Genotyping Porcine Circovirus 3 (PCV-3) Nowadays: Does It Make Sense?

The discovery of a globally distributed porcine circovirus (Porcine circovirus 3; PCV-3) has led to intense research activity and the production of a large amount of molecular data. Different research groups have proposed several, not always concordant, genotypes for this virus. While such categories could aid an easier interpretation of PCV-3 molecular epidemiology, any classification, to be useful in practical settings, must be univocal and of help in the understanding of underlying biological features and epidemiology. Based on these premises, the possibility of defining PCV-3 genotypes was evaluated on the broadest available dataset of PCV-3 complete genome (n = 357) and open reading frame 2 (ORF2, n = 653) sequences. Genetic distance and phylogenetic clustering were selected as the main objective criteria. Additional factors, including the number of within-cluster sequences, host and geographic clustering, concordance between different genomic regions, and analysis method were also taken in account to generate a classification that could be effectively applied in research and diagnostic settings. A maximum within-genotype genetic distance of 3% at the complete genome and 6% at the ORF2 levels, bootstrap support higher than 90%, and concordance between analysis methods allowed us to clearly define two clades which could be potentially defined as genotypes. Further subdivision was not suggested due to the absence of a meaningful association between PCV-3 and its biological/epidemiological features. Nevertheless, since one of the clades included two strains only, thus far we formally propose the definition of only one PCV-3 genotype (PCV-3a). The established criteria will allow us to automatically recognize other genotypes when more strain sequences are characterized.info:eu-repo/semantics/publishedVersio

An updated phylogeography and population dynamics of porcine circovirus 2 genotypes: are they reaching an equilibrium?

Introduction: Porcine circovirus 2 (PCV2) emerged more than three decades ago as one of the most impactful pathogens in the swine industry. Despite being a DNA virus, one of the hallmarks of PCV2 is its high evolutionary rate, which has led to the emergence of different genotypes, each exhibiting varying degrees of evolutionary success. Current knowledge suggests the occurrence of three main waves of genotype dominance, alternating over time (i.e., PCV2a, PCV2b, and PCV2d), alongside less prevalent genotypes. However, although PCV2d is currently the most common genotype nowadays, the others continue being circulating in the pig population. Methods: The present study reconsidered the epidemiological and evolutionary patterns of PCV2 genotypes using phylodynamic analyses, benefiting from an almost 10-fold increase in ORF2 sequence availability compared to previous studies. Additionally, a phylogeographic analysis was performed to investigate viral dispersal patterns and frequency, and the selective pressures acting on the capsid protein were estimated and compared among genotypes. Results: While successive emergence of major genotypes was confirmed, this study extends previous findings by revealing subsequent prevalence fluctuations of PCV2a and PCV2b after their initial decline. This evolutionary process may represent an example of balancing selection, specifically negative frequency-dependent selection, where a genotype fitness diminishes as it becomes more common, and vice versa. Variations in genotype- or clade-specific immunity—affected by the local prevalence of viral groups—combined with the periodic introduction of strains that have independently evolved in different regions, may have led to fluctuations in the population dynamics of major genotypes over time. These fluctuations were associated with ongoing evolution and variations in the capsid amino acid profile. Discussion: These findings have profound implications for future control strategies. Although PCV2d remains the most prevalent and widespread genotype, other genotypes should not be neglected. Control strategies should thus target the entire PCV2 population, with a focus on fostering broader and more cross-protective immunity.The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Department of Animal Medicine, Production and Health, University of Padua (grant number BIRD225455/22) and Open Access funding provided by Università degli Studi di Padova, University of Padua, Open Science Committee.info:eu-repo/semantics/publishedVersio

Genetic diversity of porcine circoviruses 2 and 3 circulating among wild boars in the Moscow Region of Russia

