Porcine Circovirus Type 2 Isolates Research Articles

Complete genome characterization and phylogenetic analysis of three distinct buffalo-origin PCV2 isolates from China

Complete genome characterization of porcine circovirus type 2 (PCV2) for bovid origins was still unclear in China. Therefore, in this study, PCV2 full-length genome of buffalo-origin was amplified and analyzed using PCR, DNAStar and MEGA 5.1. Genome size of three distinct PCV2 strains (buffalo1, buffalo2 and buffalo3) was 1767bp (48.56% G+C), 1767bp (48.67% G+C) and 1768bp (48.08% G+C), respectively. At the nucleotide level, their identity varied from 95% to 96% for complete genome, from 97% to 97.8% for ORF1, and from 90.6% to 94.4% for ORF2. At the amino acid level, their identity varied from 98.7% to 99% for ORF1, and from 88% to 94.9% for ORF2. Online Blast analysis showed that buffalo1, buffalo2 and buffalo3 had highest nucleotide identity (varied from 99.77% to 99.83%) with porcine-origin PCV2 strains. Moreover, in the phylogenetic tree, they were divided into three different clusters and belonged to the worldwide accepted genotypes of PCV2b, PCV2c and PCV2a, respectively. To summarize, this study first recorded complete genome information of PCV2 for non-porcine origins in China.

Pigs naturally exposed to porcine circovirus type 2 (PCV2) generate antibody responses capable to neutralise PCV2 isolates of different genotypes and geographic origins

Porcine circovirus type 2 (PCV2) is the essential infectious agent for PCV2-systemic disease (PCV2-SD, formerly known as postweaning multisystemic wasting syndrome) and other pathological conditions. Recent studies indicated antigenic variability amongst different PCV2 isolates and suggested that single amino acid changes within the capsid protein determine differences in the level of neutralization by specific monoclonal antibodies. The objective of the present study was to examine the cross-reactivity of PCV2 antibodies induced in the context of a natural infection against different PCV2 isolates belonging to genotypes PCV2a and PCV2b. Sera taken from several farms from animals of varying health status (PCV2-SD and age-matched healthy pigs and a set of slaughter-aged animals) were assayed for neutralizing activity against four PCV2 isolates from both predominant genotypes (PCV2a and PCV2b) and of differing geographic origins (Europe and North-America). Results showed that most of studied pigs (79 out of 82) contained neutralizing antibodies (NA) able to neutralize all four studied viral strains. Overall, pigs had significantly higher NA titres against PCV2a than against PCV2b (P < 0.001). Accordingly, studied serums were able to better neutralize Burgos390L4 and Stoon-1010 strains (PCV2a) than L-33-Sp-10-54 and MO/S-06 strains (PCV2b) (P < 0.001). No differences between capabilities of seroneutralization of viruses from different geographic origin were observed. Present data suggests that sequence differences between PCV2 isolates translate to functional antigenic differences in viral neutralization in vivo.

Genetic evolution and phylogenetic analysis of porcine circovirus type 2 infections in southern China from 2011 to 2012

Porcine circovirus type 2 (PCV2), the primary causative agent of porcine circovirus-associated diseases (PCVADs), is a serious economic problem for the swine industry worldwide. Three major PCV2 genotypes (PCV2a, PCV2b, and PCV2c), have been identified. To explore the prevalence of different subgroups of PCV2 in southern China, 66 PCV2 isolates collected during 2011–2012 were analyzed. PCV2b was the predominant genotype circulating in southern China from 2011 to 2012. Moreover, subtype 1C was the predominant subtype. Comparisons of the complete ORF2 nucleotide sequence revealed 89.3–100% homology and 87.2–100% amino acid sequence identities. The deletion at position 1042 and two nucleotide substitutions at positions T1035A or T1033C were important for the PCV2 evolution. Base-by-base of ORF2 comparison showed that the PCV2 evolution trace was PCV2a to PCV2b-1A1B to PCV2b-1C. These results contribute to the understanding of PCV2 epidemiology in southern China.

Prevalence and genetic variation of porcine circovirus type 2 in Taiwan from 2001 to 2011

Porcine circovirus type 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) in Taiwanese pig farms. We analyzed the complete genomes of 571 Taiwanese PCV2 isolates in Taiwan from 2001 to 2011 and divided the isolates into 2 distinct genotypes (PCV2a and PCV2b) with 6 clusters (1A, 1B, 1C, 2B, 2D, and 2E). Of the 571 Taiwanese PCV2 isolates, 22.9% (131/571) belonged to PCV2a and 77.1% (440/571) to PCV2b. In this study, PCV2a isolates were the most common in 2001, and then PCV2b isolates became predominate thereafter and widely distributed in pig farms since 2003. Sequence comparisons among the 571 isolates indicated that 89.6–100% had nucleotide identity for complete genome and 87.3–100% for open reading frames 2 (ORF2). The results suggest that a higher genetic variation and shift occurred among PCV2 isolates collected from 2001 to 2011 in Taiwan.

