Porcine Circovirus Type 2 Strains Research Articles

Reappearance of buffalo-origin-like porcine circovirus type 2 strains in swine herds in southern China.

Previously, we identified three porcine circovirus type 2 (PCV2) strains in buffalo meat samples from southern China. In this study, we confirmed the reappearance of those buffalo-origin-like PCV2 strains in swine herds in this region, which supported the possible cross-species infection of PCV2 between buffalos and pigs under field conditions.

Porcine circovirus type 2 (PCV2) evolution before and after the vaccination introduction: A large scale epidemiological study.

Since their commercialization, vaccines against Porcine circovirus type 2 (PCV2) have been the cornerstone control strategy. Nevertheless, the periodic emergence of new genotype waves and the recent reports of vaccine failure outbreaks have raised the question if widespread vaccination strategies could have driven viral evolution and affected different genotype fitness. To investigate this issue an in-deep analysis, based on a bioinformatics and biostatistics approach, has been implemented. ORF2 sequences from vaccinated and non-vaccinated populations (i.e. domestic pigs before and after vaccine introduction and wild boars) were considered. The action of selective forces on PCV2 strains has been analyzed and compared among groups. Remarkable differences were found in the selective forces acting on viral populations circulating in different “immune environments”. Particularly for PCV2a, a directional selection promoting a change in the viral capsid away from the vaccine specific antigenic determinants has been detected after vaccine introduction. Involved amino acids were previously reported to be part of viral epitopes whose variability is responsible of immune escape. Our findings support a change in PCV2 evolutionary pattern after widespread vaccination introduction and stress once more the compulsoriness of a continuous monitoring of PCV2 epidemiology to promptly act in response to the emergence of possible vaccine-escaping mutants.

Genotypic diversity of porcine circovirus type 2 (PCV2) and genotype shift to PCV2d in Korean pig population

Porcine circovirus type 2 (PCV2) is a causative agent of PCV2-associated disease (PCVAD), which leads to enormous economic losses in the swine industry worldwide. A high nucleotide substitution rate allows for the continuous evolution of PCV2 and the emergence of novel PCV2 strains. However, the distribution of emerging PCV2 genotypes and the co-existence of multiple genotypes in Korea have not been elucidated. The objective of this study was to determine the genetic diversity of PCV2 in Korean pig herds between 2009 and 2016. The overall prevalence of PCV2, from various samples originating from commercial pigs, was 53.8% (325/604). Two cases of a genotype shift to PCV2d at the farm level showed that the genotype shift started before 2012. In addition, genotype-specific PCR, on pen-based oral fluid samples for nationwide PCV2 surveillance in 2016, indicated that the infection pattern of PCV2 genotypes at the farm level was as follows; none (6/69), PCV2a (6/69), PCV2b (2/69), PCV2d (33/69), PCV2a/b (2/69), PCV2a/d (4/69), PCV2b/d (11/69), and PCV2a/b/d (2/69), respectively. This suggests that the genotype shift to PCV2d occurred on a nationwide scale and that the co-existence of different genotypes is common in Korean pig herds. In addition, seven sites on the capsid protein of Korean PCV2 were identified as being under positive selection pressure, all of which are related to the epitope region and neutralization activity. These data provide evidence of increased genetic diversity and shifts among Korean PCV2 isolates.

A chimeric virus created by DNA shuffling of the capsid genes of different subtypes of porcine circovirus type 2 (PCV2) in the backbone of the non-pathogenic PCV1 induces protective immunity against the predominant PCV2b and the emerging PCV2d in pigs

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Available commercial vaccines all target PCV2a subtype, although the circulating predominant subtype worldwide is PCV2b, and the emerging PCV2d subtype is also increasingly associated with PCVAD. Here we molecularly bred genetically-divergent strains representing PCV2a, PCV2b, PCV2c, PCV2d, and “divergent PCV2a” subtypes by DNA-shuffling of the capsid genes to produce a chimeric virus representing PCV2 global genetic diversity. When placed in the PCV2a backbone, one chimeric virus (PCV2-3cl14) induced higher neutralizing antibody titers against different PCV2 subtypes. Subsequently, a candidate vaccine (PCV1-3cl14) was produced by cloning the shuffled 3cl14 capsid into the backbone of the non-pathogenic PCV1. A vaccine efficacy study revealed that chimeric virus PCV1-3cl14 induces protective immunity against challenge with PCV2b or PCV2d in pigs. The chimeric PCV1-3cl14 virus is a strong candidate for a novel vaccine in pigs infected with variable PCV2 strains.