Porcine circoviruses (PCVs) are widely distributed in swine herds. PCV2, the significant swine pathogen, causes infections characterized by growth and development disorders, skin lesions, and respiratory distress. PCV3 has been circulating worldwide and can be associated with various clinical signs and disease developments. Wild boars are the main reservoir of these pathogens in wildlife and can create an alarming threat to pig farming. In Russia, three PCV2 genotypes (PCV2a, PCV2b, and PCV2d) were identified in pig farms. Additionally, PCV3 was observed in pig herds during the monitoring studies in the country. However, data considering the circulation of PCVs in herds of wild boars in Russia is scant. For this purpose, we performed PCR assays of the samples from 30 wild boars hunted in the Moscow Region of Russia in 2021–2023. The ratios of wild boars positive for PCV2, PCV3, or coinfected were 50, 10, and 13.3%, respectively. Additionally, we sequenced 15 PCV2 and four PCV3 complete genomes and conducted phylogenetic analysis, which divided PCV2 isolates into two groups: PCV2d and PCV2b. The study showed a high infection rate of PCV2 among wild boars, with PCV2d dominance. Simultaneously, PCV3 also circulates among wild boars. The obtained results can provide a basis for the development of preventive measures to support infection transmission risks between farm and wild animals

An updated phylogeography and population dynamics of porcine circovirus 2 genotypes : are they reaching an equilibrium?

Introduction: Porcine circovirus 2 (PCV2) emerged more than three decades ago as one of the most impactful pathogens in the swine industry. Despite being a DNA virus, one of the hallmarks of PCV2 is its high evolutionary rate, which has led to the emergence of different genotypes, each exhibiting varying degrees of evolutionary success. Current knowledge suggests the occurrence of three main waves of genotype dominance, alternating over time (i.e., PCV2a, PCV2b, and PCV2d), alongside less prevalent genotypes. However, although PCV2d is currently the most common genotype nowadays, the others continue being circulating in the pig population. Methods: The present study reconsidered the epidemiological and evolutionary patterns of PCV2 genotypes using phylodynamic analyses, benefiting from an almost 10-fold increase in ORF2 sequence availability compared to previous studies. Additionally, a phylogeographic analysis was performed to investigate viral dispersal patterns and frequency, and the selective pressures acting on the capsid protein were estimated and compared among genotypes. Results: While successive emergence of major genotypes was confirmed, this study extends previous findings by revealing subsequent prevalence fluctuations of PCV2a and PCV2b after their initial decline. This evolutionary process may represent an example of balancing selection, specifically negative frequency-dependent selection, where a genotype fitness diminishes as it becomes more common, and vice versa. Variations in genotype- or clade-specific immunity-affected by the local prevalence of viral groups-combined with the periodic introduction of strains that have independently evolved in different regions, may have led to fluctuations in the population dynamics of major genotypes over time. These fluctuations were associated with ongoing evolution and variations in the capsid amino acid profile. Discussion: These findings have profound implications for future control strategies. Although PCV2d remains the most prevalent and widespread genotype, other genotypes should not be neglected. Control strategies should thus target the entire PCV2 population, with a focus on fostering broader and more cross-protective immunity

