Attenuated Porcine Reproductive and Respiratory Syndrome Virus Regains Its Fatal Virulence by Serial Passaging in Pigs or Porcine Alveolar Macrophages To Increase Its Adaptation to Target Cells

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a globally important disease threatening the pork industry, and modified live-virus (MLV) vaccines are widely used for its prevention. However, PRRS MLV shows high potential for reversion to virulence, leading to a major concern about its safety. Yet the revertant mechanism is still poorly understood. Here, attenuated virus JXwn06-P80, derived from the highly pathogenic PRRS virus (PRRSV) strain JXwn06 by serial passaging in MARC-145 cells, was reversely passaged in pigs through intranasal inoculation to mimic natural infection for 13 rounds, and the pathogenicity of viruses at the 3rd, 5th, 9th, 10th, and 11th passages was evaluated in pigs. From the 9th passage, the viruses caused mortality, which was related to their increased adaptability and replication efficiency (100 times higher than those of JXwn06-P80) in porcine alveolar macrophage (PAM) target cells. Similarly, JXwn06-P80 could also regain fatal virulence through reverse passage in PAMs for 25 or more passages, indicating that the increased adaptability in PAMs directly contributes to its regained fatal virulence. Next, the full-genome sequences were analyzed to explore the genetic evolutionary processes during adaptation both in vivo and in vitro. Finally, by a reverse genetic operation, four reverse mutation sites, NSP12-W121R, ORF2b (open reading frame 2b)-H9D, ORF5-H15L, and ORF5-V189L, were finally identified to partially contribute to the ability of the virus to adapt to PAMs, which may be related to virulence reversion during reverse passage. These findings provided direct scientific evidence for the virulence reversion of PRRS MLV and provided valuable clues for exploring its molecular mechanism. IMPORTANCE Reversion to virulence of a live attenuated vaccine is a public concern; however, direct scientific evidence is limited, and the mechanism is still poorly understood. Here, we present direct evidence for the reversion to virulence of PRRS MLV after serial passaging in pigs or target cells and found a correlation between virulence reversion and increased replication fitness in primary PAMs. The genetic evolutionary process during adaptation will provide valuable clues for exploring the molecular mechanism of PRRS MLV virulence reversion and offer important implications for understanding the reversion mechanisms of other vaccines.

Keywords: attenuated strain; mutation analysis; porcine reproductive and respiratory syndrome virus; replication fitness; reverse passage; virulence reversion; whole genome.

FIG 1

Clinical scores, viremia, and PRRSV-specific antibody kinetics of pigs inoculated with different PRRSV strains passaged in vivo. (A to D) Rectal temperatures (A and B), average clinical scores (C), and average daily weight gains (D) of the inoculated pigs. Clinical scoring consisted of the gross clinical score (GCS), respiratory clinical score (RCS), and nervous sign score (NSS). Total scores for each pig were the sum of the GCS, RCS, and NSS values, ranging from 0 to 15. A score of 20 was given when a pig died. (E) Viral loads in the sera of inoculated pigs on different days after inoculation. Virus titers were determined by a microtitration infectivity assay in primary PAMs. (F) The PRRSV-specific antibody kinetics of pigs were detected by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit. The antibody level is expressed as the sample value/positive value (S/P) ratio, and a ratio of ≥0.4 is regarded as seroconversion. The data are shown as means ± SD (error bars). Statistical differences are labeled according to two-way ANOVA followed by a Bonferroni posttest. Asterisks of different colors (according to the colors of lines or bars for that group in the key) indicate a significant difference between the in vivo-passaged virus group and the JXwn06-P80 group for the average clinical scores, ADWGs, viral loads, or S/P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 2

Survival curves of pigs inoculated with different PRRSV strains passaged in vivo. The time of pig death (A) and mortality (B) for each group are shown.

FIG 3

Lung lesions and immunohistochemistry examinations of pigs inoculated with different PRRSV strains passaged in vivo. (A, D, G, and J) Representative pictures of gross lung lesions and average scores of gross lung lesions from dead pigs during the experiment (A and G) and euthanized pigs at the end of the experiment (D and J). Gross lung lesions were graded based on the percentage of the lung area affected by pneumonia. (B, E, H, and K) Microscopic lung lesions stained with hematoxylin and eosin (H&E) and average microscopic lung lesion scores from dead pigs during the experiment (B and H) and euthanized pigs at the end of the experiment (E and K). Microscopic lesions were scored based on the severity of interstitial pneumonia. Solid arrows indicate thickening of the interlobular septa or infiltration of inflammatory cells around the bronchioles. Hollow triangles indicate inflammatory cells, necrotic debris, and exfoliated epithelial cells that infiltrated the bronchioles. Solid triangles indicate hemorrhage or infiltration of inflammatory cells within alveolar septa and alveolar spaces. (C, F, I, and L) Immunohistochemistry (IHC) examinations and average scores for PRRSV antigen in the lungs of dead pigs during the experiment (C and I) and euthanized pigs at the end of the experiment (F and L). A monoclonal antibody (SDOW17) specific for the N protein of PRRSV was used for IHC examination. The macrophages stained intensely dark brown, representing PRRSV antigen-positive cells, and the numbers of positive cells in the lungs were scored. Hollow arrows indicate positive signals in macrophages within or around the alveolus and bronchus.

