Protective Efficacy of a Candidate Live-Attenuated Vaccine Derived from the SD-R Strain against NADC34-like Porcine Reproductive and Respiratory Syndrome Virus

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) strains were first detected in China in 2017 and became major circulating strains in 2021. Our previous study showed that the live-attenuated vaccine candidate SD-R strain could provide broad cross-protection against different NADC30-like PRRSVs (sublineage 1.8). However, the protective effect of SD-R against NADC34-like PRRSV is unclear. Here, a novel NADC34-like PRRSV, LNTZJ1341-2012, was isolated from a pig farm experiencing disease in 2020. Sequence analysis revealed that LNTZJ1341-2012 belonged to PRRSV-2 sublineage 1.5, exhibited the same Nsp2 amino-acid deletion characteristics as IA/2014/NADC34, and had not recombined with other strains. Additionally, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative NADC34-like strain (LNTZJ1341-2012). The control piglets in the challenge experiment displayed clinical signs typical of PRRSV infection, including transient fever, high viremia, mild clinical symptoms, and histopathological changes in the lungs and submaxillary lymph nodes. In contrast, SD-R vaccination significantly reduced serum and lung tissue viral loads, and vaccinated piglets did not show any clinical symptoms or histopathological changes. Our results demonstrated that LNTZJ1341-2012 is a mildly virulent NADC34-like PRRSV and that the live-attenuated vaccine SD-R can prevent the onset of clinical signs upon challenge with the NADC34-like PRRSV LNTZJ1341-2012 strain, indicating that SD-R is a promising vaccine candidate for the swine industry.


Introduction
Porcine reproductive and respiratory syndrome (PRRS) is a highly destructive viral disease that affects swine globally. The disease is caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and has severe economic ramifications for the pig industry worldwide [1,2]. PRRSV is an enveloped RNA virus belonging to the genus Betaarterivirus, family Arteriviridae, and order Nidovirales. PRRSV is divided into two species: Betaarterivirus suid 1 (PRRSV-1) and Betaarterivirus suid 2 (PRRSV-2) (ICTV2021). PRRSV has a genome length of approximately 15.3 kb and comprises at least ten open reading

Sample Collection and Virus Isolation
In December 2020, pigs on a farm in Liaoning Province, China, experienced NADC34like PRRSV infection, extensive miscarriage among sows, and the death of some piglets. Lung tissue samples were collected from dead piglets. The tissues were homogenized, the supernatant of the homogenate was used for viral detection by RT-PCR, the isolate was named LNTZJ1341-2012, and eight pairs of specific primers were used to determine the whole genome sequence of the virus [14]. RNA extraction, reverse transcription, primer sequence design, PCR procedures, and whole-genome sequencing were carried out in accordance with methods described in earlier studies [38]. The remaining suspensions were passed through 0.22 µm filters and inoculated onto primary alveolar macrophages (PAMs) and Marc-145 cells (an African green monkey kidney cell line) for virus isolation [39]. After three days, the cultures were harvested and preserved as viral stocks at a temperature of −80 • C. Cultures from the third passage in PAMs were utilized for animal experiments. The viral titer was evaluated using previously described methods [39].

Sequencing Analysis
All available sublineage 1.5 and sublineage 1.8 whole-genome sequences of PRRSV-2 collected from 1991 to 2022 were downloaded from GenBank and aligned with the sequences obtained in this study using MAFTT [40]. A phylogenetic tree was inferred using the neighbor-joining method and the Kimura 2-parameter substitution model (MEGA 7.0). The topology of the trees was confirmed with 1000 bootstrap replication steps [41]. Evolview (version 2.0) was used to annotate and modify the trees [42]. Deduced amino acid sequences were aligned by ClustalW in the Lasergene software (Version7.1, DNASTAR Inc., Madison, WI, USA). We utilized the Recombination Detection Program 4 (RDP4) to screen for potential recombination events in the multiple alignment of the genomes and test the role of recombination in the generation of LNTZJ1341-2012 (GenBank accession: OL516360.1) [11]. Finally, the graphics of recombination events were drawn by SimPlot v3.5.1 within a 500 bp window sliding along the genome alignment (20 bp step size).

