Xie Jiexiong, Vereecke Nick, Theuns Sebastiaan, Oh Dayoung, Vanderheijden Nathalie, Trus Ivan, Sauer Jannes, Vyt Philip, Bonckaert Caroline, Lalonde Christian, Provost Chantale, Gagnon Carl A, Nauwynck Hans
Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
PathoSense BV, 2500 Lier, Belgium.
Vaccines (Basel). 2021 Jun 3;9(6):594. doi: 10.3390/vaccines9060594.
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15, PK15). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15, PK15, MARC-145, and MARC-145) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15, PK15, MARC-145, and MARC-145. The titers in PK15 and PK15 cells were significantly correlated with virus titers in PAM for both PRRSV1 ( < 0.001) and PRRSV2 ( < 0.001) compared with MARC-145 (PRRSV1: = 0.22 and PRRSV2: = 0.03) and MARC-145 (PRRSV1: = 0.04 and PRRSV2: = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15 and PK15 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145 cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.
猪繁殖与呼吸综合征病毒（PRRSV）具有高度受限的细胞嗜性。在体内，该病毒主要感染鼻、肺、扁桃体和咽淋巴组织中的组织特异性巨噬细胞。然而在体外，MARC - 145细胞系是少数常用于体外增殖的对PRRSV敏感的细胞系之一。此前，已表明几种PRRSV非允许细胞系在表达重组进入受体后（如PK15、PK15）会变得对PRRSV感染敏感。在本研究中，我们检测了不同细胞系作为原代肺泡巨噬细胞（PAM）细胞的可能替代物用于PRRSV分离和生长的适用性。将四种不同细胞系（PK15、PK15、MARC - 145和MARC - 145）从PCR阳性血清（PRRSV1和PRRSV2）中进行PRRSV原代分离的敏感性与PAM的敏感性进行了比较。为了找到细胞嗜性与病毒基因型之间可能的相关性，对54份田间样本进行了测序，并鉴定了可能与细胞嗜性相关的氨基酸残基。关于用五种不同细胞类型获得的病毒滴度，PAM产生的平均病毒滴度最高，其次是PK15、PK15、MARC - 145和MARC - 145。与MARC - 145（PRRSV1：= 0.22，PRRSV2：= 0.03）和MARC - 145（PRRSV1：= 0.04，PRRSV2：= 0.12）相比，PRRSV1（< 0.001）和PRRSV2（< 0.001）在PK15和PK15细胞中的滴度与PAM中的病毒滴度显著相关。此外，在全基因组关联研究（GWAS）中使用PRRSV全基因组序列评估了细胞嗜性与病毒基因型之间可能的相关性。PRRSV2序列中的结构蛋白残基GP2：187L和N：28R与其在MARC - 145中的生长相关。PRRSV2的GP5：78I残基和PRRSV1的Nsp11：155F残基与在PAM上更高的复制相关。总之，PK15和PK15细胞在表型上与体内靶巨噬细胞密切相关，并且比MARC - 145/MARC - 145细胞更适合病毒分离和滴定。未来将进一步研究PRRSV蛋白中可能与细胞嗜性相关的残基。
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