was done at the Biogenouest (Nantes, France) core facility using a MiSeq HD Sequencer (Illumina). Then, bioinformatic reconstruction of the full-length genome was performed by cleaning the sample reads using Trimmomatic software (Bolger et al., 2014). A Bowtie2 2.1.0 (Langmead and Salzberg, 2012) alignment on the Sus scrofa genome eliminated most of the host reads. Remaining reads were compared against the ViPR database (Pickett et al., 2012) to extract the viral sequences. An accurate alignment afforded a first set of viral reads. These reads did not cover nucleotides 2310 to 2603 in the reference Lelystad strain full genome (GenBank accession no. M96262). A tblastn 2.2.28 (Camacho et al., 2009) on the related amino acid sequence afforded the determination of a second set of reads matching this area in our sample. Both sets were submitted to kmergenie 1.5658 (Chikhi and Medvedev, 2014) and vicuna 1.3 (Yang et al., 2012), providing us the first draft genome of Finistere strain as a single contig. The Finistere PRRSV strain sequence was deposited in GenBank under accession no. KY366411.
2.3. Vaccine and virus sequence comparison
Comparison of the genome of the immunizing strains with that of the Lena strain was performed using the Simplot 3.5.1 program (Lole et al., 1999) which plots similarity versus position based on the percentage of identity obtained by alignment of the full-genome sequences using the MUSCLE algorithm (Edgar, 2004).
2.4. Experimental setting
Forty-one, four-week-old pure Large White piglets coming from a nucleus herd (free of PRRSV, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae) were housed in our biosecurity level-3 air-filtered animal facilities. The 41 piglets were randomly assigned to five groups housed in separate rooms (Table 1). At 6 weeks of age (D27), 7 piglets were inoculated intranasally with the PRRSV Finistere strain (5 × 105 50% tissue culture infectious dose (TCID50) per piglet) (Fini group). At the same time, 9 piglets were vaccinated intramuscularly with either the MLV1 (minimum dose 104.4 TCID50/piglet) or the MLV2 vaccine (minimum dose 104.9 TCID50/piglet). At 10 weeks of age (D0), all the piglets from the Fini, MLV1 and MLV2 groups were challenged intranasally with the Lena strain (genotype 1.3, 5 × 105 TCID50/piglet). At the same time, 8 non-immunized piglets were also inoculated with the Lena strain (Lena group) and 8 non-immunized piglets were mock inoculated (control group).
2.5. Clinical monitoring, sampling and necropsy
Rectal temperature, weight gain and clinical score were monitored daily using a scoring template adapted from Weesendorp et al. (2013). Hyperthermia was recorded for rectal temperatures higher than 40 °C. Blood samples were collected without additives or with heparin to collect either serum or whole blood. Blood samples were taken just before immunization and then every week until 5 weeks after inoculation with Lena (day post-infection (dpi) −27, −20, −13, −4, 8, 14, 22, 29, 35). Half of the pigs in each group were euthanized and necropsied at the acute phase of Lena infection (between 9 and 11 dpi) and the remaining pigs at the end of the follow-up (at 42 and 43 dpi). Post-mortem examinations were carried out on each pig. Thoracic organs were thoroughly examined and pneumonia was scored as previously described in Madec and Kobisch (1982). Tissues samples were collected and frozen for further quantification of the viral genome.
2.6. Laboratory analyses
RT-PCR: RNA was purified from serum or tissue samples using the NucleoSpin RNA 8 virus kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. Before Lena challenge, viral RNA was detected using the Adiavet™ PRRS real-time RT-PCR kit (Adiagene, Saint-Brieuc, France) following the manufacturer's instructions. After Lena challenge, specific detection of the Lena strain genome was assessed by qRT-PCR as previously described (Renson et al., 2015). ELISA: antibodies against PRRSV in sera were detected using PRRS X3 Ab ELISA tests (IDEXX laboratories, Liebefeld, Switzerland) according to the manufacturer's instructions. Sample-to-positive (S/P) ratios with values equal or greater than 0.4 were considered as positive. Virus neutralization test (VNT): PRRSV neutralizing antibodies (NA) were quantified in sera on MARC145 cells against the MARC145- adapted Lena strain according to the VNT method described in (Fablet et al. 2016).
