Comparative evaluation of 4 commercial modified‐live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines against heterologous dual Korean PRRSV‐1 and PRRSV‐2 challenge

Abstract Background Four commercial porcine reproductive and respiratory syndrome virus (PRRSV) modified‐live vaccines (MLV) was compared to protect growing pigs against dual challenge of PRRSV‐1 and PRRSV‐2. Methods Two of the vaccines were based on PRRSV‐1, and two on PRRSV‐2. A total of 72 PRRSV‐naïve pigs were divided into six groups (12 pigs/group). Results Two PRRSV‐1 MLV‐vaccinated and two PRRSV‐2 MLV‐vaccinated groups reduced significantly (p < .05) genomic copies of PRRSV‐1 in their sera compared to the unvaccinated challenged group. Two PRRSV‐2 MLV‐vaccinated groups reduced significantly (p < .05) fewer genomic copies of PRRSV‐2 in their sera whereas two PRRSV‐1 MLV‐vaccinated groups were unable to reduce genomic copies of PRRSV‐2 compared to unvaccinated challenged groups. Two PRRSV‐1 MLV‐vaccinated groups induced a stronger PRRSV‐1 specific IFN‐γ‐SC response, while two PRRSV‐2 MLV‐vaccinated groups induced a stronger PRRSV‐2 specific IFN‐γ‐SC response. Two PRRSV‐2 MLV‐vaccinated groups showed significantly (p < .05) lower mean macroscopic and microscopic lung lesion scores compared to two PRRSV‐1 MLV‐vaccinated groups. Conclusions These data demonstrated that two PRRSV‐2 vaccines were efficacious and exhibited similar protection while, two PRRSV‐1 vaccines were largely ineffective against the dual challenge.


| INTRODUC TI ON
Porcine reproductive and respiratory syndrome virus (PRRSV), which belongs to the family Arteriviridae and the order Nidovirales causes great economic losses to the swine industry worldwide (Snijder, Kikkert, & Fang, 2013). Infection with PRRSV is characterized by reproductive failure (abortions, premature farrowing and weak or stillborn animals) in gilts and sows, and respiratory disease in growing pigs. PRRSV can be divided into two distinct species, PRRSV-1 (former genotype 1 from European-like strain) and PRRSV-2 (former genotype 2 from North American-like strain). PRRSV-2 was first discovered in Korea in 1994 and later PRRSV-1 was isolated in 2005 (Kim et al., 2010;Kweon et al., 1994). Since then, co-infection with both species within the same farm has become endemic (Choi, Lee, Park, Jeong, & Chae, 2015).
As co-infection with both species becomes more prevalent, controlling co-infections has rapidly become the main focus for swine producers. Theoretically, concurrent vaccination of pigs with both PRRSV-1 and PRRSV-2 vaccines has the potential to control co-infection of pigs with both species. However, a recent study has suggested that concurrent vaccination of pigs with a PRRSV-1 and a PRRSV-2 modified-live virus (MLV) vaccine may interfere with the efficacy of the PRRSV-2 vaccine . Therefore, control of both species with a single PRRSV vaccine is currently a major clinical focus. Currently, there are four commercially available PRRSV MLV vaccines in the Korean market, two based on PRRSV-1 and two based on PRRSV-2. A previous study showed that a PRRSV-2 MLV vaccine was efficacious against dual PRRSV-1 and PRRSV-2 challenge . However, to date, no comparative study has been conducted to compare all four commercially available PRRSV-MLV vaccines against heterologous dual PRRSV-1 and PRRSV-2 challenge under the same experimental conditions. Therefore, the objective of this study was to evaluate and compare the efficacy of all four commercial PRRSV MLV vaccines against respiratory disease of caused by heterologous dual PRRSV-1 and PRRSV-2 challenge in growing pigs based on clinical, virological, immunological and pathological analyses.

| Experimental design
A total of 72 PRRSV-naïve pigs from a PRRSV-free farm were selected for this study. All pigs were confirmed negative for PRRSV by serology testing and by quantitative RT-PCR on the day of vaccination. Pigs were divided into six groups (12 pigs/group) and assigned into six rooms using the random number generation function (Excel, Microsoft Corporation, Redmond, Washington, USA). Pigs in each of group were housed in same room ( At 0 dpc (63 days of age), the pigs in the Vac1A/Ch1-2, Vac1B/ Ch1-2, Vac2A/Ch1-2, Vac2B/Ch1-2 and UnVac/Ch1-2 groups were inoculated intranasally with 3 ml of PRRSV-1 (10 5 TCID 50 /mL of SNUVR090485, second passage in alveolar macrophages) and PRRSV-2 (10 5 TCID 50 /mL of SNUVR090851, second passage in alveolar macrophages) inoculum. Pigs in UnVac/UnCh were inoculated intranasally with 3 ml of PBS and served as the negative control group. Oral fluids were collected from all pigs prior to challenge (Prickett et al., 2008).
Upon challenge with PRRSV-1 and PRRSV-2, pigs in the Vac1A/Ch1-2, Vac1B/Ch1-2, Vac2A/Ch1-2, Vac2B/Ch1-2 and UnVac/Ch1-2 groups were randomly assigned into five of six rooms using the random number generation function (Excel; Microsoft Corporation). Each room had 12 pens and pigs were housed individually in each pen. The pigs in the UnVac/UnCh group were randomly placed into 12 pens in the one remaining room. Following PRRSV challenge, the physical condition of each pig was monitored daily and rectal temperatures were recorded.
From each group, six pigs were randomly selected using the random number generation function (Excel; Microsoft Corporation) at 7 and 14 dpc. Pigs were, sedated by an intravenous injection of sodium pentobarbital, euthanized by electrocution and necropsied (Beaver et al., 2001). F I G U R E 1 Phylogenetic analysis. Open reading frame 5 genome from the challenge PRRSV and the vaccine viruses. An unrooted neighbour-joining tree was constructed from aligned nucleotide sequences

| Clinical observation
Clinical respiratory observations were also recorded daily using scores ranging from 0 (normal) to 6 (severe dyspnoea and abdominal breathing) (Halbur et al., 1995). Observers were blinded to vaccination and challenge status. Rectal temperatures were recorded daily at the same time by same personnel.