Phylogenetic analysis of porcine circovirus type 2 (PCV2) isolates from China with high homology to PCV2c.

Porcine circovirus type 2 (PCV2) is an important emerging pathogen that has been causatively associated with multifactorial disease syndromes in pigs and other species. It has a worldwide distribution and causes significant economic losses in the swine industry. Its genome is dynamically evolving through recombination and mutation, and the circulating genotypes of PCV2 strains in Asia are PCV2a, PCV2b and PCV2d. In this study, 12 PCV isolates were evaluated and identified by amplification, sequencing, and phylogenetic analysis, and the results revealed a new monophyletic group of PCV in China. More importantly, three of these isolates shared high homology within the ORF1 region with a strain of genotype PCV2c that was detected only in Denmark. Phylogeographic analysis of these isolates suggested that the isolates may have arisen in Denmark and that they were then transported to China.

Production of Escherichia coli-based virus-like particle vaccine against porcine circovirus type 2 challenge in piglets: Structure characterization and protective efficacy validation

We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.

Genetic and phylogenetic analysis of a new porcine circovirus type 2 (PCV2) strain in China.

Porcine circovirus type 2 (PCV2) is the etiological agent associated with several pig diseases that are collectively referred to as porcine circovirus-associated disease (PCVAD). Unfortunately, PCV2 has had a serious economic impact on the swine industry. In this study, we report the genome sequence of a novel PCV2 isolate (JS2015) identified in pigs in Jiang Su, China. The complete DNA sequence was 1766 nucleotides long with an A+T content of 52.7%. It lacked a guanine (G) at nucleotide position 1045 compared to other reference PCV2 strains with a sequence length of 1766 nucleotides. Genetic characterization and phylogenetic analysis showed that the isolate JS2015 was most closely related to members of the PCV2d (AY181946) lineage. Our data provide insight into the epidemiology of porcine circovirus and may facilitate further study of the origin and evolution of PCV2.

Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d.

The oldest porcine circovirus type 2 (PCV2) sequence dates back to 1962 and is among several hundreds of publicly available PCV2 sequences. Despite this resource, few studies have investigated the global genetic diversity of PCV2. To evaluate the phylogenetic relationship of PCV2 strains, 1680 PCV2 open reading frame 2 (ORF2) sequences were compared and analysed by methods of neighbour-joining, maximum-likelihood, Bayesian inference and network analysis. Four distinct clades were consistently identified and included PCV2a, PCV2b, PCV2c and PCV2d; the p-distance between PCV2d and PCV2b was 0.055±0.008, larger than the PCV2 genotype-definition cut-off of 0.035, supporting PCV2d as an independent genotype. Among the 1680 sequences, 278-285 (16.5-17 %) were classified as PCV2a, 1007-1058 (59.9-63 %) as PCV2b, three (0.2 %) as PCV2c and 322-323 (19.2 %) as PCV2d, with the remaining 12-78 sequences (0.7-4.6 %) classified as intermediate clades or strains by the various methods. Classification of strains to genotypes differed based on the number of sequences used for the analysis, indicating that sample size is important when determining classification and assessing PCV2 trends and shifts. PCV2d was initially identified in 1999 in samples collected in Switzerland, now appears to be widespread in China and has been present in North America since 2012. During 2012-2013, 37 % of all investigated PCV2 sequences from US pigs were classified as PCV2d and overall data analysis suggests an ongoing genotype shift from PCV2b towards PCV2d. The present analyses indicate that PCV2d emerged approximately 20 years ago.

Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2-systemic disease (PCV2-SD), previously known as post-weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2-SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.

Genetic characterisation of Porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: An opportunity to reconstruct the history of PCV2 evolution

Since its discovery, Porcine circovirus type 2 has emerged as one of the most relevant swine infectious diseases, causing relevant economic losses for the pig industry. While four genotypes were identified, only three (PCV2a, PCV2b and PCV2d) are currently circulating and display a worldwide distribution. Another genotype, PCV2c, has been described only once in Danish archive samples collected between 1980 and 1990. In addition to commercial pigs, PCV2 has been demonstrated to infect wild boars and other wild species, which can potentially serve as a reservoir for domestic populations. In this study, eight sequences obtained from feral pigs in the Pantanal region (Mato Grosso do Sul State, Brazil) were compared with reference sequences and other Brazilian sequences, and the results revealed remarkable genetic diversity, with all four genotypes currently recognised being detected (PCV2a, PCV2b, PCV2c and PCV2d). This finding represents a remarkable discovery, as it is the first detection of PCV2c since 1990 and the first-ever detection of PCV2c in live animals. The peculiar population history and ecological scenario of feral pigs in the Pantanal coupled with the complex, and still only partially known relationship of feral pigs with other PCV2 susceptible species (i.e., domestic pigs, wild boars and peccaries), open exciting questions concerning PCV2 origin and evolution. Overall, the results of the present study led us to form the following hypothesis: the PCV2 strains found in feral pigs may be the last descent of the strains that circulated among European pigs in the past, or they may have infected these feral pigs more recently through a bridge species.

Differential recognition of the ORF2 region in a complete genome sequence of porcine circovirus type 2 (PCV2) isolated from boar bone marrow in Korea

Porcine circovirus type 2 (PCV2) is the causative agent of post-weaning multisystemic wasting syndrome (PMWS) in swine. Here, a phylogenetic tree was constructed using PCV2 nucleotide sequences derived from the bone marrow of Korean boar and previously reported PCV2 sequences isolated from various countries. PCV2 from Korean boar bone marrow (KC188796) was classified into the group containing PCV2a-Canada and other PCV2 strain from Korea. While the ORF1 region of the PCV2 genome was highly conserved, ORF2 (the capsid protein coding region) was relatively variable. The nucleotide sequences for bone marrow-derived PCV2 were 93.4–99.0% homologous to the other reference sequences. The deduced amino acid sequences for the ORF1 and ORF2 coding regions were 97.4–99.3% and 84.5–97.4% homologous with the other reference strains, respectively, indicating that KC188796 did not differ markedly from the other PCV2 strains. Phylogenetic analysis demonstrated that bone marrow-derived PCV2 was highly similar to PCV2a from Canada and may be related to persistent PCV2 infections in swine.

International trades, local spread and viral evolution: The case of porcine circovirus type 2 (PCV2) strains heterogeneity in Italy

Porcine circovirus type 2 is one of the most widespread and economically relevant infections of swine. Four genotypes have been recognized, but currently, only three (PCV2a, PCV2b and PCV2d) are effectively circulating. The widespread livestock trade and rapid viral evolution have contributed to determining the high heterogeneity of PCV2 and the dispersal of potentially more virulent strains. Italian swine farming and the related processing industry are relevant in the national economy. Despite the noteworthy losses associated with direct and control measure costs, no data are currently available on the molecular epidemiology of PCV2 in Italy. Our study, which was intended to fill this gap, considered 75 completed genome PCV2 sequences, which were obtained from samples collected from the highly densely populated area of Northern Italy between 2007 and 2014. Phylogenetic analysis and comparison with reference sequences demonstrated the co-circulation, with different prevalences, of PCV2a, PCV2b and PCV2d within the national borders, with PCV2b being the most prevalent. Recombination between different genotypes was also proven to be frequent. Phylogeographic analysis demonstrated that the marked variability of Italian PCV2 strains can be attributable to multiple introduction events. The comparison of the phylogenetic analysis results, the location of different haplotypes and the international commercial routs of live pigs allow the speculation of several links as well as the role of Italy as both an importer and exporter of PCV2 haplotypes, mainly from and to European and Asian countries. A similarly intricate contact network was demonstrated within national borders, with different haplotypes being detected in the same province and different provinces harbouring the same haplotype. Overall, this paper represents the first description of PCV2 in Italy and demonstrates that the high variability of circulating Italian strains is due to multiple introduction events, wide circulation within national boundaries and rapid viral evolution.