Animals and Experimental Design
A total of thirteen 28-day-old piglets were obtained from a commercial pig herd in Harbin with PRRSV-, ASFV-, PCV2-, PRV-, and CSFV-free status. The piglets were blindly divided into three groups: 5 piglets in group A were used for SD-R immunization and LNTZJ1341-2012 inoculation; 5 piglets in group B were used for LNTZJ1341-2012 inoculation; and 3 piglets in group C were used as negative controls (Table 1). To ensure separation, each group of piglets was housed in an individual room. Piglets belonging to group A were immunized intramuscularly with a 2 mL dose of SD-R (dose: 10 6.2 TCID 50 /mL). At 28 days post-vaccination (dpv), the piglets in groups A and B were infected intramuscularly (2 mL) and intranasally (2 mL), respectively, with third-passage PAMs LNTZJ1341-2012 (1 × 10 5.0 TCID 50 /mL). The three piglets in group C were intramuscularly (2 mL) and intranasally (2 mL) administered Dulbecco's modified Eagle's medium (DMEM) ( Table 1). The rectal body temperatures and clinical signs of the piglets were recorded daily throughout the experiment, and body weight was measured every week.

Serological Testing
A commercial ELISA kit from IDEXX, Inc. (Westbrook, ME, USA) was employed to measure PRRSV-specific antibodies, following the manufacturer's instructions. The PRRSVspecific antibody titer was determined by calculating the sample-to-positive (S/P) ratio, and serum samples were classified as positive if the S/P ratio was ≥0.4. To conduct serum neutralization (SN) assays, all sera collected at 28 dpv and 21 dpi were subjected to heat inactivation at 56 • C for 30 min. Subsequently, serial two-fold dilutions of each serum sample were prepared using DMEM as the diluent. Suspensions containing 100 TCID50 PRRSV per 100 µL were then prepared, and 100 µL aliquots were added to each serum dilution. The serum-viral mixtures were incubated for 1 h in a 37 • C water bath. Next, the mixtures were dispensed onto Marc-145 cells in 96-well plates. The plates were further incubated at 37 • C in a humidified atmosphere with 5% CO 2 for 7 days. Duplicate samples were analyzed to determine the cytopathic effect (CPE) every day after inoculation. The neutralization titer of serum was calculated by the Reed-Muench method. A titer of ≥1:4 was considered positive. Three independent tests were performed for each serum sample.

Macroscopic and Histopathological Lesions
At 21 days post-infection (dpi), all piglets were humanely euthanized for pathological examination. Lung and submaxillary lymph node samples were collected from each piglet and fixed in a 4% formaldehyde solution for subsequent histopathological analysis using hematoxylin and eosin staining.

Statistical Analysis
Significant differences between two groups were determined using the t test (and nonparametric tests) in GraphPad 5.0 (San Diego, CA, USA). The level of significance was set at p < 0.05.

Virus Isolation and Identification
To evaluate the pathogenicity of the newly emerging NADC34-like LNTZJ1341-2012 strain and the protective effect of the vaccine candidate strain SD-R against it, tissue homogenates of samples positive for the LNTZJ1341-2012 strain were added to PAMs and Marc-145 cells. PRRSV N-protein expression was observed in PAMs and Marc-145 cells that were inoculated with the LNTZJ1341-2012 strain during IFA testing. These findings suggested that the virus isolates replicated successfully in both PAMs and Marc-145 cells ( Figure 2). branches represent Chinese strains, and blue branches represent strains from other countries. The green branch represents PRRSV-1 strain. (B) The NSP2 deletion pattern in SD-R and LNTZJ1341-2012: 131 aa discontinuous deletions are labeled yellow; 100 aa continuous deletions are labeled red. (C) Characterization of the recombination events between representative PRRSVs of each lineage and the LNTZJ1341-2012 isolate.

Virus Isolation and Identification
To evaluate the pathogenicity of the newly emerging NADC34-like LNTZJ1341-2012 strain and the protective effect of the vaccine candidate strain SD-R against it, tissue homogenates of samples positive for the LNTZJ1341-2012 strain were added to PAMs and Marc-145 cells. PRRSV N-protein expression was observed in PAMs and Marc-145 cells that were inoculated with the LNTZJ1341-2012 strain during IFA testing. These findings suggested that the virus isolates replicated successfully in both PAMs and Marc-145 cells ( Figure 2).