| Pathology
The severity of macroscopic lung lesions was scored to estimate the percentage of the lung affected by pneumonia. The scoring was done by two pathologists at the institution where this study was performed. For the entire lung, 100 points were assigned as follows; 10 points each to the right cranial lobe, right middle lobe, left cranial lobe, and left middle lobe, 27.5 points each to the right caudal lobe and left caudal lobe and 5 points to the accessory lobe (Halbur et al., 1995).
TA B L E 1 Experimental design and means (with standard deviation) of lung lesion score in pigs from various groups with 7 and 10 days post challenge (dpc)

| D ISCUSS I ON
In this study, we compared the efficacy of four commercial vaccines against respiratory disease in growing pigs after heterologous dual It is important to note that according to a previous study, the same PRRSV-2 challenge strain used here caused gross and microscopic lung lesions comparable to dual challenge .
PRRSV-1 viraemia was also similarly reduced in the dual challenge compared to PRRSV-1 infection alone after vaccination . Therefore, we believe that the PRRSV-1 strain used in the current study does not contribute to respiratory disease severity in a dual infection. Our preliminary data using combinations of three other strains of each PRRSV-1 and PRRSV-2 showed similar results (data not shown). This again suggests that PRRSV-2 may play a more prominent role than PRRSV-1 in respiratory disease and pathological lung lesions from a dual infection with Korean PRRSV-1 and PRRSV-2 strains. For this reason, the effect of PRRSV-2 vaccine on PRRSV-1 virus may be inconclusive, as the PRRSV-2 virus was dominate over the PRRSV-1 virus in dually infected pigs. In the previous single challenge study, the same PRRSV-2 MLV vaccines used in this study can protect against both PRRSV-1 and PRRSV-2 challenge (Oh et al., 2019). Therefore, the PRRSV-2 MLV appeared to be effective against heterologous dual challenge.
Altogether, this suggests that the PRRSV detected in the serum of the vaccinated pigs after the dual challenge is the challenge strains.
Neutralizing antibodies and T-cell responses typically play an important role in reducing PRRSV viraemia and controlling PRRSV infection (Madapong et al., 2017). However, there is recent evidence from studies with single PRRSV-1 or PRRSV-2 infection that PRRSV viraemia is often reduced even before neutralizing antibodies are detected in infected and vaccinated pigs (Madapong et al., 2017;Mateu & Diaz, 2008;Mengeling, Lager, Vorwald, & Clouser, 2003;Nelson, Christopher-Hennings, & Benfield, 1994). Vaccination with all four vaccines used in this study did not generate any detectable neutralizing antibodies until at least 14 days post single challenge with either PRRSV-1 or PRRSV-2 Kim et al., 2015;Park et al., 2014). Taken together, this suggests that neutralizing antibodies are not essential for PRRSV clearance (Kimman, Cornelissen, Moormann, Rebel, & Stockhofe-Zurwieden, 2009;Mateu & Diaz, 2008). In contrast, T-cell responses as measured by an increase in the number of IFN-γ-SC directly correlates with the reduction of PRRSV viraemia (Correas, Osorio, Steffen, Pattnaik, & Vu, 2017;Meier et al., 2003). In the present study, vaccination of pigs with either of the two PRRSV-1 MLV vaccines or the two PRRSV-2 MLV vaccines elicited equal numbers of PRRSV-1 specific IFN-γ-SC.
In contrast, vaccination of pigs with either of the two PRRSV-2 MLV vaccines elicited higher frequency of PRRSV-2 specific IFN-γ-SC F I G U R E 5 Mean (with standard deviation) of PRRSV-1 specific IFN-γ-SC (a) and PRRSV-2 specific IFN-γ-SC (b) in peripheral blood mononuclear cells of pigs from six groups. Different letters (a, b and c) at days post challenge indicate significant differences among groups compared to vaccination of pigs with either of the two PRRSV-1 MLV vaccines. These data can explain why PRRSV-1 MLV vaccines are able to reduce PRRSV-1 viraemia only while PRRSV-2 MLV vaccines are able to reduce both PRRSV-1 and PRRSV-2 viraemia.
To date, in Korea, there are four PRRSV MLV vaccines that have been licensed for commercial use. Two are based on PRRSV-1 and two on PRRSV-2. Therefore, it was important to compare the efficacy of all four under the same experimental conditions. Control of both species of PRRSV by a single vaccine is the number one goal for swine producers because the number of pigs that are co-infected with both species is rapidly increasing. Different levels of cross protection between PRRSV-1 and PRRSV-2 MLV vaccines can provide swine practitioners and producers with significant clinical information on how to select the proper vaccines to protect their livestock against respiratory disease caused by co-infection with both species of PRRSV.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests with respect to their authorship or the publication of this article.

E TH I C A L S TATEM ENT
All of the methods were previously approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee.
Sample collection was carried out according to the animal welfare code of Korea.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.