Phylodynamic analysis of porcine circovirus type 2: Methodological approach and datasets.
ABSTRACT: Since its first description, PCV2 has emerged as one of the most economically relevant diseases for the swine industry. Despite the introduction of vaccines effective in controlling clinical syndromes, PCV2 spread was not prevented and some potential evidences of vaccine immuno escape have recently been reported ("Complete genome sequence of a novel porcine circovirus type 2b variant present in cases of vaccine failures in the United States" (Xiao and Halbur, 2012) , "Genetic and antigenic characterization of a newly emerging porcine circovirus type 2b mutant first isolated in cases of vaccine failure in Korea" (Seo et al., 2014) ). In this article, we used a collection of PCV2 full genomes, provided in the present manuscript, and several phylogentic, phylodynamic and bioinformatic methods to investigate different aspects of PCV2 epidemiology, history and evolution (more thoroughly described in "PHYLODYNAMIC ANALYSIS of PORCINE CIRCOVIRUS TYPE 2 REVEALS GLOBAL WAVES of EMERGING GENOTYPES and the CIRCULATION of RECOMBINANT FORMS"). The methodological approaches used to consistently detect recombiantion events and estimate population dymanics and spreading patterns of rapidly evolving ssDNA viruses are herein reported. Programs used are described and original scripts have been provided. Ensembled databases used are also made available. These consist of a broad collection of complete genome sequences (i.e. 843 sequences; 63 complete genomes of PCV2a, 310 of PCV2b, 4 of PCV2c, 217 of PCV2d, 64 of CRF01, 140 of CRF02 and 45 of CRF03.), divided in differnt ORF (i.e. ORF1, ORF2 and intergenic regions), of PCV2 genotypes and major Circulating Recombinat Forms (CRF) properly annotated with respective collection data and country. Globally, all of these data can be used as a starting point for further studies and for classification purpose.
Project description:Porcine circovirus type 2 (PCV2) is an important pathogen that causes significant economic losses in the swine industry worldwide. Five major PCV2 genotypes have been identified, including PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. To investigate the prevalence and phylodynamics of the different PCV2 genotypes in Taiwan, 214 PCV2 ORF2 sequences from Taiwan and other countries were analyzed. Genotypic differences were observed among PCV2a, 2b, and 2d at amino acid position 89 in ORF2, with isoleucine (I), arginine (R), and leucine (L), respectively. Similar to other countries, a genotypic shift was also observed in Taiwan, where the predominant genotype shifted from PCV2b to 2d after 2010. The estimated nucleotide substitution rate of Taiwanese strains in the ORF2 region was 8.467?×?10-4 substitutions per site per year. This rapid evolution rate of PCV2 may lead to the genotypic shift observed in Taiwan. The times to the most recent common ancestor (TMRCA) for PCV2a, -2b, and -2d-2 was dated to 1970, 1992 and 2004, respectively. Thus, the PCV2a, -2b, and -2d genotypes were already present in Taiwan before the introduction of the PCV2 vaccine.
Project description:BACKGROUND:Porcine circovirus-associated diseases (PCVAD), caused by porcine circovirus type 2 (PCV2), threaten the pig industry worldwide. Five genotypes of PCV2 were recently identified: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. In addition, a novel porcine circovirus from a case of a sow with dermatitis, nephropathy syndrome and reproductive failure has been identified based on metagenomic analysis and classified as porcine circovirus type 3 (PCV3). Therefore, the current study was conducted to determine the prevalence and genetic characteristics of PCV2 and PCV3 in clinical samples. RESULTS:A total of 471 samples (161 tissue samples of lungs and lymph nodes from 34 farms and 310 serum samples from 47 farms) were tested for PCV2. Among them, 171 samples from 59 farms that had been positive for PCV2 were genotyped. Another 690 samples (296 tissue samples of lungs and lymph nodes from 91 farms, 108 samples of aborted foetuses from 26 farms, and 286 serum samples from 47 farms) were tested for PCV3. Based on PCV2 genotyping results, PCV2d was the most prevalent genotype (107 of 171 samples), and co-infections with combinations of PCV2a, 2b and 2d were identified in 48 samples from 17 farms. A total of 14 samples from 11 farms were also positive for both PCV2 and PCV3. For PCV3, 57 samples (9.8%) from 32 farms (23.2%) were positive. Among the 108 aborted foetuses from 26 farms, only 2 samples were positive for PCV3. Based on sequence comparisons, PCV2d shares 89.6-91.0% and 93.2-94.3% homology with PCV2a and PCV2b, respectively; 98.6-100% homology is shared among PCV2d strains. The PCV3 strains identified in this study share 98.0-99.5% homology. CONCLUSIONS:Our study concludes that PCV2d has become the most predominant genotype in Korea. PCV3 was also identified in clinical samples, though no significant association with clinical symptoms was observed in PCV3-positive cases.
