Sustaining Interferon Induction by a High-Passage Atypical Porcine Reproductive and Respiratory Syndrome Virus Strain

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) strain A2MC2 induces type I interferons in cultured cells. The objective of this study was to attenuate this strain by serial passaging in MARC-145 cells and assess its virulence and immunogenicity in pigs. The A2MC2 serially passaged 90 times (A2MC2-P90) retains the feature of interferon induction. The A2MC2-P90 replicates faster with a higher virus yield than wild type A2MC2 virus. Infection of primary pulmonary alveolar macrophages (PAMs) also induces interferons. Sequence analysis showed that the A2MC2-P90 has genomic nucleic acid identity of 99.8% to the wild type but has a deletion of 543 nucleotides in nsp2. The deletion occurred in passage 60. The A2MC2-P90 genome has a total of 35 nucleotide variations from the wild type, leading to 26 amino acid differences. Inoculation of three-week-old piglets showed that A2MC2-P90 is avirulent and elicits immune response. Compared with Ingelvac PRRS^® MLV strain, A2MC2-P90 elicits higher virus neutralizing antibodies. The attenuated IFN-inducing A2MC2-P90 should be useful for development of an improved PRRSV vaccine.

Figure 1. A2MC2-P90 induces synthesis of interferons.

(A) Interferon bioassay in Vero cells. Dilutions of cell culture supernatant of MARC-145 cells infected with A2MC2-P90 (A2P90) or A2MC2-P9 (A2P9) were used to treat Vero cells. Treatment with 1000 U IFN-α was included as a control. At 12 h after the treatment, the Vero cells were inoculated with NDV-GFP. At 24 h post-inoculation of NDV, the cells were observed under fluorescence microscopy. (B) Elevation of RIG-I in Vero cells treated with A2MC2-P90 supernatant. The Vero cells were treated with culture supernatant from MARC-145 cells infected with VR-2385 (VR) or A2MC2-P90 (A2) and harvested 24 and 48 h post treatment for immunoblotting. (C) Treatment with A2MC2-P90 supernatant leads to elevation of RIG-I and MDA5 expression in Vero cells detected by real-time PCR. Relative levels of transcripts are shown in folds in comparison to treatment with supernatant from mock-infected MARC-145 cells. Significant difference from treatment with supernatant of mock-infected cells is denoted by “*”, which indicates P < 0.05 and “***” for P < 0.001.

Figure 2. Growth properties of A2MC2-P90 in MARC-145 cells.

(A) Multi-step growth curve of A2MC2-P90 in MARC-145 cells. The cells were inoculated with A2MC2 virus at a multiplicity of infection (MOI) of 0.01, 0.1 or 1 per cell. Inoculation of A2MC2-P9 at an MOI of 0.1 was included as a control. Virus yields at different time points after inoculation were titrated by an immunofluorescence assay. Error bars represent variation of three repeated experiments. “***”Denotes significant difference (P < 0.001) in virus yields between A2MC2-P90 and A2MC2-P9. (B) Plaque assay in MARC-145 cells. The cells were infected with diluted A2MC2-P9 and A2MC2-P90 and overlaid with agarose. Plaques were observed 4 days post-infection and photographed for comparison.

Figure 3. Illustration of sequence variation of A2MC2-P90 (GenBank accession number: KU318406) in comparison to VR-2332 (GenBank accession number: U87392), MLV (GenBank accession number: AF066183) and wild type A2MC2 (GenBank accession number: JQ087873).

The top line indicates the genomic sequence of VR-2332 and the numbers above the line indicate nucleotide positions in the genome. The nucleotide variations in comparison with VR-2332 are indicated by vertical bars. The wide bars indicate the unique nucleotides identified earlier for A2MC2 in comparison with VR-2332 and MLV. A deletion (Del) in A2MC2-P90 from nt2994 to 3536 is indicated. For a list of non-synonymous nucleotide variations in A2MC2-P90 genome compared to wild type A2MC2, please refer to Table 1. For a full list of all nucleotide variations in A2MC2-P90 genome compared to wild type A2MC2, VR-2332 and Ingelvac PRRS^® MLV, please refer to Supplemental Table 1.

Figure 4. Identification of the initial A2MC2 passage that carries the nsp2 deletion in genome.

(A) PCR detection of the nsp2 deletion in A2MC2 passages. The passages P30 to P80 were tested. Wild type A2MC2 (P9) was included as a control. The PCR product from the genome without deletion is 1.262 kb and it is 0.719 kb from the genome with the nsp2 deletion. (B) Identification of the initial A2MC2 passage that has the nsp2 deletion. The passages P60 to P63 were tested.

Figure 5. A2MC2-P90 induces IFN synthesis in PAM cells.

(A) IFN bioassay in CRL2843 cells, in comparison with wild type A2MC2. IFN-α was included as a positive control. (B) Multi-step growth curve in PAM cells. The cells were inoculated with A2MC2-P90 at an MOI of 0.5. Virus yields were titrated on MARC-145 cells. Error bars represent variation of three repeated experiments.

Figure 6. Lung lesions in pigs infected with PRRSV A2MC2-P9, A2MC2-P75, A2MC2-P90 and MLV at 14 days post-infection.

Four pigs from each group were necropsied. Mock-infected pigs (PBS) were included as controls. (A) Average macroscopic lung lesion scores. Error bars represent standard errors of the scores among the four pigs in each group. A2: A2MC2. (B) Average microscopic lung lesion scores. Significant differences between the group of A2MC2-P9-infected pigs and each of the rest groups are denoted by “**”, which indicates P < 0.01.

Figure 7. Serological testing of serum samples from pig studies.

Four pigs from each group were infected. Mock-infected pigs (PBS) were included as controls. (A) ELISA of PRRSV antibodies in serum samples of 35 days post-infection (DPI). The S/P ratio above 0.4 is considered positive. Error bars represent standard errors of the ratios among the four pigs in each group. A2: A2MC2. (B) Virus-neutralization antibody assay against PRRSV VR-2332. The virus neutralization (VN) titers are shown as reciprocals of serum dilutions shown VN activity. Significant differences between the A2MC2 groups and the group of MLV-infected pigs are denoted by *and **, which indicate P < 0.05 and P < 0.01, respectively.