Clinical Reactions after Immunization and Challenge
After immunization with SD-R, no piglets in group A showed any clinical signs of PRRS, in contrast with those in group B and group C. After the LNTZJ1341-2012 challenge, some piglets in group B (3/5) showed the typical clinical symptoms of PRRSV infection, such as loss of appetite, lethargy, sleepiness, and cough. Two pigs in group B had only a transient fever (≥40.5 °C), showing fever symptoms up to 41.2 °C at 1 dpi (1/5) and 2 dpi (2/5), and then their body temperature returned to the normal range at 3 dpi, which was maintained until the end of the experiment ( Figure 3A). Piglets in group A and group C did not display any clinical symptoms throughout the entire experiment. Weekly measurements of the body weight of the piglets were taken, and the results showed that there was no significant difference in daily weight gain among group A, group B, and group C ( Figure 3B). Every piglet in the three groups survived until the end of the experiment without any fatalities.

Clinical Reactions after Immunization and Challenge
After immunization with SD-R, no piglets in group A showed any clinical signs of PRRS, in contrast with those in group B and group C. After the LNTZJ1341-2012 challenge, some piglets in group B (3/5) showed the typical clinical symptoms of PRRSV infection, such as loss of appetite, lethargy, sleepiness, and cough. Two pigs in group B had only a transient fever (≥40.5 • C), showing fever symptoms up to 41.2 • C at 1 dpi (1/5) and 2 dpi (2/5), and then their body temperature returned to the normal range at 3 dpi, which was maintained until the end of the experiment ( Figure 3A). Piglets in group A and group C did not display any clinical symptoms throughout the entire experiment. Weekly measurements of the body weight of the piglets were taken, and the results showed that there was no significant difference in daily weight gain among group A, group B, and group C ( Figure 3B). Every piglet in the three groups survived until the end of the experiment without any fatalities.

Antibody Responses in Immunized or Challenged Piglets
PRRSV-specific antibodies were measured using IDEXX ELISA, which showed that all the immunized piglets in group A seroconverted at 14 dpv ( Figure 3C). In group B, a total of two out of five piglets seroconverted by 7 dpi, and the remaining piglets seroconverted by 10 dpi ( Figure 3C). During the experiment, piglets from group C showed negative serological results ( Figure 3C).

Antibody Responses in Immunized or Challenged Piglets
PRRSV-specific antibodies were measured using IDEXX ELISA, which showed that all the immunized piglets in group A seroconverted at 14 dpv ( Figure 3C). In group B, a total of two out of five piglets seroconverted by 7 dpi, and the remaining piglets seroconverted by 10 dpi (Figure 3C). During the experiment, piglets from group C showed negative serological results ( Figure 3C).

Macroscopic and Histopathological Lesions
All piglets were euthanized at 21 dpi (after the LNTZJ1341-2012 challenge). In contrast to the piglets in groups A and C, some piglets in group B showed lesions typical of PRRS, such as hemorrhaging in the submaxillary lymph nodes (2/5) ( Figure 4B,E). Histopathology revealed extensive inflammatory cell infiltration with alveolar epithelial cell proliferation and moderate alveolar diaphragm widening in the lungs (5/5) ( Figure 4H) and scattered bleeding in the paracortical region of the submaxillary lymph nodes (5/5) ( Figure 4K) in group B, in contrast with that in pigs in groups A and C. No macroscopic or histopathological changes were observed in piglets in groups A and C.

Macroscopic and Histopathological Lesions
All piglets were euthanized at 21 dpi (after the LNTZJ1341-2012 challenge). In contrast to the piglets in groups A and C, some piglets in group B showed lesions typical of PRRS, such as hemorrhaging in the submaxillary lymph nodes (2/5) ( Figure 4B,E). Histopathology revealed extensive inflammatory cell infiltration with alveolar epithelial cell proliferation and moderate alveolar diaphragm widening in the lungs (5/5) ( Figure 4H) and scattered bleeding in the paracortical region of the submaxillary lymph nodes (5/5) ( Figure 4K) in group B, in contrast with that in pigs in groups A and C. No macroscopic or histopathological changes were observed in piglets in groups A and C.