Project description:Porcine circovirus type-2b (PCV2b) is recognized as the etiological agent of the various clinical manifestations of porcine circovirus-associated disease (PCVAD). Previous studies have demonstrated effectiveness of chimeric PCV1-2 vaccines against PCV2b challenge. In this study, the efficacy of inactivated and live-attenuated (2?×?10(3.5) or 2?×?10(4.0) 50% tissue culture infective dose [TCID50] dose) chimeric PCV1-2b vaccines was compared side-by-side in conventional pigs.Twenty-seven non-PCV2 viremic pigs without PCV2-specific antibody were randomly divided into six groups, including four vaccinated and challenged groups, a nonvaccinated challenged group, and a mock group. All pigs except those in the mock group were challenged at 28 days post vaccination (DPV) using PCV2b.Both inactivated and live-attenuated chimeric PCV1-2b vaccines induced a robust antibody responses, and significantly decreased microscopic lesion and lower viral loads in serum or superficial inguinal lymph nodes (SILN) compared with that in the nonvaccinated challenged group. PCV2 antibody titers decreased after 7 days post challenge (DPC) in pigs administered the inactivated PCV1-2b vaccine and they were lower than those in pigs inoculated with live-attenuated PCV1-2b on the day of necropsy. Moreover, no viremia was present in pigs inoculated with live-attenuated PCV1-2b vaccine at 21 DPC regardless of the dose difference.The results demonstrated that both inactivated and live-attenuated chimeric PCV1-2b vaccines were effective to induce protective immunity against PCV2b infection.
Project description:Porcine circovirus type 2 (PCV2) is a very small, non-enveloped and icosahedral virus, with circular single stranded DNA genome. This virus is the most ubiquitous and persistent pathogen currently affecting the swine industry worldwide. PCV2 has been implicated as the major causative agent of postweaning multisystemic wasting syndrome (PMWS), a disease which is characterized by severe immunosuppressive effects in the porcine host. Worldwide PCV2 isolates have been classified into four different genotypes, PCV2a, PCV2b, PCV2c and PCVd. The goal of this work was to conduct the first phylogenetic analysis of PCV2 in Chile.PCV2 partial ORF2 sequences (462 nt) obtained from 29 clinical cases of PMWS in 22 Chilean intensive swine farms, covering over the 90% of the local pork-production, were analyzed.14% and 52% of sequences belonged to the genotypes PCV2a and PCV2b, respectively. Surprisingly, 34% of sequences were PCV2a/PCV2d recombinant viruses.Our findings suggested that a novel cluster of Chilean sequences emerged resulting from intergenotypic recombination between PCV2a and PCV2d.
Project description:BACKGROUND: Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms. RESULTS: Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc). Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples. CONCLUSIONS: LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.
Project description:Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Here, we report the complete genome sequence of PCV2 strain HB-MC1, which belongs to PCV2d.
Project description:The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-?-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.
Project description:Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease. Here, we first report the complete genome sequence of PCV2 strain JSTZ, which was isolated from piglet stool samples and is highly prevalent in China. It will help in understanding the epidemiology and molecular characteristics of PCV2.
Project description:The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P < 0.05) decreased amounts of PCV2 in tissues and sera and significantly (P < 0.05) reduced macroscopic and microscopic lesions. The results of this study indicate that the experimental live-attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure.
Project description:Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894-0.998, p < 0.001), 100% (n = 93, 95% CI 0.966-1.000, p < 0.001), 100% (95% CI 0.953-1.000, p < 0.001), 97.9% (95% CI 0.921-0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (? = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ~3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.