PCV2 Isolates Research Articles

Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the U.S. concurrently infected with PPV2

Porcine circovirus (PCV) type 2b (PCV2b) emerged in North America in 2005–2006. During May of 2012, PCVAD occurred in 10–18-week-old pigs in two farms within a production system that routinely vaccinated against PCV2. Both farms received replacement gilts from the same multiplier. A mutant PCV2b strain not previously present in North America was identified. The strain was found to be 99.9% identical to a recently described mutant PCV2 isolate reported in China in 2010 and thought to be more virulent than classical PCV2a or PCV2b strains. It is possible that the current PCV2a-based commercial vaccines are not fully protective against this new strain. In addition, emerging porcine parvovirus type 2 (PPV2) was detected in 55% of the serum samples (73/132), perhaps implying that PPV2 could be a cofactor in cases of PCVAD.

Genetic characterization of Porcine Circovirus 2 found in Malaysia

BackgroundPorcine circovirus type 2 is the primary etiological agent associated with a group of complex multi-factorial diseases classified as Porcine Circovirus Associated Diseases (PCVAD). Sporadic cases reported in Malaysia in 2007 caused major economic losses to the 2.2 billion Malaysian ringgit (MYR) (approximately 0.7 billion US dollar) swine industry. The objective of the present study was to determine the association between the presence of PCV2 and occurrences of PCVAD.ResultsThis study showed that 37 out of 42 farms sampled were positive for PCV2 using PCR screening. Thirteen whole genome of PCV2 isolates from pigs with typical PCVAD symptoms were successfully sequenced. These isolates shared 98.3-99.2% similarities with sequences of isolates from the Netherlands. All thirteen isolates fell into the same clade as PCV2b isolates from other countries. Amino acid sequence analysis of the putative capsid protein (ORF2) of the PCV2 revealed that there are three clusters found in Malaysia, namely cluster 1C and 1A/1B. Of interest, three of the isolates (isolates Mal 005, Mal 006 and Mal 010) had a proline substitution for arginine or isoleucine encoded at nt. position 88-89. Eight of the isolates had mutations at the C terminus of the putative capsid protein suggestive of higher pathogenicity which may account for the high reports of PCVAD clinical symptoms in 2007.ConclusionPhylogenetic study suggests that there may be a link between movements of animals by import of breeders into the country being the route of entry of the virus. While it is not possible to eradicate the virus from commercial pigs, the swine industry in Malaysia can be safeguarded by control measures implemented throughout the country. These measures should include improved biosecurity, disease surveillance; vaccination as well as enforcement of regulations formulated to control and prevent the spread of this disease on a national scale.

A candidate inactivated chimeric vaccine PCV1-2 constructed based on PCV1 and PCV2 isolates originating in China and its evaluation in conventional pigs in regard to protective efficacy against PCV2 infection

A chimeric PCV1-2 clone containing the PCV2 capsid gene cloned into the backbone of the nonpathogenic PCV1 genome was recently generated based on PCV2 and PCV1 strains isolated in China. The efficacy of this available candidate inactivated vaccine was evaluated by subjecting conventional pigs to intramuscular immunization with the inactivated chimeric PCV1-2 virus, followed by challenge with wild-type PCV2 strain. By 35 days post-vaccination (DPV), all vaccinated pigs had developed seroconversion, having high indirect immunofluorescence assay (IFA) titers of antibody and neutralizing antibody against PCV2. By 21 days post-challenge, gross and microscopic lesions of lymph nodes and lungs in non-vaccinated but challenged pigs were significantly more severe than those found in the vaccinated group. PCV2 viral copy loads detected in the tracheobronchial lymph nodes or serum samples of vaccinated pigs were significantly smaller than those in non-vaccinated but challenged pigs (P ≤ 0.05). The results suggest that inactivated PCV1-2 is effective in inducing protective immunity against PCV2 infection.

Genetic characterization of porcine circovirus type 2 in piglets from PMWS-affected and -negative farms in Thailand

Porcine circovirus type 2 (PCV2) is the major swine pathogen associated with Porcine circovirus associated disease (PCVAD) including post-weaning multisystemic wasting syndrome (PMWS). Currently, there are 4 subtypes of PCV2 (PCV2a, b, c and d) and some epidemiological evidences demonstrated that virulence of PCV2 may relate to its subtypes. Recently, PMWS was observed more frequently in swine farms in Thailand; however, the information regarding to PCV2 subtype involved was limited. Therefore, this study was aimed to determine the association between occurrence of PMWS and PCV2 subtypes as well as genetically characterize PCV2 in Thailand. PCV2 DNA was isolated from faecal swabs and whole blood of piglets from PMWS-affected and -negative farms. The full length ORF2 sequences were compared using multiple alignment. The results showed that PCV2 DNA was detected more frequently in PMWS-affected farms. The nucleotide identities of the ORF2 from 9 PCV2 isolates representing each PMWS-affected farm and one from the negative farm ranged from 92.4 to 99.5% suggesting that there is some genetic variation of PCV2 in Thai swine. The 10 PCV2 isolates were classified into 2 clusters, in which the 7 isolates from PMWS-positive farms were in PCV2b cluster 1 A/B. The remaining isolates were separated in the new subtype called PCV2e. The results suggest the presence of new PCV2 subtypes in addition to PCV2a and PCV2b in Asian swine population. However, correlation between subtypes and virulence of PCV2 infection is not conclusive due to limited number of the PCV2 sequences from PMWS negative farms.