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets

BackgroundPost-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS.MethodsIn the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 107 TCID50 of a virulent PCV2 isolate.ResultsThe vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group.ConclusionsThe obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.

Evaluation of the transmission of porcine circovirus type 2 (PCV-2) genogroups a and b with semen from infected specific-pathogen-free boars

The porcine circovirus type 2 (PCV-2) is associated with several diseases including reproductive failure. This syndrome has been experimentally reproduced twice with two PCV-2 isolates representative of each major PCV-2 genogroup, i.e. PCV-2a and PCV-2b (Cariolet et al., 2002; Rose et al., 2007). In these two previous studies, the sows were infected by intra-uterine inoculation at insemination with 104.3 and 103.18 TCID50 of PCV-2a and PCV-2b, respectively, corresponding to 1.2×1011 and 3×1010 genome copies, respectively.The aim of this present study was to quantify viral shedding in semen from specific-pathogen-free (SPF) boars infected with isolates from the two major PCV-2 genogroups a and b. We studied the transmission of the PCV-2 virus through contaminated semen to SPF sows and their offspring. The four inoculated boars developed sub-clinical PCV-2 infections and PCV-2 genomes were occasionally detected in semen after nasal infection of boars, with up to 1.2×106copies/mL in the sperm-rich fraction. When PCV-2-contaminated semen was inoculated in SPF sows at artificial insemination, the sows and their offspring did not show any signs of PCV-2 infection or PCV-2 antibodies or genomes. In the present study, sows were inoculated with a maximal dose of 1.7×107 viral genome copies, which is lower than the genomic loads (i.e. 1.2×1011 and 3×1010 genome copies) that have been shown to induce reproductive troubles in intra-uterine inoculated sows. Our results together with the previous experiment findings suggest that PCV-2-induced reproductive disorders depend on the infectious dose inoculated to sows by the intra-uterine route.

Genetic variation and phylogenetic analysis of porcine circovirus type 2 infections in central China

Porcine circovirus type 2 (PCV2) infection causes postweaning multisystemic wasting syndrome and porcine circovirus-associated diseases in many regions. A total of 77 sequences, including 31 sampled from Henan province of China, were retrieved from GenBank and subjected to amino acid variation and phylogenetic analyses. The two PCV genotypes prevailing in Henan were PCV-2a and PCV-2b with PCV-2b accounting for 93.5% (29/31) of the Henan isolates. The 31 Henan isolates all shared between 92.7 and 100% sequence similarity. Amino acid variation analysis of the capsid protein revealed that Henan PCV2 strains tended to accumulate more substitutions within epitopic regions-a substitution pattern consistent with host immune system-mediated selection of virus immune escape variants. The analysed PCV sequences carry evidence of at least six unique recombination events. Selective pressure analysis of the relative recombination-free ORF2 sequences of these viruses revealed evidence of sites that are likely evolving in response to host-driven immune pressures-a finding that coupled with information on the prevalent diversity in Henan PCV2 isolates of known immunoreactive genomic loci will aid in future studies aiming to assess the evolutionary responses of PCV2 in China to the widespread deployment of anti-PCV vaccines in the country.

Porcine circovirus type 2 infection before and during an outbreak of postweaning multisystemic wasting syndrome on a pig farm in the UK

The presence of porcine circovirus type 2 (PCV-2) and other pathogens before and during an outbreak of postweaning multisystemic wasting syndrome (PWMS) in pigs is evaluated in this study. At...

Detection and molecular characterization of porcine circovirus type 2 (PCV2) from piglets with exudative epidermitis in Uruguay

Porcine circovirus type 2 (PCV2) is an economically important emerging pathogen associated with distinct syndromes and diseases in swine, collectively known as porcine circovirus associated diseases (PCVAD).The main purpose of this study was to investigate the presence of PCV2 in piglets affected with exudative epidermitis (EE) in Uruguay. In addition we aimed to analyze the phylogenetic relationships of the isolated strains.In June 2011 an outbreak of EE detected in a small herd was reported. Piglets presented skin lesions compatible with EE and symptoms associated with postweaning multisystemic wasting syndrome (PMWS) were also observed. Sera from affected and healthy animals were tested for the presence of viral DNA.Exclusively, diseased piglets were infected with PCV2. Phylogenetic analysis showed that PCV2 isolates belonged to PCV2b genotype.We report the detection and molecular characterization of PCV2 strains for the first time in Uruguay.