ELISPOT: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from heparinized whole blood using Ficoll-Paque plus density gradient media (GE Healthcare, Little Chalfont, UK) and LeucoSep centrifuge tubes (Greiner Bio One, Les Ulis, France). PRRSV-specific IFNγ-secreting cells (IFNγ-SCs) were quantified in triplicate as previously described (Fablet et al., 2016), using a 16 h PRRSV stimulation of 4 × 105 PBMC with a multiplicity of infection of 0.2 for either the Lena strain, the Finistere strain, the MLV1 vaccine strain or the MLV2 vaccine strain (obtained as described above). The number of spots per well were counted using an ImmunoSpot S5 UV Analyzer (CTL, Shaker Heights, OH, USA). The number of IFNγ-SCs was calculated by subtracting the mean number of spots obtained for the triplicate vaccine stimulation from the mean of potential non-specific spots obtained for the triplicate negative stimulation (cell culture medium), then expressed per 106 PBMCs.
2.7. Ethics statement
The animal experiment was authorized by the French Ministry for Research (project no. 2015060113297443_v1) and approved by the national ethics committee (authorization no. 07/07/15-3).
2.8. Statistical tests
The weekly average daily weight gain was calculated. An analysis of variance was used to compare growth performance between groups (p > 0.05). Post hoc pairwise comparisons were then performed using the Holm test to adjust the p-values of these comparisons according to the number of tests conducted (p > 0.05). The daily rectal temperatures, the viral load (at 8, 14, 22 and 29 dpi) and the number of PRRSVspecific IFNγ-SCs (from −27 to 35 dpi) in the experimental groups were compared with the Kruskal–Wallis test (p > 0.05). When significant, the Holm test was then used to adjust the p-values of pairwise comparisons according to the number of tests conducted (p > 0.05). The relationship between the blood genomic viral load and the number of IFNγ-SCs for all the Lena inoculated animals during the postchallenge follow-up period was assessed with a Spearman correlation test (p > 0.05).
3. Results
3.1. Genetic relationship with Lena is higher for Finistere and MLV1 than for MLV2
To gain a comprehensive overview of the genetic proximity of the immunizing strains (Finistere or MLV1 and MLV2 vaccine strains) with the Lena challenge strain, we used the complete genome sequences for each strain to generate a genetic similarity graph. As shown in Fig. 1A, there is a close relationship between the Finistere and MLV1 strains because both strains belong to genotype 1, subtype 1. Full-genome alignment of the Finistere and MLV1 strains showed a sequence identity of 86.7% and 88.1% between the two strains for the full genome and for ORF5, respectively. However, similarity of these two strains with the Lena strain (genotype 1, subtype 3) was lower (full-genome sequence identity with the Lena strain of 79.6% or 79.8% for the Finistere and theMLV1 strains; respectively; ORF5 sequence identity of 82.8% and82.5%, respectively). Regarding the MLV2 strain (Fig. 1B), as expected for a genotype 2 strain, the level of genetic similarity with Lena was much lower than for the genotype 1 strains (full-genome sequence identity of 62.8% and ORF5 sequence identity of 62.9%).
3.2. Immunization with both genotype 1 or genotype 2 strains reduced the duration of hyperthermia in Lena infected pigs
After vaccination, the piglets from the MLV1 and MLV2 groups displayed very limited increases in rectal temperature and a low number of cases of hyperthermia as described in the vaccine Summary of Product Characteristics. After infection with Finistere, the piglets in the Fini group displayed a slight and transient increase in rectal temperature, with 5 out of 7 animals displaying hyperthermia on day 3 after challenge with the Finistere PRRSV strain (data not shown).
After infection with Lena, the non-immunized animals (Lena group) showed a rapid and persistent increase in rectal temperature between 2 and 11 dpi with a mean rectal temperature above 40.5 °C during this period (Fig. 2). In the Fini group, the hyperthermia period was shortened compared with the Lena group, with a significantly lower rectal temperature between 8 and 11 dpi. In the MLV1 group, the decrease in rectal temperature was also significant at 10 and 11 dpi, whereas for the MLV2 group the difference was only significant at 11 dpi (Fig. 2).
Regarding clinical scores (CS), higher symptoms (reduced motility, mild dyspnea, some sneezing and cough) were observed after Lena infection in the non-immunized group compared with the immunized groups but there were no significant differences. Interestingly, during the second week after infection, 4 of 8 animals from the Lena group displayed redness on the ears or the body. During the same period, none of the immunized animals showed these symptoms.
