DISCUSSION
Lineage 1, especially NADC30-like PRRSV, has become the most prevalent PPRSV lineage in North America and Asia (de Avellar and Markus, 1993; Kawabori et al., 1983; Savary and Ferron, 1982; Sun et al., 2020). In 2013, a new PRRSV strain called NADC30-like PRRSV, which has a unique 131-aa deletion within its NSP2 protein, was isolated from diseased piglets in China (Zhao et al., 2015; Zhou et al., 2015). This PRRSV originated in the United States and has become one of the major endemic strains in China since 2016 (Guo et al., 2019). The pathogenicity of NADC30-like PRRSVs ranges from moderate (Sun et al., 2016; Wang et al., 2018; Zhang et al., 2019a) to high (Chen et al., 2021; Liu et al., 2017b; Zhao et al., 2015), and most of them are moderately pathogenic. However, the current commercial vaccines, Ingelvac PRRS MLV/RespPRRS MLV(Sun et al., 2018; Wei et al., 2019), CH-1a(Li et al., 2022), HuN4-F112(Bai et al., 2016), JXA1-P80(Sun et al., 2018), R98(Zhang et al., 2016), TJM-F92(Bai et al., 2016) and GDr180(Bai et al., 2016; Zhang et al., 2019a), do not provide completely effective protection against NADC30-like PRRSVs. Here, we selected the moderately pathogenic NADC30-like PRRSV SD strain and described a newly developed lineage 1 PRRSV vaccine candidate, SD-R, which is efficacious in the prevention of clinical infection caused by NADC30-like PRRSVs.
Unlike CH-1a or HP-PRRSV, NADC30-like PRRSVs have lower levels of whole-genome similarity (Guo et al., 2019; Zhang et al., 2019a) and a wider variety of recombination patterns (Yu et al., 2020; Zhang et al., 2019a). Almost all NADC30-like PRRSVs are recombinant viruses (Yu et al., 2020; Zhang et al., 2019a). Although recombination breakpoints are relatively random, statistical analysis reveals that recombination hotspots range from nucleotide positions of approximately 7,900 to 8,100 and 12,400 to 13,500 (Yu et al., 2020). We speculated that NADC30-like PRRSVs with two recombination regions may be more stable and important for viral survival. Therefore, we selected an NADC30-like PRRSV SD with the above two recombination regions (7365-7661 in the NSP9 region and 12305-12773 in the GP2a-GP3 region) for passage in Marc-145 cells.
In the present study, SD-R, a genetically stable attenuated viral strain, was obtained by serial passaging in Marc-145 cells with the lineage 1 PRRSV SD strain. All amino acid mutations related to SD were observed before the 125th passage. No nucleotide and amino acid mutations were observed between the 125thand 150th passages. This indicated that the SD strain at the 125th passage had adapted to the Marc-145 cells and was subsequently stably passaged. The HP-PRRSV vaccines JXA1-R, TJM, HuN4-F112 and GDr180 were obtained through passaging in Marc-145 cells for 80, 92, 112 and 180 passages, respectively (Leng et al., 2012; Liu et al., 2015; Tian et al., 2009; Yu et al., 2015). Because the pathogenicity of SD is far lower than that of HP-PRRSV, we believe that SD-R (the 125th passage in Marc-145 cells) is safer for piglets than commercial HP-PRRSV vaccines. Furthermore, both high-dose and repeated-dose tests based on SD-R were safe for all piglets (data not shown).
In this study, piglets immunized with SD-R developed a rapid and effective humoral response and were effectively protected against NADC30-like PRRSV challenge. Indeed, PRRSV vaccines have poor cross-protection effects (Bai et al., 2016; Yu et al., 2021). However, surprisingly, SD-R could provide better cross-protection, even though the genomic nucleotide similarity of SD and HLJWK108-1711 was only 89.9%. All the piglets immunized with SD-R and then challenged with SD or HLJWK108-1711 survived without any major clinical signs at any point in the experimental period. Piglets in the immunized and challenge groups were healthier than those in the challenge control group based on clinical signs, body temperature, body weight, viremia and viral loads in tissues. Altogether, these results suggested that the SD-R candidate vaccine is effective against infections caused by different NADC30-like PRRSVs. However, the detailed molecular basis of cross-protection induced by the SD-R vaccine and its attenuation mechanism remain unclear. Furthermore, the cross-protection against other types of PRRSVs, such as NADC34-like PRRSV, QYYZ-like PRRSV and HP-PRRSV, should be studied further.
In conclusion, we developed the first attenuated lineage 1 PRRSV candidate vaccine strain, SD-R. Furthermore, SD-R was sufficiently attenuated and antigenic enough to confer protection against homologous and heterologous NADC30-like PRRSV challenge.