Porcine Circovirus 2 Genotypes, Immunity and Vaccines: Multiple Genotypes but One Single Serotype

Identified for the first time in the 1990s, Porcine circovirus 2 (PCV-2) should not be considered an emerging virus anymore. Nevertheless, many aspects of its biology and epidemiology are still controversial. Particularly, its high evolutionary rate has caused the emergence of several variants and genotypes, alternating on the worldwide proscenium. The biological and practical implications of such heterogenicity are unfortunately largely unknown. The effectiveness of currently available vaccines against new genotypes that have emerged over time has been the topic of an intense debate and often inconclusive or contradictory results between experimental, field, and epidemiological studies have been gathered. The challenge in establishing an effective PCV-2 disease model, the peculiarities in experimental design and settings and the strains involved could justify the observed differences. The present work aims to summarize and critically review the available knowledge on PCV-2 genetic heterogeneity, immunity, and vaccine efficacy, organizing and harmonizing the available data from different sources, shedding light on this complex field and highlighting current knowledge gaps and future perspectives. So far, all vaccines in the market have shown great efficacy in reducing clinical signs associated to diseases caused by PCV-2, independently of the genotype present in the farm. Moreover, experimental data demonstrated the cross-protection of PCV-2a vaccines against the most widespread genotypes (PCV-2a, PCV-2b, and PCV-2d). Therefore, despite the significant number of genotypes described/proposed (PCV-2a to PCV-2i), it seems one single PCV-2 serotype would exist so far.info:eu-repo/semantics/publishedVersio

Phylodynamic analysis of porcine circovirus type 2: Methodological approach and datasets

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) [1], “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) [2]). 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”[3]). 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

Phylodynamic analysis of porcine circovirus type 2 reveals global waves of emerging genotypes and the circulation of recombinant forms

Since the first description of Porcine circovirus type 2 (PCV2), four genotypes (PCV2a, PCV2b, PCV2c and PCV2d) have been recognized and three of them have been shown to exhibit worldwide distribution. Here, the population dynamics of PCV2 has been reconstructed over time and the factors that have shaped its evolution determined. The results obtained confirm that PCV2 originated approximately at the beginning of the 20th century. The most recent common ancestor of genotypes PCV2a, PCV2b, PCV2c and PCV2d circulated in the 1950s, 1980s, 1960s and 1950s, respectively, and the population sizes of the individual genotypes remained low until the mid 90s, coinciding with the identification of PCV2 as a major pathogen of the pig industry. The population dynamics of PCV2 have been characterized by the appearance of periodic waves of distinct genotypes that, after an initial rise, spread following major swine commercial routes and were then superseded by subsequent emerging genotypes. Various recombinant forms displayed comparable population dynamics and spreading routes to those of major genotypes, suggesting that recombinant strains are able to compete with parental ones. The capsid gene is subjected to immune selection and evasion of the host immune response seems to be a major force for the emergence and spread of new genotypes. In contrast, the evolution of other genes appears to be constrained by the particular genomic organization of PCV2. In summary, obtained results suggest that changes in farming strategies, international trade, host population immunity, recombination and the constraints imposed by genome organization have all played a major role in the evolutionary dynamics of PCV2

Further insight into the molecular epidemiology and evolutionary dynamics of rapidly evolving RNA and ssDNA viruses

