Two Amino Acid Mutations in the Capsid Protein of Type 2 Porcine Circovirus (PCV2) Enhanced PCV2 Replication In Vitro and Attenuated the Virus In Vivo

Abstract

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 10 4.9 50% tissue culture infective doses (TCID 50 ) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 10 4.9 TCID 50 of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (P < 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (P ‫؍‬ 0.0032 and P ‫؍‬ 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.

Evidence of Breed-dependent Differences in Susceptibility to Porcine Circovirus Type-2-associated Disease and Lesions

“…Previously, singular PCV2 infection with the same isolate at a similar cell culture passage and in approximately the same dose as used in the purebred pigs of this current study did not result in expression of clinical disease or presence of microscopic lesions consistent with PMWS in 264 crossbred pigs that were part of nine studies. [21][22][23][24]35,[52][53][54][55] This further supports our conclusion that the purebred Landrace pigs used in this study were more susceptible to PCV2-associated diseases.…”

“…To better ensure infection of all pigs at the time of inoculation, each pig received 1 ml of the PCV2 inoculum intranasally and 0.5 ml intramuscularly in the right side of the neck as described. 22,23 On the day of PCV2 inoculation, the pigs were 5-8 weeks old. Mean 6 SE age was 43.5 6 1.8 days for the Duroc, 48.6 6 2.0 days for the Landrace, and 46.0 6 2.0 days for the Large White pigs.…”

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

“…The established real-time PCR for PCV2 DNA quantitation were found to be in the (3~11)-log10 dynamic range with excellent linearity (Fig.3). This compares favorably with other methods described previously: a competitive PCR (Liu et al, 2000) or real-time PCR based on SYBR Green I (Fenaux et al, 2004;Gilpin et al, 2003). The former have given linear ranges of 4-to 6-log10, whereas the latter have not presented the range.…”

“…But all of them used a hybridization probe method in the TaqMan system and are mostly applied on serum samples and the primers were mostly based on their local PCV2 strains. Although some papers (Gilpin et al, 2003;Fenaux et al, 2004) used an SYBR Green I to quantify the PCV2 DNA, but a regression lines between the C T values and the input concentrations were not given.…”

Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2

“…Recent studies have suggested that ORF2-and ORF3-related proteins play important roles in the pathogenesis of PCV2. A two-aminoacid (aa) mutation or nucleotide deficiency in the capsid protein can alter the virulence of PCV2 (17,18), while ORF3-deficient mutant PCV2 may be attenuated in vivo (19,20). However, contradictory results have also been found.…”