Genetic indicators for disease resilience in pigs

Infectious swine diseases have the potential to decimate the health and productivity of swine farms. One of the most economically concerning diseases is caused by the Porcine Reproductive and Respiratory Syndrome (PRRS) virus. While swine producers can implement vaccines, medications, or antibiotics and antiviral drugs, many infectious pathogens such as the PRRS virus have shown these strategies to be ineffective. One complimentary strategy would be to select pigs for increased disease resistance or resilience, where disease resilience is defined as an animal’s ability to maintain performance when infected. However, the elite populations that are used for genetic improvement are typically kept in high health conditions, making it difficult and impractical for swine breeders to use phenotypic selection in an environment with exposure to disease to select for increased disease resilience. Previous research has shown that host response to PRRS virus infection has a sizable genetic component and revealed a Quantitative Trait Locus (QTL) for host response to PRRS virus infection on Sus Scrofa Chromosome (SSC) 4. A putative causative mutation in the GBP5 gene was identified for this QTL. This mutation was determined to be in complete linkage disequilibrium with the single nucleotide polymorphism (SNP) WUR10000125 (WUR) that was included on commercial SNP panels. However, this was based on data from one genetic source. The overall objective of this thesis was to determine if the WUR SNP and phenotypes obtained from in-vitro mitogen stimulation assays (MSA) of peripheral blood mononuclear cells (PBMCs) from young healthy nursery pigs can be used as genetic indicators to select for disease resilience. The two requirements for a genetic indicator are that it must be heritable and have a sizeable genetic correlation with the trait of interest, in this case, disease resilience. Data from experimental PRRS virus infection trials from the PRRS Host Genetics Consortium (PHGC) and a polymicrobial Natural Disease Challenge Model (NDCM) of grow-finish pigs were used to address these objectives. Data and SNP genotypes, including for the WUR SNP and the putative causative mutation in the GBP5 gene, were available on 1414 pigs from eight PHGC trials of ~200 commercial crossbred nursery pigs per trial from six unrelated populations. Results showed that the WUR and GBP5 SNPs were not in complete linkage disequilibrium (r2 = 0.94). Discordant genotypes were determined to be the result of recombination, rather than genotyping errors. Although it was previously speculated that the GBP5 gene is a major gene responsible for host response to PRRS, there were small but non-significant differences between the effect of GBP5 and WUR on PRRS viral load and weight gain post-infection. These results indicate that either GBP5 or the WUR SNP can be used for marker-assisted selection to increase resistance to PRRS. In the NDCM, data from 3139 crossbred nursery barrows that were genotyped using a 650 K SNP Panel (Affymetrix) were used. The 650 K panel included the WUR SNP but not the GBP5 SNP. In the NDCM, pigs were entered through a batch system of 60 or 75 pigs per batch into a facility that was seeded with multiple infectious pathogens, including PRRS, to maximize the expression of disease resilience. Disease resilience traits, including growth, feed intake, and treatment and mortality rates were recorded. Based on these data, it was determined that the favorable G allele for the WUR SNP was significantly associated with greater average daily gain (p=0.02) and lower numbers of treatments in the challenge nursery (p=0.05) and across the challenge nursery and finisher (p=0.01), establishing the effect of the SSC4 QTL on resilience to a polymicrobial disease challenge. For the MSAs, PBMCs were isolated from blood samples of 882 pigs from 19 batches of the NDCM, taken at 27 or 35 days of age and prior to their entry in the disease challenge. For the MSAs, PBMCs were stimulated with five unique mitogens: Concanavalin A (Con A), Phytohemagglutinin (PHA), Poke Weed Mitogen (PWM), Lipopolysaccharide (LPS), and Phorbol Myristate Acetate (PMA), and evaluated for counts of proliferated cells after 48, 72, and 96 hours compared to unstimulated samples (restcount). Proliferated cell counts were adjusted for restcount in two ways: 1) by dividing the average cell count of the stimulated wells by the average cell count of the non-stimulated wells, to compute a Blastogenic Index Score (BIS), and 2) by including the average cell count of the non-stimulated wells as a covariate in the model for analysis of the average cell count of the stimulated wells. Data on BIS and stimulated means at each time point were analyzed separately for each mitogen. For pigs that had data at all three time points for a mitogen, data across these time points were incorporated into a single phenotype called the Area Under the Curve (AUC). Differences between pairs of time points for a given mitogen (delta = 72 – 48 hrs, 96 – 72 hrs, and 96 - 48 hrs) were also analyzed as phenotypes. Genetic parameters (heritabilities and genetic correlations) were estimated for the MSA phenotypes. In general, MSA phenotypes based on BIS versus stimulated means adjusted for restcount had similar estimates of genetic parameters. Heritability estimates for the Con A, PHA, and PMA MSA phenotypes were moderate, ranging from 0.13 +0.09 to 0.37 +0.10 for Con A, from 0.10 +0.07 to 0.34 +0.09 for PHA, and from 0.05 +0.06 to 0.30 +0.10 for PMA. Heritability estimates for the PWM and LPS MSA phenotypes were low, ranging from 0.00 +0.00 to 0.15 +0.09. Disease-related phenotypes collected on these same pigs in the NDCM were then used to estimate genetic correlations of the MSA phenotypes with disease resilience phenotypes. Phenotypic correlations between MSA and disease resilience phenotypes were low. Phenotypes derived from the Con A, PHA, and PMA MSAs, however, had moderately high estimates of genetic correlations with several disease resilience traits, although none were significantly different from zero due to large standard errors. However, genetic correlation estimates were generally in the expected direction, with pigs with higher MSA response having better resilience at the genetic level. Overall, Con A presented itself as the most promising mitogen to use as a genetic indicator for disease resilience, although further studies are recommended to validate its potential and to determine the ideal time point or MSA phenotype to use. In conclusion, the use of a genetic indicator to indirectly select for increased disease resilience in swine is a viable approach. The two indicators investigated in this thesis, i.e. genotype at an SNP on chromosome 4 and results of an in vitro mitogen stimulation assay on immune cells derived from the blood of young healthy piglets, are suitable genetic indicators for disease resilience to a polymicrobial disease challenge