Viremia and Viral Tissue Distribution
The viral load in serum samples from individual piglets was estimated at 0, 7, 14, 21, and 28 dpv and at 3, 5, 7, 10, 14, 17, and 21 dpi. The results showed that only two of the five piglets in group A had low levels of viremia at 14 dpv ( Figure 5A). From 0 to 10 dpi, piglets infected with LNTZJ1341-2012 (groups A and B) showed a gradual increase in viral load, reaching a peak at 10 dpi. Subsequently, the viral load gradually decreased. No viremia was observed in group C ( Figure 5A). One piglet in group B still had detectable viremia at 21 dpi. The average viral loads at 7~14 dpi in group A were significantly lower than those in group B ( Figure 5A). Moreover, the number of piglets with viremia in group A was also less than that in group B.
The hearts, livers, spleens, lungs, kidneys, stomachs, small intestines, brains, submaxillary lymph nodes, and tonsils were collected from individual piglets and analyzed by qPCR. The viral load was found to differ in different tissues in groups A and B ( Figure   Figure 4. Gross and histological lesions of the lungs and submaxillary lymph nodes from group A, group B, and group C. The lungs in group B (B) showed no obvious lesions compared with those in groups Aand C (A,C). Hemorrhage in the submaxillary lymph nodes in group B (E) was observed, in contrast with groups A and C (D,F). In contrast to those in groups A and C (G,I), extensive inflammatory cell infiltration with alveolar epithelial cell proliferation and moderate alveolar diaphragm widening in the lungs were observed in group B (H). In contrast to that in groups A and C (J,L), scattered bleeding in the paracortical region of the submaxillary lymph nodes was observed in group B (K). Scale bar = 200 µm. The location of the lesion is indicated by an arrow.

Viremia and Viral Tissue Distribution
The viral load in serum samples from individual piglets was estimated at 0, 7, 14, 21, and 28 dpv and at 3, 5, 7, 10, 14, 17, and 21 dpi. The results showed that only two of the five piglets in group A had low levels of viremia at 14 dpv ( Figure 5A). From 0 to 10 dpi, piglets infected with LNTZJ1341-2012 (groups A and B) showed a gradual increase in viral load, reaching a peak at 10 dpi. Subsequently, the viral load gradually decreased. No viremia was observed in group C ( Figure 5A). One piglet in group B still had detectable viremia at 21 dpi. The average viral loads at 7~14 dpi in group A were significantly lower than those in group B ( Figure 5A). Moreover, the number of piglets with viremia in group A was also less than that in group B.
Vaccines 2023, 11, 1349 9 of 14 5B). The highest viral load was detected in the tonsils, followed by the submaxillary lymph nodes and the lungs. The average viral loads of ten tissues in group A were lower than those in group B. Notably, group A pigs had significantly lower average viral loads in their lung tissue than group B pigs ( Figure 5B). Throughout the study, no viremia or viral loads were detected in the samples from group C. The samples were confirmed to contain the original virus by sequencing a portion of the ORF7 gene. The PRRSV viral load in tissues and serum from each group was determined using qPCR. Tissue samples were collected at 21 dpi, while serum was collected at 0, 7, 14, 21, and 28 dpv and at 3, 5, 7, 10, 14, 17, and 21 dpi. The data are presented as means ± SDs (error bars). Groups A, B, and C are labeled green, purple, and orange, respectively. An asterisk (*) indicates a significant difference between the groups (ns: no significant difference; *: p < 0.05; **: p < 0.01). The numbers represent the number of piglets with detectable viral loads and the number of piglets in each group.