Genotypic Change and Phylogenetic Analysis of Porcine Circovirus Type 2 in Taiwanese Pig Herds

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome. Two major PCV2 genotypes, PCV2a and PCV2b, have been identified. To explore the prevalence of different subgroups of PCV2 in Taiwan, 37 PCV2 isolates collected during 2002-2011 were analyzed. The genotypes of the PCV2 isolates collected before 2007 belonged to either PCV2a or PCV2b. However, all of the isolates collected after 2008 were PCV2b. Most of the isolates obtained since 2008 have been classified into a novel genotype within a subgroup of PCV2b based on complete ORF2 sequence analysis. Moreover, analysis of the PCV2 isolates from the same pig farm but from different years revealed that the viruses shifted from a PCV2b genotype to a novel subgroup of the PCV2b genotype. Collectively, PCV2b was the dominant PCV2 genotype in Taiwan currently, and the viruses have shifted into a new emerging subgroup of the PCV2b genotype.

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) in Serbia

Porcine circovirus type 2 (PCV2) is the main causative agent of postweaning multisystemic wasting syndrome (PMWS). To characterize and determine the genetic diversity of PCV2 in the porcine population of Serbia, nucleotide and deduced amino acid sequences of the open reading frame 2 (ORF2) of PCV2 collected from the tissues of pigs that either had died as a result of PMWS or did not exhibit disease symptoms were analyzed. Sequencing and phylogenetic analysis showed considerable diversity among PCV2 ORF2 sequences and the existence of two main PCV2 genotypes, PCV2b and PCV2a, with at least three clusters, 1A/B, 1C and 2D. In order to provide further proof that the 1C strain is circulating in the porcine population, the whole viral genome of one PCV2 isolate was sequenced. Genotyping and phylogenetic analysis using the entire viral genome sequences confirmed that there was a PMWS-associated 1C strain emerging in Serbia. Our analysis also showed that PCV2b is dominant in the porcine population, and that it is exclusively associated with PMWS occurrences in the country. These data constitute a useful basis for further epidemiological studies regarding the heterogeneity of PCV2 strains on the European continent.

First construction of infectious clone for newly emerging mutation porcine circovirus type 2 (PCV2) followed by comparison with PCV2a and PCV2b genotypes in biological characteristics in vitro

BackgroundPorcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem in the swine industry. Different genotypes (PCV2a, PCV2b and PCV2d) of the virus are present in the clinical cases in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. In this study, four strains of different genotypes were isolated, two were ordinary strains and another two were mutation strains, which there are one and two amino acids elongation in the capsid protein (Cap) of PCV2, respectively. Representative strains of different genotypes of the virus were constructed by infectious molecular clone and biological characterization of the rescued viruses were identified in vitro.ResultsFour PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 105.7 TCID50/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.ConclusionsConclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10(-5) ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Genetic Characterization of Porcine Circovirus Type 2 from Pigs with Porcine Circovirus Associated Diseases in Argentina

Porcine circovirus type 2 (PCV-2) has been associated with syndromes grouped by the term porcine circovirus associated diseases (PCVAD). The PCV-2 isolates have been grouped into two major groups or genotypes according to their nucleotide sequence of whole genomes and/or ORF-2: PCV-2b, which have, in turn, been subdivided into three clusters (1A–1C), and PCV-2a, which has been subdivided into five clusters (2A–2E). In the present study, we obtained 16 sequences of PCV-2 from different farms from 2003 to 2008, from animals with confirmatory diagnosis of PCVAD. Since results showed an identity of 99.8% among them, they were grouped within a common cluster 1A-B. This preliminary study suggests a stable circulation of PCV-2b among the Argentinean pig population.

Porcine circovirus-associated disease in weaner pigs in Western Australia

To report the occurrence and pathology of porcine circovirus type 2 (PCV2)-associated disease (PCVAD) of postweaning pigs in two Australian pig herds. Mortality data from two commercial piggeries that experienced higher than normal postweaning illthrift and mortalities were examined. Gross and histopathological examinations were performed on the index cases, and at weekly intervals thereafter for a period of 10 weeks. Specimens were submitted to the laboratory for routine diagnostic testing and for exclusion of porcine reproductive and respiratory syndrome virus (PRRSV). The genomes of two strains of PCV2 isolated during testing were sequenced. Mortality rates in weaned, 5-12-week-old pigs spiked significantly during mid to late 2007. This increase in the mortalities was mainly attributed to salmonella-associated diarrhoea and illthrift. Salmonellosis was diagnosed in 73/110 cases inclusive of both piggeries. Many pigs also had chronic granulomatous lymphadenitis and diffuse histiocytic interstitial pneumonia consistent with PCVAD and associated with varying amounts of PCV2 antigen and inclusion bodies. All samples tested for PRRSV were negative. Sequence analysis of the PCV2 isolates showed strain differences between piggeries. This report describes the first outbreaks of PCVAD in growing pigs in Western Australia (WA) and describes lesions not previously seen in this laboratory. It also describes the first isolation of a PCV2 group 1 virus in WA associated with PCVAD. Although the outbreaks of PCVAD occurred with concurrent salmonellosis, the two diseases were unrelated. Neither of the outbreaks met the Australian case definition for the diagnosis of postweaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV2): genetic variation and newly emerging genotypes in China

BackgroundPorcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem for the swine industry in China. In this study, we investigated the genetic variation of PCV2 in China using strains isolated from 2004-2008. Viruses were isolated from samples collected from pigs with multi-systemic lesions and clinical signs of PMWS from different regions of China, and the genomes of these viruses were sequenced. The assembled sequences were used to define the genotypes of these strains; PCR-RFLP methodology was used to distinguish isolates and capture ELISA was used to demonstrate the antigenic changes resulted from ORF2 gene mutation of the isolates.ResultsWe identified 19 PCV2 isolates, including four newly emerging PCV2 mutant strains. The 19 isolates were designated into three genotypes (PCV2a, PCV2b and PCV2d). PCV2d represented a novel genotype and a shift from PCV2a to PCV2b as the predominant genotype in China was identified. This is the first report of 1766 nt PCV2 harboring a base deletion at other new different positions. Amino acid sequence analysis identified two novel ORF2 mutations (resulting in ORF2 sequences 705 and 708 nt in length) in three deletion strains (1766 nt) and one strain with a genome 1767 nt in length. Finding of two amino acids elongation of the ORF2-encoded Cap protein is firstly observed among PCV2 strains all over the world. The isolates were distinguished into different genotypes by PCR-RFLP methodology and antigenic changes were present in Cap protein of mutation isolates by capture ELISA.ConclusionsThe results of this study provide evidence that PCV2 is undergoing constant genetic variation and that the predominant strain in China as well as the antigenic situation has changed in recent years. Furthermore, the PCR-RFLP method presented here may be useful for the differential identification of PCV2 strains in future studies.

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates ( n = 10) into two known genotypes, PCV-2a ( n = 1) and PCV-2b ( n = 9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.

Differential Effects of Porcine Circovirus Type 2 (PCV2) Vaccination on PCV2 Genotypes at Japanese Pig Farms

Infection of pigs with porcine circovirus type 2 (PCV2) causes a variety of disorders collectively referred to as porcine circovirus associated diseases (PCVADs). PCV2 isolates can be classified into two major types: PCV2a and PCV2b. In the present study, effects of vaccination on antibody titers in sera, PCV2 viremia, and shedding of PCV2 in feces were studied on Japanese commercial pig farms where vaccination of piglets against PCV2 was performed using commercially available vaccines. The effectiveness of vaccination against various PCV2 genotypes was also assessed. Among the 16 farms studied, 10 and 6 had been infected with PCV2a and PCV2b, respectively. PCV2a was further subdivided into PCV2a-1 and PCV2a-2. PCV2a-1 and PCV2a-2 prevailed on 6 and 4 farms, respectively, among the 10 farms infected with PCV2a. The PCV2 vaccines were effective in reducing PCV2 infection on commercial pig farms. Mean mortality rates were significantly decreased over 8 months after the start of the PCV2 vaccination program as compared to those before the start of the PCV2 vaccination program on farms infected with PCV2a-2 (20.8% vs. 12.1%) and PCV2b (26.5% vs. 13.7%). On the farms with PCV2a-1 infected pigs, there was no significant difference in the mean mortality rate before versus after the start of the vaccination program (14.7% vs. 14.1%). Mortality rate reduction with the PCV2 vaccination might depend on the genetic types of PCV2.

Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009: genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). In the present study, we obtained sequences of 31 PCV2 isolates from different farms of 11 provinces of eastern China and analyzed the genetic characterization of 136 eastern China-derivate PCV2 isolated during 2001-2009. The results showed that these PCV2 isolates could be divided into two groups, PCV2b (108 of 1A/1B, 19 of 1C) and PCV2a (1 of 2A, 2 of 2D, 6 of 2E). Among the 9 PCV2a isolates, eight were found before the year 2005. Meanwhile, three major heterogenic regions were observed in amino acid positions 53-91, 121-151, and 190-210; a few specific substitution patterns were found in each subgroup and several variant or conserved epitopes were also observed in the Cap protein. It indicated that PCV2b predominated in PMWS occurrences in eastern China and a few recombinants of different genotypes existed in nature.

Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10 5.55 TCID 50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.