Rapidly evolving RNA and ssDNA viruses represent a fascinating field characterized by a strict interconnection between the “speculative” study of viral evolution and its practical implications in everyday veterinary medicine. This thesis has been thought as a collection of manuscripts that aimed to investigate different aspects and levels of viral evolution while still maintaining a focus on practical repercussions. Even if different infectious diseases and etiological agents are considered, they are all functional to the study of different aspects and implications of rapid virus evolution. Considering the heterogeneous nature of the manuscripts, they have been organized according to a “crescent” scale, starting from the lowest scale of viral evolution and progressing to broader scales. The manuscript “Viral subpopulations in aMPV vaccines are unlikely to be responsible for reversion to virulence.” addresses a fine-level analysis of the population structure of the AMPV subtype B live attenuated vaccine and its potential role in the previously demonstrated phenomenon of reversion to virulence. The widespread administration of live attenuated vaccines, despite their obvious advantages in terms of reducing disease prevalence, clinical signs and economic losses, is associated with costs that are not limited to the risk of reversion to virulence or to direct economic costs. Based on a wide collection of Italian samples, “Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype’s disappearance” reports the impact of these vaccines in complicating the diagnostic process and, as a consequence, the interpretation of the epidemiological scenario in the absence of known vaccine markers. Obviously, updated knowledge of the strains currently circulating in a particular area is of great relevance for the implementation of proper control strategies. With this aim in mind, a field survey, which is published in “Field survey of Avian Metapneumovirus in Northern Italy”, was conducted on hundreds of Italian farms to estimate and characterize the AMPV strains circulating in our country. To further support frequent and extensive surveys, an assay that is able to detect, quantify and genotype the two AMPV subtypes currently circulating in Italy was developed and validated. Because economic constraints often represent a major limit, efforts were made to reduce the assay costs compared with other real-time RT-PCR methods while still guaranteeing comparable or superior performances (“A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B”). Unfortunately, the diagnosis of rapidly evolving RNA viruses is itself an arduous task that requires a continuous evaluation and updating of diagnostic tools, even in laboratory that receive samples from limited geographic areas. The manuscripts entitled “Observation of high recombination occurrence of Porcine reproductive and respiratory syndrome virus in field condition” and “Phylodynamic analysis of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy: action of selective pressures and interactions between different clades.” address the study of the molecular epidemiology of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy considering the evolutionary forces driving PRRSV evolution at the local scale (i.e., high substitution rate, recombination, interaction between different clades and action of selective pressures). The high heterogeneity of PRRSV in the national contest was then evaluated with respect to the challenges that it poses in the development and validation of RT-PCR- and qRT-PCR-based diagnostic methods (“Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus”) and assessing its impact on diagnostic accuracy (“The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances”). Similarly, “International trades, local spread and viral evolution: the case of Porcine circovirus type 2 (PCV2) strains heterogeneity in Italy” investigates the genetic variability of PCV2 within national borders and compares it with the knowledge of its molecular epidemiology available from other countries. This study provides evidence regarding the role of both “in loco” evolution and importation of different genotypes and strains from foreign countries in determining the Italian PCV2 genetic heterogeneity. The crescent amount of PCV2 sequences deposited in publically available databases has revealed its marked variability and challenged the current classification criteria. Nevertheless, at least a superficial knowledge of the PCV2 molecular epidemiology is mandatory for the planning and evaluation of control strategies. “Revisiting the Taxonomical classification of PCV2: still a real challenge” proposes new criteria for the classification of PCV2 into different genotypes. Our aim was to provide a scheme that both accounts for the constraint imposed by the biological proprieties of this virus and allows a rapid, practical and easy way to classify PCV2 strains even during routine diagnostic activity. Last, “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” investigates more speculative issues inherent to the PCV2 origin. The discovery of a PCV2c genotype, which, to date, was believed to be extinct, in a feral pig population characterized by a peculiar population history and by a complex, and still partially known, relationship network with other PCV2-susceptible species opens exciting scenarios concerning the history and origin of PCV

Genomic and structural investigation on dolphin morbillivirus (DMV) in Mediterranean fin whales (Balaenoptera physalus).

Dolphin morbillivirus (DMV) has been deemed as one of the most relevant threats for fin whales (Balaenoptera physalus) being responsible for a mortality outbreak in the Mediterranean Sea in the last years. Knowledge of the complete viral genome is essential to understand any structural changes that could modify virus pathogenesis and viral tissue tropism. We report the complete DMV sequence of N, P/V/C, M, F and H genes identified from a fin whale and the comparison of primary to quaternary structure of proteins between this fin whale strain and some of those isolated during the 1990-'92 and the 2006-'08 epidemics. Some relevant substitutions were detected, particularly Asn52Ser located on F protein and Ile21Thr on N protein. Comparing mutations found in the fin whale DMV with those occurring in viral strains of other cetacean species, some of them were proven to be the result of diversifying selection, thus allowing to speculate on their role in host adaptation and on the way they could affect the interaction between the viral attachment and fusion with the target host cells

Multilocus Sequence Typing of Commensal and EnteropathogenicEscherichia Colifrom Domestic and Wild Lagomorphs in Italy

The aim of the study was to determine the multilocus sequence types of Escherichia coli from diseased farm rabbits and apparently healthy wild lagomorphs, and the genetic relatedness among them. Fifty-five enteropathogenic E. coli from reared rabbits and 32 from wild rabbits and hares were characterised by multilocus sequence typing (MLST) according to the Michigan State University EcMLST scheme. Isolates were differentiated into 37 sequence types (STs), which were grouped into 8 clonal complexes (CCs). The most common ST was ST140 (CC31), followed by ST238 and ST119 (CC17). MLST analysis revealed 22 novel STs. Phylogenetic analyses showed a heterogeneous distribution of STs into 3 clusters of genetically related strains. The genetic relationship among STs of different origin and the detection of new, as well as previously described STs as human pathogens, indicate a widespread distribution and adaptability of particular lineages to different hosts. These findings highlight the need for further research to improve the knowledge about E. coli populations colonising the gut of lagomorphs and their zoonotic potential

Porcine circovirus 2 (PCV-2) genotype update and proposal of a new genotyping methodology