Serum-Neutralizing Antibody Detection
To evaluate the protective immune responses induced by SD-R immunization, SN Abs against the SD-R and LNTZJ1341-2012 strains were measured. All control wells (100 TCID50) displayed CPE on day 3 of cell inoculation. On day 7 of cell inoculation, no further changes were observed in the experimental wells. Therefore, this study chose to present the results from the 3-and 7-day post-inoculation of cells. The homologous SN Ab could be detected beginning at 28 dpv in group A, and all pigs showed higher titers of neutralizing antibodies against SD-R at 21 dpi (Table 3). In the SN assay against the LNTZJ1341-2012 strain, at 28 dpv, neither group A nor group B developed SN Abs against the LNTZJ1341-2012 strain either (Table 4). Interestingly, serum from group B pigs at 21 dpi could delay the appearance of CPE on the third day of the SN assay, and two pigs were able to completely neutralize the LNTZJ1341-2012 strain on day 7 (Table 4). In contrast, higher neutralization titers were detected in group A pigs both on day 3 and day 7 of cell inoculation (Table 4). The PRRSV viral load in tissues and serum from each group was determined using qPCR. Tissue samples were collected at 21 dpi, while serum was collected at 0, 7, 14, 21, and 28 dpv and at 3, 5, 7, 10, 14, 17, and 21 dpi. The data are presented as means ± SDs (error bars). Groups A, B, and C are labeled green, purple, and orange, respectively. An asterisk (*) indicates a significant difference between the groups (ns: no significant difference; *: p < 0.05; **: p < 0.01). The numbers represent the number of piglets with detectable viral loads and the number of piglets in each group.
The hearts, livers, spleens, lungs, kidneys, stomachs, small intestines, brains, submaxillary lymph nodes, and tonsils were collected from individual piglets and analyzed by qPCR. The viral load was found to differ in different tissues in groups A and B ( Figure 5B). The highest viral load was detected in the tonsils, followed by the submaxillary lymph nodes and the lungs. The average viral loads of ten tissues in group A were lower than those in group B. Notably, group A pigs had significantly lower average viral loads in their lung tissue than group B pigs ( Figure 5B). Throughout the study, no viremia or viral loads were detected in the samples from group C. The samples were confirmed to contain the original virus by sequencing a portion of the ORF7 gene.

Serum-Neutralizing Antibody Detection
To evaluate the protective immune responses induced by SD-R immunization, SN Abs against the SD-R and LNTZJ1341-2012 strains were measured. All control wells (100 TCID50) displayed CPE on day 3 of cell inoculation. On day 7 of cell inoculation, no further changes were observed in the experimental wells. Therefore, this study chose to present the results from the 3-and 7-day post-inoculation of cells. The homologous SN Ab could be detected beginning at 28 dpv in group A, and all pigs showed higher titers of neutralizing antibodies against SD-R at 21 dpi (Table 3). In the SN assay against the LNTZJ1341-2012 strain, at 28 dpv, neither group A nor group B developed SN Abs against the LNTZJ1341-2012 strain either (Table 4). Interestingly, serum from group B pigs at 21 dpi could delay the appearance of CPE on the third day of the SN assay, and two pigs were able to completely neutralize the LNTZJ1341-2012 strain on day 7 (Table 4). In contrast, higher neutralization titers were detected in group A pigs both on day 3 and day 7 of cell inoculation (Table 4).