Due to the high hyperthermia level detected after challenge with the Lena strain, the average daily weight gain (ADWG) dropped significantly for all Lena-infected groups during the first week after infection (D0–D7, Fig. 3). During the second week post-infection (wpi) (D7–D14), the ADWG was still significantly lower than the controls for the Lena, MLV1 and MLV2 groups. In contrast, the animals in the Fini group showed an ADWG that was statistically the same as that of the control group. From the third wpi, the ADWG of the four Lena-infected groups reached the levels of the control group.
To evaluate the impact of a previous PRRSV immunization on Lenainduced lung lesions, 3 to 4 animals in each group were euthanized between 9 and 11 dpi. Surprisingly, the level of macroscopic lung lesions was very low with only one animal in the Lena group obtaining a score of 3 out of 28 and no differences between the groups (data not shown).
3.3. Immunization with both genotype 1 or genotype 2 strains reduced the viremia in Lena infected pigs
After immunization with the Finistere strain or the MLV1 or MLV2 vaccines, all piglets displayed a detectable level of PRRSV viremia (RTPCR). At 1 week after immunization, the Ct values for the Fini group were lower than for MLV1 or MLV2 groups (Fini group: 27.3 ± 1.7, MLV1 group: 32.4 ± 2.7, MLV2 group: 32.6 ± 3.1), indicating higher viremia in the Fini group.
To assess if a previous PRRSV immunization affects Lena viremia, we then quantified the Lena genomic viral load in sera with a specific qRT-PCR. As shown in Fig. 4, the non-immunized piglets from the Lena group showed a peak of viremia at 8 dpi with a mean viral load of 5.76 ± 0.37 log10 equivalent (eq) TCID50/ml of serum. At the same time, animals from the Fini and MLV2 groups exhibited a significantly lower viral load than the Lena group (Fini: 4.45 ± 0.48; MLV2: 4.84 ± 0.58 log10 eqTCID50/ml). The mean viremia level for MLV1 group was also lower than for the Lena group (5.35 ± 0.41 log10 eqTCID50/ml), but not significantly. At 14 dpi, the viral load dropped for all groups. For the Fini group, the viral load was still significantly lower than Lena group (1.14 ± 1.33 versus 3.63 ± 1.69 log10 eqTCID50/ml respectively), which was not the case for MLV2 group (3.97 ± 0.80 log10 eqTCID50/ml) and MLV1 group (3.04 ± 0.68 log10 eqTCID50/ml). Finally, from 22 dpi, the viral load became undetectable in the Fini group, whereas viremic animals persisted in the other immunized groups until 29 dpi (1/5 in MLV1 group, 3/5 in MLV2 group and 2/4 in Lena group).
The Lena strain genome was also quantified in lung and tonsil tissues collected in the animals euthanized at 9–11 or 42–43 dpi. In the lung, all piglets were PCR positive at 9–11 dpi whereas they were all negative, except one MLV2 animal, at 42–43 dpi. In the tonsil, all piglets were PCR positive at 9–11 and 42–43 dpi with a slight decrease in viral load over time (data not shown). No significant differences in tissue viral load were observed in lung or tonsil between the different groups at 9–11 or at 42–43 dpi.
3.4. Cell-mediated immune response is correlated with control of Lena viremia
Regarding the humoral immune response to PRRSV, all the animals from Fini, MLV1 and MLV2 seroconverted (ELISA) 2 weeks after immunization, with a slightly faster seroconversion for the Fini group (2 out of 7 seropositive animals in the Fini group versus 0 out of 9 animals for MLV1 and MLV2 groups at −20 dpi) (Fig. 5). For the animals in the Lena group, seroconversion occurred at 8 dpi. Regarding neutralizing antibodies directed against the Lena strain, no detectable levels were observed in any piglet during the follow-up period (data not shown).
The cell-mediated immunity to the immunizing or the challenge strain was evaluated using an ELISPOT IFNγ assay. After immunization with Finistere strain or the MLV1 and MLV2 vaccine, a specific IFNγ response to the immunizing strain was detected from 2 weeks postimmunization (−13 dpi, Fig. 6A). The number of IFNγ-SCs then increased until 14 dpi. At 14 dpi, the number of IFNγ-SCs was significantly higher in the MLV1 group compared with the MLV2 group. Regarding the IFNγ-SCs specific to the Lena strain (Fig. 6B), their number was lower than that directed toward the immunizing strains with no significant differences between the groups.