Deep sequencing of PRRSV isolates: rapid and large-scale characterization of viral genomes

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single stranded, positive sense RNA virus with a genome size of approximately 15 kb. Much of the genetic characterization or viral genotyping of PRRSV isolates is limited to one or two viral genes only (ORF5 and/or ORF7) for a number of reasons, for example: (1) characterizing one or two ORFs is sufficient for diagnostics; (2) genome characterization is laborious because traditional (Sanger) sequencing yields only a single sequence of 800-1000 bases per reaction; (3) large-scale genome characterization is time-consuming and costly. Collectively, this hinders the study of PRRSV genomic evolution at different levels (host, regional, and global). We demonstrate here the use of 454 technology to rapidly sequence PRRSV genomic nucleic acid from different sources (cell culture and swine tissue), genotypes (type 1 and type 2), and genome structure (non-deletion vs. deletion variants). Samples (n=16) were multiplexed to bring down cost per genome sequence. Assembly of sample specific reads resulted in a single contig in almost all instances (15 out of 16). Average genome coverage was 96.7% with reference to prototype isolates (Lelystad virus for type 1 and ATCC VR2332 for type 2). Average sequence depth was 405 reads per nucleotide position. This high sequence depth allowed characterization of variants from quasispecies that occurred at frequencies even lower than 1%. In summary, next generation sequencing technology offers unparalleled opportunity to quickly and efficiently characterize near complete length PRRSV genomes in an economical manner. This allows experiments to be designed with considerations to viral genomic evolution rather than those with limited insights from select viral genes only.postprin

Metagenomic, Viral and Host Genetic Analyses of Congenital Tremor in Pigs

The Pestivirus genus contains several viral species having a major impact on the livestock species. While there were only four major pestiviral species for a time, recent metagenomic sequencing approaches identified additional species, such as atypical porcine pestivirus (APPV). Congenital tremor was first identified almost a hundred years ago and since still has an impact on swine health. There are two main types of congenital tremor, type A and B, with type A congenital tremor further subcategorized based on causative agent. Until recently, type A-II congenital tremor did not have a known cause. Recent viral sequencing of affected samples, including our research, revealed APPV to be the predominant viral species in congenital tremor piglets, providing evidence of a causative agent for type A-II congenital tremor. Various strains of APPV have been identified across the globe. Through sequence comparison, it was found most of the viral APPV genome is not highly conserved. This can lead to misdiagnosis or false-negative results due to variations in the APPV genome sequence when testing of congenital tremor samples is based on qPCR assays. While degenerate primers are an option to combat this, the high degree of variation across the strains and rapid evolution of the viral genome will eventually lead to qPCR assays not recognizing all strains. Whole sequence comparison of full-length APPV genomes demonstrated the 5’ untranslated region (5’UTR) is highly conserved (85%) between 20 worldwide species. This high degree of conservation makes the 5’UTR an ideal candidate region for a universal qPCR assay to detect any strains of APPV. The role of host genetics in disease susceptibility and severity have been documented for several swine viruses. The swine leukocyte antigen class II complex (SLAII) is involved in the antigen presentation during viral infection. The highly polymorphic DQB1 gene in this region was partially sequenced to provide haplotype profiling to a group of sows subjected to an APPV-based exposure leading to litters with various degrees of congenital tremor. Relationships between DQB1 haplotypes and incidence of congenital tremor and the rate of pre-weaning mortality were investigated. Future more detailed research will need to better quantify the role of host genetics in APPV susceptibility and disease progression. Advisor: Daniel Cioban