Discussion
NADC34-like PRRSV was first reported in the United States and has had a severe impact on the American pig industry, causing a phenomenon described as an "abortive storm" [44]. American researchers found that the enhancement of the pathogenicity of NADC34-like PRRSV was very obvious, and the IA/2014/NADC34 prototype was highly pathogenic to piglets [13]. There have been many reports on the pathogenicity of NADC34like PRRSV in China, indicating the mild pathogenicity of the HLJDZD32-1901 strain [21], the moderate pathogenicity of the PRRSV-ZDXYL-China-2018-1 strain [26], and the high pathogenicity of the JS2021NADC34 strain [25]. In contrast to those reports, only some of the pigs challenged with the LNTZJ1341-2012 strain (group B) showed clinical symptoms typical of PRRSV (transient fever, cough, etc.), slight histopathological changes, and no significant difference in weight gain (Table 3). Therefore, the LNTZJ1341-2012 strain exhibited mild pathogenicity in piglets.
Interestingly, LNTZJ1341-2012 shared high genomic similarity with the strains HLJDZD32-1901 (95.4%), PRRSV-ZDXYL-China-2018-1 (95.1%), and JS2021NADC34 (94.1%), without any recombination with other PRRSV strains, and shared the same pattern of deletion in Nsp2 (100 aa continuous deletion). However, the pathogenicity of these viruses varies greatly, with the JS2021NADC34 strain causing a fatality rate of 75%, while the HLJDZD32-1901 and LNTZJ1341-2012 strains cause only mild clinical symptoms (Table 3). A high viral load is one of the most typical findings for highly pathogenic strains [45,46]. The LNTZJ1341-2012 strain can generate a higher serum viral load compared to the IA/2014/NADC34 strain, induce a longer duration of viremia than the JS2021NADC34 strain, and have the lowest pathogenicity (Table 5). Studies have found that there is no positive correlation between viremia and the pathogenicity of NADC34-like PRRSV strains prevalent in the United States [13], so the use of viremia to determine the pathogenicity of NADC34-like PRRSV strains does not seem to be applicable. There are two sublineages of lineage 1 PRRSV-2 circulating in China, including NADC30-like (sublineage 1.8) and NADC34-like (sublineage 1.5) strains. These strains were first detected in China in 2012 and 2017, respectively [12,47]. Lineage 1 PRRSV-2 has become the most prevalent subtype of PRRSV strain in China [17]. Since the first PRRSV vaccine was released in China in 2005, China has used vaccines to prevent and control PRRSV. At present, many experiments have proven that vaccines, such as Ingelvac PRRS MLV/RespPRRS MLV [33,36], CH-1a [34], JXA1-P80 [36], TJM-F92 [35], HuN4-F112 [35], GDr180 [35], and R98 [37], available in the Chinese market, can provide only partial protection against NADC30-like (sublineage 1.8) strains. In a previous study, we created an attenuated lineage 1 PRRSV vaccine (SD-R) that has been proven to offer full protection against both homologous and heterologous NADC30-like PRRSV challenges [27].
To our knowledge, only one vaccine (Ingelvac PRRS MLV) has been evaluated for protection against NADC34-like PRRSV, and it provides incomplete protection against the PRRSV/CN/FJGD01/2021 strain [22]. In this study, the vaccine candidate SD-R significantly reduced the average serum viral load from 7 dpi to 14 dpi and prevented fever in the vaccinated challenged group (group A); at the same time, vaccinated piglets showed significantly lower levels of the virus in their lungs than unvaccinated piglets (group B) on the 21st day post-infection.
Previous studies have indicated that serum-neutralizing antibodies (SN Abs) play a critical role in the development of protective immunity against PRRSV [2,48]. Normally, neutralizing antibodies induced by PRRSV vaccines only provide homologous neutralizing activity and show lower/zero neutralizing titers against heterogenic strains [49,50]. In this study, the neutralizing titers against the LNTZJ1341-2012 strain were undetectable in all 28 dpv sera. Notably, sera from pigs in the challenged group at 21 dpi exhibited the ability of the LNTZJ1341-2012 strain to effectively hinder the infection of Marc-145 cells. In the vaccinated challenged group, the numbers and neutralization titers of pigs that could produce neutralizing antibodies were higher than those in the challenged group. Consequently, even though neutralizing titers were not detected at 28 dpv, prophylactic immunization with the SD-R vaccine could help animals generate heterologous neutralizing antibodies in the later stages, reducing the impact of the LNTZJ1341-2012 strain on pigs. Interestingly, pig 083 in the vaccinated group exhibited higher neutralizing titers against LNTZJ1341-2012 (1:10) compared to SD-R (1:6.32) at 21 dpi, which may be caused by LNTZJ1341-2012 inducing homologous neutralizing antibody production in infected pigs at 21 dpi.
The protective effect of SD-R against the heterologous strain LNTZJ1341-2012 regarding viremia and organ viral load was not as obvious as that of the homologous strain SD [27]. However, the vaccinated challenged pigs did not show any clinical symptoms, and pathological analyses revealed that SD-R was effective in preventing the development of gross and microscopic lung lesions in the vaccinated challenged piglets. Taken together, these findings indicate that the PRRSV vaccine candidate SD-R has the ability to provide cross-protection against NADC34-like PRRSV LNTZJ1341-2012 infection. Meanwhile, only two of the five immunized piglets presented low viral loads at 14 dpv, which further demonstrated that SD-R showed higher safety.

Conclusions
In summary, our findings indicate that LNTZJ1341-2012 is a mildly virulent NADC34like PRRSV, and the vaccine candidate SD-R prevents the onset of clinical signs against the LNTZJ1341-2012 strain. Therefore, the vaccine candidate strain SD-R can effectively prevent and control lineage 1 PRRSV-2 infection.

Conflicts of Interest:
The authors declare no potential conflict of interest with respect to the research, authorship, or publication of this article.