To evaluate if the cell-mediated immune response is involved in the control of Lena viremia, we investigated the correlation between the genomic viral load in the blood and the number of IFNγ-SCs for all the Lena-inoculated animals during the post-challenge follow-up period. There was a low but significant negative correlation between the number of IFNγ-SCs and the Lena blood viral load (r = −0.24, p = 0.03). Interestingly, the correlation was higher when considering only the Lena-specific IFNγ response (r = −0.44, p > 0.0001).
4. Discussion
The risk of introduction of highly pathogenic PRRSV strains from Eastern to Western Europe swine-producing countries is a real threat to the swine industry. To prepare for the possible emergence of genotype 1.3 PRRSV strains, swine practitioners from Western Europe need information on the level of protection provided by previous contact with a field or an attenuated vaccine PRRSV strain. To address this issue, the present study evaluated and compared the protective effect of a new commercial PRRS MLV vaccine for genotype 1, an MLV vaccine for genotype 2 and immunization with a genotype 1.1 field strain upon challenge with the highly pathogenic genotype 1.3 Lena strain.
In non-immunized pigs, Lena infection induced a marked and longlasting hyperthermia associated with a sharp drop in weight gain during the first 2 weeks after infection. These results are consistent with the previous studies conducted in our laboratory and elsewhere to qualify this strain as highly pathogenic compared with a common genotype 1.1 PRRSV strain circulating in France (Karniychuk et al., 2010; Renson et al., 2015). In terms of lung lesions, surprisingly and despite some respiratory troubles, almost no macroscopic lesions were observed in the animals necropsied regardless of the time elapsed after challenge with the Lena strain (9–11 or 42–43 dpi) and whether previously immunized or not. These results contrast with a previous study (Trus et al., 2014) that reported that 15% of the lung surface showed pneumonia-related lesions in non-vaccinated, Lena-infected pigs. One possible explanation for this low lesion level is the absence of respiratory co-infection in the piglets in our study, because the animals came from a nucleus herd with a very high respiratory health status (free of Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae). At the microscopic level, the histological analysis conducted in a limited set of samples in each group nevertheless showed typical lesions related to PRRSV infection (data not shown).
To date, only two studies have evaluated the effect of MLV (genotype 1) vaccines upon challenge with the Lena strain. As in the present study, the other studies (Trus et al., 2014); (Bonckaert et al., 2016) reported partial protection in vaccinated animals, with a decrease in clinical and virological parameters. Although direct comparison of the efficacy of vaccines tested in different studies is difficult, it appears that the tested MLV vaccines provided virtually the same partial protection toward Lena infection from a clinical point of view with a reduction in the duration of fever period in all studies. Regarding virological parameters, Trus et al. (2014) showed a significant reduction in viremia with the DV vaccine strain. In the present study, a significant decrease in viremia was also demonstrated for the MLV2 vaccine (at 8 dpi), but the decrease was not significant for the MLV1 vaccine, probably due to the limited number of animals followed after 10 dpi. Interestingly, Bonckaert et al. (2016) reported results very similar to those of our MLV1 group with a significant impact of vaccination on clinical signs, but no significant decrease in viremia.
The differences in the vaccine virological efficacy measured in these studies can be attributed to various factors such as the breed or age of the animals, the antigenic homology between vaccine and challenge strain, but also the delay between vaccination and challenge. Regarding this last point, relatively higher virological vaccine efficacy (Trus et al., 2014) occurred for a vaccine-challenge delay of 6–8 weeks, associated with induction of NAs in the vaccinated animals. In contrast, in the Bonckaert study (Bonckaert et al., 2016) and the present study, slightly lower vaccine efficacy was associated with a shorter vaccine-challenge interval (4 weeks) and the absence of NAs in the vaccinated animals. Although the role of NAs in the control of PRRSV viremia is still subject of debate (Lopez and Osorio, 2004), these observations suggest that the lack of NAs related to a short vaccine-challenge interval contributes to lower vaccine efficacy for the control of Lena viremia.