Genetic variation analysis of Chinese strains of porcine circovirus type 2

Forty Chinese PCV2 strains collected between 2004 and 2008 were sequenced and their genetic variations were analyzed together with nine previous PCV2 isolates. Phylogenetic analysis indicated that these Chinese PCV2 strains could be divided into four genotypes (PCV-2a, PCV-2b, PCV-2d and PCV-2e), and the genotype PCV-2c defined in Denmark was not found. PCV-2d and PCV-2e were two genotypes firstly determined in our study. Variation analysis of amino acids of capsid protein revealed that Chinese PCV2 strains clustered within PCV-2d had four amino acid marker positions and the isolates within PCV-2e had seven unique amino acid mutations. Our analysis also showed that PCV-2b became dominating in China in recent years. These data contribute to the understanding of PCV2 molecular epidemiology.

Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus Type 2

Type 2 porcine circovirus (PCV2) is associated with post-weaning multisystemic wasting syndrome in pigs. In this study, three monoclonal antibodies (mAbs) against the capsid protein (Cap) of PCV2, eight mAbs to Cap of type 1 porcine circovirus (PCV1) and five mAbs specific for Cap of both PCV1 and PCV2, were generated and used to finely map the antigenic sites of PCV1 and PCV2, and to identify the antigenic phenotype of PCV2 with different length of genome. Five linear B-cell epitopes, including the residues 231–233 and 195–202 specific for PCV2, residues 92–103 specific for PCV1, and residues 156–162 and 175–192 shared between PCV1 and PCV2, were finely defined with synthetic peptides, and the critical residue in epitope 231–233 and 156–162 was located at residues 233 ( 233Proline) and 156 ( 156Tyrosine), respectively. The conformational epitopes recognized by mAbs with neutralizing activity against both PCV1 and PCV2 were detected in transfected PK-15 and the residues 231–233 also participated in the formation of conformational epitopes. Analysis of antigenic diversity on these epitopes exhibited three antigenic phenotypes of PCV2, 1766PCV2, 1767PCV2 and 1768PCV2 using mAbs. The results from this study first demonstrated the different antigenic phenotype between PCV2 isolates.

Examination for a viral co-factor in postweaning multisystemic wasting syndrome (PMWS)

In order to test the hypothesis that a putative co-factor for the development of postweaning multisystemic wasting syndrome (PMWS) in pigs could be of viral origin, we performed extensive virological examinations on organ material from pigs diagnosed with PMWS originating from within a Danish PMWS-transmission study. Virus isolation attempts were carried out on a large panel of different cell types including primary pig kidney cells and lung macrophages, primary rabbit kidney cells and seven established cell lines (MARC-145, ST117, PK15, BHK21, HeLa, Vero, and MDCK). Although these represent cells with susceptibility to a wide range of known viruses, the results did not provide evidence for a specific virus other than PCV2 contributing to the development of PMWS. Furthermore, in order to test whether specific genotypes of PCV2 may trigger the switch from PCV2 infection to clinical disease, we compared complete DNA genome sequences of PCV2 derived from PMWS-positive as well as PMWS-negative pigs. On the basis of the DNA sequences, the PCV2 isolates were divided into two groups. Group 1 consisting of one isolate originating from a herd unaffected by PMWS, with group 2 consisting of nine isolates originating from four PMWS-affected herds, four PMWS-positive pigs plus one unaffected herd. The PCV2 genomes from the two groups showed 95.5% identity. Alignment analyses of the sequences encoding the replicase and capsid protein from group 1 and group 2 PCV2 isolates showed two amino acid differences encoded in the replicase protein, while 19 amino acid differences were predicted among the capsid protein sequences. The PCV2 DNA sequence analysis supports recent observations from studies in USA as well as Europe, which suggest that strain variations may influence the clinical outcome of PCV2 infection.

