A single amino acid substitution in porcine reproductive and respiratory syndrome virus glycoprotein 2 significantly impairs its infectivity in macrophages

Authors: CHAUDHARI, JAYESHBHAI - University Of Nebraska; LEME, RAQUEL - University Of Nebraska; MARTINEZ, KASSANDRA - University Of Nebraska; SILLMAN, SARAH - University Of Nebraska; Workman, Aspen; VU, HIEP - University Of Nebraska

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2022
Publication Date: 12/18/2022
Citation: Chaudhari, J., Leme, R.A., Durazo-Martinez, K., Sillman, S., Workman, A.M., Vu, H.L.X. 2022. A single amino acid substitution in porcine reproductive and respiratory syndrome virus glycoprotein 2 significantly impairs its infectivity in macrophages. Viruses. 14(12). Article 2822. https://doi.org/10.3390/v14122822.
DOI: https://doi.org/10.3390/v14122822

Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in many swine-producing countries, causing substantial economic losses to the industry. Modified-live virus (MLV) vaccines are widely used to protect pigs from disease; however, substantial genetic variation between circulating variants of PRRSV undermine vaccine efficacy. Therefore, MLV vaccines based on locally circulating PRRSV strains are in continuous demand. Virulent field strains of PRRSV can be attenuated by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Passaging on MARC-145 cells introduces several changes in the PRRSV genome that collectively enhance its ability to replicate in MARC-145 cells and reduces its ability to replicate in porcine alveolar macrophages (PAMs), the primary target for PRRSV replication in pigs. Thus, this method is commonly used to generate live-attenuated vaccines. In the present study, the virulent PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, followed by three consecutive rounds of plaque purification. These plaque-purified viral clones were then tested to see if they had reduced replication in PAMs. One passage 95 viral clone had nearly completely lost its ability to infect PAMs. Genomic analysis of different NCV1 passages and clones revealed a single amino acid change in the viral protein GP2 was unique to this clone that had lost its ability to infect PAMs. Specifically, this clone had a Lysine (K) to Isoleucine (I) substitution in GP2 at residue 160 (K160I). Reverse genetics techniques were used to revert this amino acid back to Lysine, and this restored infectivity in PAMs. To substantiate these results, we introduced this K160I substitution in the GP2 of another highly virulent PRRSV strain, NCV13, and tested the ability of this variant to infect PAMs and cause disease in pigs. This single amino acid substitution in NCV13 reduced its ability to infect PAMs and pigs inoculated with this virus exhibited significantly lower viremia levels and pathological lesions than those inoculated with the wild-type NCV13 strain. Thus, this study demonstrated that the K160 residue in GP2 of PRRSV is one of the determinants for PRRSV infectivity in PAMs.

Technical Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.