Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2

Abstract

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Increasing evidence indicates that a variant strain of porcine circovirus (PCV), designated type 2 PCV (PCV-2), is responsible for PMWS. To determine the extent of genetic heterogeneity of PCV-2 isolates, the complete genomes of six PCV-2 isolates from different regions of North America were amplified by PCR and sequenced. Sequence and phylogenetic analyses confirmed that two distinct genotypes of PCV exist: nonpathogenic genotype PCV-1 and PMWS-associated genotype PCV-2. However, within the PCV-2 genotype, several minor branches that have been identified appear to be associated with geographic origins. The genomic sequences of two French PCV-2 isolates diverge the most from those of other PCV-2 isolates and form a distinct branch. Other minor but distinguishable branches have also been identified for a Taiwan PCV-2 isolate and two of the Canadian PCV-2 isolates. All the U.S. PCV-2 isolates are closely related, but the Canadian isolates vary, to some extent, in their genomic sequences. The data from this study indicate that although the genome of PCV-2 is generally stable among different isolates, PCV-2 isolates from different geographic regions vary in their genomic sequences. This variation may have important implications for PCV-2 diagnosis and research. On the basis of genetic analyses of available PCV strains, a universal PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and differentiate between infections with PCV-1 and PCV-2. This PCR-RFLP assay should be useful for studying the pathogenesis of PCV-2, for detecting PCV-2 infection in pigs from different geographic regions, and for screening donor pigs for use in xenotransplantation.

FIG. 1

Genome organization of PCV-2. The origin of replication (0), the putative capsid gene (ORF2), the PCR-RFLP fragment, and the two overlapping PCR fragments used to determine the complete genome of PCV-2 are indicated in the circular map. The relative positions of the oligonucleotide primers used in this study are indicated by arrows with respective numbers: 1, CV1; 2, CV2; 3, CV3; 4, CV4; 5, CV1-1; 6, CV1-2; 7, CV2-1; 8, CV2-2; 9, CV3-1; 10, CV3-2; 11, CV4-1; 12, CV4-2; 13, MCV1; 14, MCV2. The sequences and designations of these primers are listed in Table 2.

FIG. 2

Nucleotide sequence alignment of the region amplified in the PCR-RFLP assay. The regions from which the consensus PCR primers (MCV1 and MCV2) were chosen are underlined. The unique NcoI restriction enzyme site that is present in all PCV-2 isolates is indicated by asterisks. The sequence of PCV-2 isolate 26606 from this study is shown on top, and only differences from that sequence are indicated for the other isolates. The sequences used in the alignment are cited in the text.

FIG. 3

Amino acid sequence alignment of the putative capsid protein (ORF2) of PCV-1 and PCV-2 isolates sequenced thus far. Deletions are indicated by hyphens. Amino acid sequence differences are indicated with asterisks above the alignment. The sequences used in the alignment are cited in the text.

FIG. 4

Phylogenetic tree based on the complete genomic nucleotide sequences of all PCV isolates. The tree was constructed with the aid of the PAUP program. Branch-and-bound searching and midpoint rooting options were used to produce a consensus tree. A scale bar that represents the numbers of character-state changes is shown. Branch lengths are proportional to the numbers of character-state changes. The geographic locations of the isolates are also indicated with the usual state abbreviations and the following country abbreviations: CAN, Canada; TAI, Taiwan; FR, France; GER, Germany; IRE, Ireland. BOV, bovine.

FIG. 5

Detection and differentiation of PCV infections by a PCR-RFLP assay. (A) Results of PCR amplification of a 243-bp fragment from tissue samples that contained PCV isolates (both PCV-1 and PCV-2) but not from a negative control liver tissue sample (lane 1). (B) Results of RFLP analysis of the PCR products. Lane L, 50-bp DNA ladder; lane 1, a sample of liver tissue from a control specific-pathogen-free pig; lanes 2 to 11, tissue samples from 10 pigs with PMWS, respectively; lane 12, PK15 cells containing PCV-1; lane 13, a sample containing both PCV-1 and PCV-2. The expected PCR fragment (A) and three RFLP fragments of 243, 168, and 75 bp, respectively (B), are indicated with arrows.