Genetic characterisation and phylogenetic analysis of PCV2 isolates from India: Indications for emergence of natural inter-genotypic recombinants

Porcine circovirus type 2 (PCV2), the necessary agent in pathogenesis of porcine circovirus diseases (PCVDs), has a worldwide distribution and is considered as one of the most important emerging viral pathogens of economic importance. PCV2 has been divided into four major genotypes namely PCV2a with five clusters or subtypes (2A–2E), PCV2b with three clusters (1A–1C), PCV2c and PCV2d, based on capsid (cap) gene analysis. PCV2 genome is rapidly evolving through events of recombination and mutation. Though, PCV2a was the predominant genotype initially, PCV2b shared majority of PCV2 sequences submitted to GenBank since 2003. In India, data regarding molecular characterisation of PCV2 is scant or absent. In the present study, we thoroughly analysed genetic heterogeneity of PCV2 strains circulating in Indian pig population. The results revealed that pigs in this region harboured PCV2 viruses of different genotypes including PCV2a–2D, PCV2b–1C and PCV2d. More interestingly, two isolates (PCV2Izn-89-13 and PCV2Izn-218-13) were classified as recombinant strains. Further detailed analysis suggested that these strains evolved from inter-genotypic recombination between PCV2a–2C and PCV2b–1C genotypes within cap gene. This study reports for the first time, the emergence of recombinant PCV2 strains in the Indian pig population.

Identification of natural recombinants derived from PCV2a and PCV2b.

Porcine circovirus type 2 (PCV2) is considered to be the main pathogen in PC-associated diseases, and significantly affects the global pig-producing industry. PCV2 continuously evolves by point mutations and genome recombinations. In the present study, we aimed to further identify recombinant PCV2 strains. We used polymerase chain reaction to detect PCV2 in the carcasses of pigs with suspected infections from different regions of Guangdong Province in China. DNA was extracted from samples with confirmed infection and full- genome amplification, sequencing, phylogenetic tree construction, gene recombination detection, and sequence alignment were performed in gene recombination analysis. Our results show that recombination occurred between the strains SHC (DQ104421) and ZhuJi2003 (AY579893). The recombination resulted in three recombinants: GD003 (KM503044), GD005 (KM487708), and GD008 (KM487709). Further analyses revealed that these novel recombinants appeared to result from recombination between the PCV2a and PCV2b strains, with crossover regions located in ORF2. This study was a comprehensive analysis that used several different methods, which demonstrated that a cluster of PCV2 strains resulted from the same type of inter-genotypic recombination pattern, with a breakpoint in the structural protein coding region. The results of our study provide both information on the recombination mechanism and disease pathogenesis and useful data for the prevention of PCV2 in the swine industry.

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.

Recombination in truncated genome sequences of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the causal agent of a serious disease found in pigs. Here, we report the first detection of truncated genome sequences of PCV2 strain ZJ-R, with the genomic region encoding part of Rep and Cap with a nonviral insertion. To our knowledge, the genome of ZJ-R represents the first PCV2 DNA with a coding insertion. The PCV2 ZJ-R genome is 694 nucleotides long and has two main open reading frames. The whole genome sequence of ZJ-R may facilitate further study of the origin and evolution of PCV2.

First molecular detection of porcine circovirus type 2 in bovids in China.

