Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2

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

“…PAM-KNU (an immortalized PAM cell line) and MARC-145 cells were cultured and maintained as described previously [ 22 27 ]. The previously reported PRRSV CA-2 strain was plaque-purified and propagated in MARC-145 or PAM-pCD163 cells [ 6 22 ]. The high-passage derivative of CA-2, namely CA-2-P100, was obtained by continuous passaging for 100 times in MARC-145 cells, as previously described [ 22 ].…”

“…The previously reported PRRSV CA-2 strain was plaque-purified and propagated in MARC-145 or PAM-pCD163 cells [ 6 22 ]. The high-passage derivative of CA-2, namely CA-2-P100, was obtained by continuous passaging for 100 times in MARC-145 cells, as previously described [ 22 ]. CA-2-P100 was serially passaged in PAM-KNU cells as described elsewhere [ 23 27 ].…”

“…Pigs were weighed at 0, 3, 6, 8, 14, 21, 28, and 35 days post-inoculation (dpi), and blood samples were collected at the same times. All animals were necropsied at 35 dpi and subjected to post-mortem examinations as described previously [ 10 22 24 ].…”

“…PRRSV levels in serum samples were assessed by virus titration using MARC-145 or PAM-pCD163 cells as described previously [ 6 22 ]. PRRSV-specific antibodies were detected by using a commercial PRRSV antibody enzyme-linked immunosorbent assay (ELISA) kit (HerdChek PRRS X3; IDEXX Laboratories, USA), according to the manufacturer's protocols, and a serum neutralization test as described previously [ 22 ].…”

“…We found that strain CA-2-P100 (100th passage of CA-2) exhibited an attenuated phenotype in inoculated pigs and had numerous aa mutations distributed throughout its genome. However, some pigs challenged with CA-2-P100 remained viremia-negative and seronegative to PRRSV throughout the trial, implying that the virus might be over-attenuated [ 22 ]. Since PRRSV is a pathogenic macrophage-tropic arterivirus of swine, sequentially passaging PRRSV over 100 times in a non-host cell line may have caused it to lose its tropism to porcine alveolar macrophages (PAMs), which are targeted by the virus during infection in the natural host.…”