Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

Evaluation of the efficacy of an attenuated live vaccine based on virulent porcine reproductive and respiratory syndrome virus 2 in young pigs

“…We have previously reported the virulence, immunogenicity, and genetic characteristics of the CA-2-MP120 virus strain that was obtained by sequentially passing the virulent Korean PRRSV-2 lineage 1 strain CA-2 through the African green monkey kidney epithelial cell line MARC-145 (100 passages) and cultured porcine alveolar macrophage (PAM) (additional 20 passages) cells [8]. In this present study, we assessed the efficacy of CA-2-MP120 as a genetically stable, live-attenuated vaccine strain against PRRSV-2 lineage 2 in host animals.…”

“…After one week of acclimatization, the pigs in *Corresponding author Tel: +82-53-950-7365, Fax: +82-53-955-5522 E-mails: changhee@knu.ac.kr (C Lee), sykang@cbu.ac.kr (SY Kang) Group 1 (n = 6) were immunized intramuscularly with 2 mL of CA-2-MP120 at a 50% tissue culture infective dose per mL (TCID 50 /mL) of 10 6.0 , whereas the challenge control (Group 2; n = 6) and strictly negative control (Group 3; n = 4) received only cell culture medium as a placebo. At 57 days post-immunization (dpi), each piglet (except the negative control pigs from Group 3) was challenged intramuscularly with 2 mL (10 6.0 TCID 50 /mL) of the CA-2-P20 strain [2,8]. Clinical parameters, such as appetite, activity level, and respiratory signs were monitored daily throughout the study.…”

“…The body weight and rectal temperature were measured at 0, 3, 6, 8, 14, 21, 28, 35, 54, 61, and 71 dpi, and blood as well as oral, nasal, and fecal swabs were collected at the same time points. All the animals were euthanized at 14 days post-challenge (dpc) for post-mortem examination as described previously [8].…”

“…To determine if the animals were viremic during vaccination and challenge, PRRSV levels in the sera of pigs were determined by virus titration using MARC-145 or PAM-KNU cells as described previously [2,8]. PRRSV-specific antibodies in serum samples collected from the infected and controls animals were determined using a commercial PRRSV antibody ELISA kit (HerdCheck PRRS X3; IDEXX Laboratories, USA) according to the manufacturer's instructions and a serum neutralization test on PAM-KNU cells using a homogeneous CA-2 strain as previously described [2,8].…”