For the worldwide pig industries, porcine circovirus type 2 (PCV2) is an economically important pathogen. At present, the prevalence of PCV2 is common in Chinese swine herds. However, there is little information on PCV2 prevalence in non-porcine animals in China, such as bovids. Therefore, the goal of this study is to obtain the firsthand prevalence data of PCV2 in bovids in China. Two hundred and eighty serum and muscle samples from dairy cows (n = 180), buffalo (n = 50), and yellow cattle (n = 50) were analyzed by PCR. The detection results show that PCV2 infections (16 %, 8/50) only exist in buffaloes. In addition, there are different PCV2 viral DNAs identified by differential PCR in the same buffalo sample. Nucleotide sequencing and phylogenetic analysis results based on partial ORF1 and ORF2 sequences suggest that PCV2 strains have genetic diversity in buffaloes and they are divided into three different genotypes (PCV2b, PCV2d, and PCV2e, respectively). Moreover, to our knowledge, the PCV2d and PCV2e genotypes have not been previously reported in bovids. Through this study, the firsthand data of PCV2 prevalence in bovids in China was documented.

A recombinant porcine circovirus type 2 expressing the VP1 epitope of the type O foot-and-mouth disease virus is infectious and induce both PCV2 and VP1 epitope antibodies

Porcine circovirus type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome, a disease that causes huge economic damage in swine industry. A recombinant PCV2 expressing the neutralizing VP1 epitope (aa 141-160) of the foot-and-mouth disease virus (FMDV) was rescued using an infectious cloning technique. The PCV2 antigen and FMDV-VP1 antigenic epitope of the cloned strain recPCV2-CL-VP1 were confirmed by an immunoperoxidase monolayer assay (IPMA) and a capture enzyme-linked immunosorbent assay (ELISA). The morphological features of the recPCV2-CL-VP1 were not discernibly different from those of its parental strain (PCV2-CL). However, the recombinant virus could be differentiated from its parental virus by PCR and capture ELISA. The recPCV2-CL-VP1 was demonstrated to replicate stably in PK-15 cells through ten passages. An infection experiment using BALB/c mice showed that both recPCV2-CL-VP1 and PCV2-CL could replicate in the mice, cause various pathological changes, and induce a high level of anti-Cap antibodies. The recombinant virus emulsified with Freund's adjuvant was used to immunize BALB/c mice and induced antibodies against the FMDV-VP1 epitope. Hence, the recombinant PCV2 strain, which expressed the neutralizing FMDV-VP1 epitope, provides a valuable platform to develop novel genetic vaccines.

Phylogenetic comparison of porcine circovirus type 2 (PCV2) and porcine reproductive respiratory syndrome virus (PRRSV) strains detected in domestic pigs until 2008 and in 2012 in Croatia.

BackgroundPorcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) have been present for the last 2 decades in Croatia, causing large economical losses in the pig production. The clinical features of the infections are mostly manifested by the development of respiratory problems, weight loss and poor growth performance, as well as reproductive failure in pregnant sows. Even though the infections are continuously recognized in some regions in Croatia, the heterogeneity of the detected viral strains from 2012 has not yet been investigated. The objective of this study was to compare virus strains of PCV2 and PRRSV detected until 2008 in Croatia with strains isolated in 2012 to gain a better epidemiological understanding of these two infections.ResultsPCV2 and PRRSV strains detected in 2012 in fattening pigs from regions where these two diseases have been previously described were compared to strains that have been detected in the same regions within the past two decades. The phylogenetic analysis revealed that the circulating PCV2 and PRRSV strains are distantly related to the previously described Croatian viral strains. However, when compared to known isolates from the GenBank a high genetic identity of PRRSV isolates with isolates from Hungary, Denmark and the Netherlands was found.ConclusionThe results of this study reveal that even though PCV2 and PRRSV are constantly present in the investigated regions in Croatia, the viral strains found in 2012 genetically differ from those detected in earlier years. This indicates that new entries into the pig population appeared with regard to both infections, probably as a result of pig trade.

Differentiation of PCV1 and PCV2 by a multiplex real-time PCR assay

Porcine circovirus (PCV), belonging to Circoviridae family, is a small, non-enveloped, icosahedral virus containing a circular single-stranded DNA genome (Tischer and others 1982). PCV is divided into two genotypes, PCV...