LV vaccine upon challenge with the Lena strain. This evaluation is particularly relevant because MLV2 vaccines are now licensed and used in numerous Western Europe countries. In our study, we showed that the MLV2 vaccine has the same level of partial protection as the MLV1 vaccine. In light of the low level of genetic similarity between the Lena and MLV2 strains, our results were quite unexpected. Although some studies have demonstrated that the genotype 2 MLV vaccine affords significant protection in a challenge with a genotype 1 PRRSV strain (Park et al., 2015), protection after a homologous challenge is generally greater than after a heterologous challenge (Labarque et al., 2003; van Woensel et al., 1998). Our results thus offer further support that for PRRSV vaccines, the level of cross-protection is not always linked to genetic similarity between the vaccine and the challenge strain (Murtaugh, 2012).
The third immunizing strain evaluated in this study was the Finistere strain, a low pathogenic genotype 1.1 PRRSV strain from France (Renson et al., 2015). Similar to the vaccine strains, this strain offered partial clinical and virological protection upon challenge with the Lena strain. Compared with the MLV vaccines, the Finistere strain displayed a slightly higher protection with a shorter fever period, a positive effect on growth performance and a better control of viremia. From a genetic point of view, the better protection provided by the Finistere strain compared with the MLV1 vaccine was unexpected because the MLV1 and Finistere strain are very similar (Fig. 1) and have practically the same level of genetic similarity with the Lena strain. Therefore, our results are in line with those from a previous study (Labarque et al., 2003) where, in a homologous challenge model, the virological protection provided by a non-attenuated genotype 1 strain is better than the protection induced by the attenuated strain.
Immunization with a non-attenuated strain thus appears to be somewhat more efficient than that of a closely related attenuated strain, but this type of immunization is also more hazardous. Although genotype 1.1 strains are generally low virulent strains, they have the ability to spread easily from infected to naive animals (Rose et al., 2015) and can induce reproductive failure or respiratory disorders when associated with other respiratory pathogens (Fablet et al., 2012). In contrast, vaccine strains generally have largely decreased transmission efficiency and do not induce clinical signs (Martinez-Lobo et al., 2013). Accordingly, in field conditions, Linhares (Linhares et al., 2012) demonstrated that after exposure to live-resident virus, the control of PRRS circulation is reached sooner than when using a MLV vaccine, but the herds using MLV vaccine recover production performance earlier.
To further understand the level of protection elicited by the three immunizing strains, we investigated the cell-mediated immune response through the level of circulating IFNγ-SCs. Our results indicate that the best protection observed in the Fini group may be linked to a higher level of IFNγ-SCs (specific to the Lena strain) among the animals of this group. Although the exact basis of this enhanced immune response for the Fini strain is unknown, we can nevertheless highlight two hypotheses: first, the level of viral replication for the Finistere strain was higher than for the vaccine strains as indicated by higher post-immunization Ct values for the vaccine strains which probably result from the attenuation of the virulence of the vaccine strains by MARC-145 cell culture adaptation; second, the route of immunization for the Finistere strain was intranasal whereas the vaccine strains used the intramuscular route. One or both of these differences in the immunization process may have led to better Lena immunity for the Fini group. More generally, we also found a significant negative correlation between the number of IFNγ-SCs and the Lena blood viral load, indicating that the cell-mediated immune response is an important component in the control of Lena viremia. These data are in accordance with a previous study (Park et al., 2015) that showed a reduction in viremia associated with a high level of IFNγ-SCs in piglets vaccinated with a genotype 2 MLV vaccine and then challenged with a genotype 1 strain. These results underscore that determination of the cell-mediated immune response is an interesting tool to evaluate and predict the vaccine efficacy upon homologous as well as heterologous challenge.
In conclusion, this study demonstrates that MLV vaccines of genotype 1 or 2, as well as genotype 1.1 field strains can provide partial clinical and virological protection upon a challenge with the genotype 1.3 Lena strain. The cross-protection level induced by the immunizing strains was not related with the genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is associated with a higher level of cell-mediated immune response.
Conflict of interest statement
None.
Acknowledgments
This work was supported by the Regional Council of Brittany, the Union des Groupements de Producteurs de Viande de Bretagne (UGPVB), Boehringer Ingelheim France and INAPORC.
The authors thank Claire de Boisséson for preparing the NGS libraries and Angélique Moro and Jean-Marie Guionnet for their assistance with animal care.
We are most grateful to the Genomics platform at the Nantes (Biogenouest Genomics) core facility for its technical support in NGS sequencing and to Jennifer Richardson (UMR Virologie Anses-InraEnva, Maisons-Alfort) and Sandy Peltier (Seppic, Puteaux) for their assistance in ELISPOT reading.
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