FIG 4

Growth kinetics of the in vivo reversely passaged PRRSV strains in primary PAMs. PIG-R-3-, PIG-R-5-, PIG-R-9-, PIG-R-10-, PIG-R-11-, and JXwn06-P80-infected primary PAMs at an MOI of 0.01 are shown. Virus titers were determined by microtitration infectivity assays in primary PAMs. The data are shown as means ± standard deviations (SD) (error bars) from three independent experiments. Statistical differences are labeled according to two-way ANOVA followed by a Bonferroni posttest. Asterisks of different colors indicate a significant difference between the in vivo-passaged virus group and the JXwn06-P80 group (*, P < 0.05; ***, P < 0.001).

FIG 5

Growth kinetics of the in vitro reversely passaged PRRSV strains in primary PAMs. PAM-R-25-, PAM-R-60-, and JXwn06-P80-infected primary PAMs at an MOI of 0.01 are shown. Virus titers were determined by microtitration infectivity assays in primary PAMs. The data are shown as means ± SD (error bars) from three independent experiments. Statistical differences are labeled according to two-way ANOVA followed by a Bonferroni posttest. Asterisks of different colors indicate a significant difference between the in vivo-passaged virus group and the JXwn06-P80 group (***, P < 0.001).

FIG 6

Clinical scores, viremia, and PRRSV-specific antibody kinetics of pigs inoculated with different PRRSV strains passaged in vitro. (A to C) Rectal temperatures (A), average clinical scores (B), and average daily weight gains (C) of the inoculated pigs. (D) Serum viral loads in pigs inoculated with the in vitro reversely passaged PRRSV strains. Virus titers were determined by a microtitration infectivity assay in primary PAMs. (E) Serum antibody kinetics of pigs in different groups. The data are shown as the means ± standard deviations (SD) (error bars), except for only one pig left in the JXwn06-infected group at 21 dpi. Asterisks of different colors indicate a significant difference between the in vitro-passaged virus group and the JXwn06-P80 group for the average clinical scores, ADWGs, viral loads, or S/P values (*, P < 0.05; ***, P < 0.001).

FIG 7

Survival curves of pigs inoculated with different PRRSV strains passaged in vitro. The time of pig death (A) and mortality (B) for each group are shown.

FIG 8

Lung lesions and immunohistochemistry examinations of pigs inoculated with different PRRSV strains passaged in vitro. (A, D, G, and J) Representative pictures of gross lung lesions and average scores of gross lung lesions from dead pigs during the experiment (A and G) and euthanized pigs at the end of the experiment (D and J). Gross lung lesions were graded based on the percentage of the lung area affected by pneumonia. (B, E, H, and K) Microscopic lung lesions stained with H&E and average microscopic lung lesion scores for dead pigs during the experiment (B and H) and euthanized pigs at the end of the experiment (E and K). Microscopic lesions were scored based on the severity of interstitial pneumonia. Solid arrows indicate thickening of the interlobular septa or infiltration of inflammatory cells around the bronchioles. Hollow triangles indicate inflammatory cells, necrotic debris, and exfoliated epithelial cells infiltrating the bronchioles. Solid triangles indicate hemorrhage or infiltration of inflammatory cells within alveolar septa and alveolar spaces. (C, F, I, and L) IHC examinations and mean scores for PRRSV antigen in the lungs of dead pigs during the experiment (C and I) and euthanized pigs at the end of the experiment (F and L) in each group. Hollow arrows indicate positive signals in macrophages within or around the alveolus and bronchus. Statistical differences are labeled according to a two-tailed untailed t test with Welch’s correction. Asterisks in different colors (according to the colors of lines or bars for that group in the key) indicate a significant difference between the in vitro reversely passaged virus-infected group and the JXwn06-P80-infected group (**, P < 0.01).

FIG 9

Growth kinetics of the rescued virus with reverse mutations in primary PAMs. (A) Strategy for the construction of the full-length cDNA clone of JXwn06-P80. Capital letters (A, B, C, and D) represent four fragments amplified from the JXwn06-P80 genome, with unique restriction enzyme cleavage sites in overlapping regions. SwaI was introduced into the 5′ end of fragment A, PacI was introduced into the 3′ end of fragment D, and BstBI was introduced between fragments A and B. Four fragments were assembled into the vector pWSK29-CMV-MCS-HDV-SV40 in the order D, C, B, and A. The full-length infectious clone plasmid was named pCMV-JXP80. HDVRz, hepatitis delta virus ribozyme. (B) Growth kinetics of the rescued virus with reverse mutations in primary PAMs. The rescued viruses or the attenuated JXwn06-P80 virus infected primary PAMs at an MOI of 0.01. Virus titers at different time points were determined by microtitration infectivity assays in primary PAMs. The data are shown as means ± SD (error bars) from three independent experiments. Statistical differences are labeled according to two-way ANOVA followed by a Bonferroni posttest. Asterisks indicate a significant difference in the virus titers between RvJXP80-4BM and JXwn06-P80 (***, P < 0.001).