First description of postweaning multisystemic wasting syndrome (PMWS) in wild boar ( Sus scrofa ) in Croatia and phylogenetic analysis of partial PCV2 sequences

This report describes the first case of postweaning multisystemic wasting syndrome (PMWS) in wild boar in Croatia. During the winter season of 2004, eight wild young piglets (of approximately 2 to 5 months of age) were found dead in a fenced hunting area. Polymerase chain reaction (PCR) was carried out on mesenteric lymph nodes and all animals yielded positive results. In one of these animals diagnosis of PMWS was established based on the three key diagnostic criteria including the clinical manifestation, moderate lymphoid lesions consisting of lymphocyte depletion and granulomatous inflammation, and detection of the presence of PCV2 genome within the lymphoid lesions by in situ hybridisation (ISH). Three additional wild piglets had also mild PMWS-like lesions and a low amount of PCV2 was also found. No PMWS-like lesions or PCV2 genome were detected in the rest of the wild piglets studied. Three PCR-positive isolates were partially sequenced, which confirmed the diagnosis of PCV2 and demonstrated that the three sequences were genetically identical. The phylogenetic analysis of a representative PCV2 isolate indicated that its sequence (DQ875444) is grouped in a separate branch with Hungarian isolate (AY256460) and differs from any of the annotated sequences.

Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2

Post-weaning multisystemic wating syndrome (PMWS) associated with PCV2 was one of the most costly diseases currently faced by the swine industry. In order to develop a vaccine to control this disease, we previously constructed a recombinant adenovirus expressing the capsid of PCV2. Here, we examined the protection of swine against PMWS by the recombinant adenovirus. Eighteen 32-day-old pigs were assigned to three groups each with six. Group 1 was vaccinated subcutaneously with rAd-Cap and boosted 2 weeks later. Thirty-seven days after first vaccination, Groups 1 and 2 were oronasally challenged with virulent PCV2 isolate, 4 and 7 days later, intramuscularly exposed to keyhole limpet hemocyanin (KLH). Group 3 remained unchallenged but with KLH. The results showed that high level of PCV2-specific ELISA antibody and neutralizing antibody could be induced at 37 days after first vaccination. After challenge, pigs in vaccinated group had no clearly clinical signs, although some of them had increased rectal temperatures (≥40 °C) for short time. The pyrexic phase in vaccinated group was significantly lighter than that in challenge-control group ( P < 0.05). The relative daily weight gain in vaccinated-challenged group was similar to that in empty control group. But it was significantly high compared to the challenge-control group ( P < 0.05). Mean while the pathological lesions and virema presented in vaccinated group were milder than those in control group. It indicated that the recombinant adenovirus was able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PCV2 challenge, even though it could not provide complete virological protection. The recombinant adenovirus might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.

Serological Investigation and Genomic Characterization of PCV2 Isolates from Different Geographic Regions of Zhejiang Province in China

Sera collected from 46 swine farms in Zhejiang province were evaluated for the presence of antibodies to PCV2 using an indirect-fluorescent antibody procedure. In addition PCV2 isolated from superficial inguinal lymph node samples collected from 40-to 90-day-old pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) using the PK-15 cell line were sequenced and compared. Overall seroprevalence of PCV2 antibody averaged 58.34% for all samples. Breakdown of serology by groups was as follows: 59.38% for sows, 57.41% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalence of Landrace sows was higher than that of Yorkshire and Duroc sows, but non-significant (p > 0.05). Serological analysis also showed that seroprevalence of PCV2 antibody was a negative correlation to that of PRRSV antibody. The complete genomes of five PCV2 isolates identified in the herds with PMWS consisted of 1767nt, containing the 11 potential ORFs. Genome of the virus isolates shared 93.8% to 99.8% identity with PCV2 reference strains from GenBank, 76.6% to 77.9% identity with PCV1. Phylogenetic analysis indicated that there were two subgenotypes within PCV2: subgenotype I (1767 nt) and subgenotype II (1768 nt).

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

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.

Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs

PMWS is a new emerging disease in swine herds worldwide. Field isolates of PCV-2, a putative major causative agent of PMWS, were isolated and genetically characterized. Viral genome of two field isolates (PC201DJ and PC201SS) from pigs showing typical PMWS was sequenced. The nucleotide sequence homology with other PCV-2 isolates was ranging from 95% to 99% in complete viral genomic sequence. The highly conserved nonanucleotide motif of replication origin was identical to that of other PCV-2 isolates. To determine the genetic heterogeneity of PCV-2 isolates, the phylogenetic tree based on the complete genome of PCV-2 isolates were constructed. Two PCV-2 field isolates were closely related to Canadian isolates of PCV-2. PCV-2 isolated from field may have an origin of North America and is possibly originated from importation of breeding stocks. The result indicates that although the genome of PCV-2 is relatively stable in general, minor genetic variations exist among PCV-2 isolates from the different geographic locations. These differences of viral genome might have an important implication for genetic characteristics of PCV-2 infection. Three major immunorelevant epitopes of capsid protein showed variations in amino acid sequences. Also, the variance of amino acid sequence in antigenic epitope existed between two Korean PCV-2 isolates.

Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection.

Summary. Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B-133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13–14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B-133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2.

The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described.

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.

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.

Characterisation of PCV-2 isolates from Spain, Germany and France

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.