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.Facultad de Ciencias Veterinaria

Investigating CRESS DNA Viruses in Carnivores with emphasis on the family Circoviridae: Insights and Implications

The present thesis provides original data on the study of the molecular, epidemiological and phylogenetic patterns of CRESS DNA viruses and in particular of the members of the Circoviridae family in carnivores. The thesis was structured into chapters, in the format of a ‘thesis by publication’, encompassing articles published in international peer reviewed journals. Chapter 1 focuses on the investigation of CRESS DNA viruses in domestic carnivores. In Section 1.1 we describe a wide genetic diversity of Rep sequences of CRESS DNA viruses identified from feline samples. CanineCV-like sequences were identified from feline serum samples suggesting virus replication able to sustain viremia in cats. Moreover, the identification of a novel circovirus species named as feline Circovirus 1 (FeCV-1) was reported in cats in Italy. In Section 1.2 we evaluate the circulation of canine circovirus in dogs infected with canine parvovirus in Iran. Phylogenetic analysis showed that the Iranian CanineCV strains were more closely related to strains detected in Turkey. We provide new insights on the CanineCV’s molecular epidemiology and its role as a co-infection agent. Chapter 2 focuses on the detection of CRESS DNA viruses in wild carnivores. In Section 2.1 we describe the CAdV-1 and CanineCV circulation in wild carnivore populations in Italy. We report the detection of CanineCV in wolves and badgers and the first detection of cycloviruses in wolves in co-infection with CanineCV. In Section 2.2 we report the identification of a novel circovirus in the Iberian lynx population from different areas of Spain. The virus, termed ILCV-1, was repeatedly identified in the spleen samples, suggesting virus ability to replicate and spread actively in the host.The present thesis provides original data on the study of the molecular, epidemiological and phylogenetic patterns of CRESS DNA viruses and in particular of the members of the Circoviridae family in carnivores. The thesis was structured into chapters, in the format of a ‘thesis by publication’, encompassing articles published in international peer reviewed journals. Chapter 1 focuses on the investigation of CRESS DNA viruses in domestic carnivores. In Section 1.1 we describe a wide genetic diversity of Rep sequences of CRESS DNA viruses identified from feline samples. CanineCV-like sequences were identified from feline serum samples suggesting virus replication able to sustain viremia in cats. Moreover, the identification of a novel circovirus species named as feline Circovirus 1 (FeCV-1) was reported in cats in Italy. In Section 1.2 we evaluate the circulation of canine circovirus in dogs infected with canine parvovirus in Iran. Phylogenetic analysis showed that the Iranian CanineCV strains were more closely related to strains detected in Turkey. We provide new insights on the CanineCV’s molecular epidemiology and its role as a co-infection agent. Chapter 2 focuses on the detection of CRESS DNA viruses in wild carnivores. In Section 2.1 we describe the CAdV-1 and CanineCV circulation in wild carnivore populations in Italy. We report the detection of CanineCV in wolves and badgers and the first detection of cycloviruses in wolves in co-infection with CanineCV. In Section 2.2 we report the identification of a novel circovirus in the Iberian lynx population from different areas of Spain. The virus, termed ILCV-1, was repeatedly identified in the spleen samples, suggesting virus ability to replicate and spread actively in the host