Porcine circovirus 2 (PCV-2) is one of the most widespread viral infections of swine, causing a remarkable economic impact because of direct losses and indirect costs for its control. As other ssDNA viruses, PCV-2 is characterized by a high evolutionary rate, leading to the emergence of a plethora of variants with different biological and epidemiological features. Over time, several attempts have been made to organize PCV-2 genetic heterogeneity in recognized genotypes. This categorization has clearly simplified the epidemiological investigations, allowing to identify different spatial and temporal patterns among genotypes. Additionally, variable virulence and vaccine effectiveness have also been hypothesized. However, the rapid increase in sequencing activity, coupled with the per se high viral variability, has challenged the previously established nomenclature, leading to the definition of several study-specific genotypes and hindering the capability of performing comparable epidemiological studies. Based on these premises, an updated classification scheme is herein reported. Recognizing the impossibility of defining a clear inter-cluster p-distance cut-off, the present study proposes a phylogeny-grounded genotype definition based on three criteria: maximum intra-genotype p-distance of 13% (calculated on the ORF2 gene), bootstrap support at the corresponding internal node higher than 70% and at least 15 available sequences. This scheme allowed defining 8 genotypes (PCV-2a to PCV-2h), which six of those had been previously proposed. To minimize the inconvenience of implementing a new classification, the most common names already adopted have been maintained when possible. The analysis of sequence-associated metadata highlighted a highly unbalanced sequencing activity in terms of geographical, host and temporal distribution. The PCV-2 molecular epidemiology scenario appears therefore characterized by a severe bias that could lead to spurious associations between genetic and epidemiological/biological viral features. While the suggested classification can establish a “common language” for future studies, further efforts should be paid to achieve a more homogeneous and informative representation of the PCV-2 global scenario.info:eu-repo/semantics/publishedVersio

Porcine circovirus 3 (PCV-3) as a causal agent of disease in swine and a proposal of PCV-3 associated disease case definition

Porcine circovirus 3 (PCV-3) was discovered in 2015 using next-generation sequencing (NGS) methods. Since then, the virus has been detected worldwide in pigs displaying several clinical-pathological outcomes as well as in healthy animals. The objective of this review is to critically discuss the evidence existing so far regarding PCV-3 as a swine pathogen. In fact, a significant number of publications claim PCV-3 as a disease causal infectious agent, but very few of them have shown strong evidence of such potential causality. The most convincing proofs of disease association are those that demonstrate a clinical picture linked to multisystemic lymphoplasmacytic to lymphohistiocytic perivascular inflammation and presence of viral nucleic acid within these lesions. Based on these evidence, individual case definitions for PCV-3-reproductive disease and PCV-3-systemic disease are proposed to standardize diagnostic criteria for PCV-3-associated diseases. However, the real frequency of these clinical-pathological conditions linked to the novel virus is unknown, and the most frequent outcome of PCV-3 infection is likely subclinical based on its worlwide distribution.info:eu-repo/semantics/publishedVersio

Genomic and structural investigation on Dolphin Morbillivirus (DMV) in Mediterranean fin whales (Balaenoptera physalus)

Dolphin Morbillivirus (DMV) has been deemed as one of the most relevant threats for fin whales (Balaenoptera physalus) being responsible for a mortality outbreak in the Mediterranean Sea in the last years. Knowledge of the complete viral genome is essential to understand any structural changes that could modify virus pathogenesis and viral tissue tropism. We report the complete DMV sequence of N, P/V/C, M, F and H genes identified from a fin whale and the comparison of primary to quaternary structure of proteins between this fin whale strain and some of those isolated during the 1990-‘92 and the 2006-‘08 epidemics. Some relevant substitutions were detected, particularly Asn52Ser located on F protein and Ile21Thr on N protein. Comparing mutations found in the fin whale DMV with those occurring in viral strains of other cetacean species, some of them were proven to be the result of diversifying selection, thus allowing to speculate on their role in host adaptation and on the way they could affect the interaction between the viral attachment and fusion with the target host cells

Porcine circovirus 3 (PCV-3) as a causal agent of disease in swine and a proposal of PCV-3 associated disease case definition

Altres ajuts: Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria RTA2017-00007-00-00Porcine circovirus 3 (PCV-3) was discovered in 2015 using next-generation sequencing (NGS) methods. Since then, the virus has been detected worldwide in pigs displaying several clinical-pathological outcomes as well as in healthy animals. The objective of this review is to critically discuss the evidence existing so far regarding PCV-3 as a swine pathogen. In fact, a significant number of publications claim PCV-3 as a disease causal infectious agent, but very few of them have shown strong evidence of such potential causality. The most convincing proofs of disease association are those that demonstrate a clinical picture linked to multisystemic lymphoplasmacytic to lymphohistiocytic perivascular inflammation and presence of viral nucleic acid within these lesions. Based on these evidence, individual case definitions for PCV-3-reproductive disease and PCV-3-systemic disease are proposed to standardize diagnostic criteria for PCV-3-associated diseases. However, the real frequency of these clinical-pathological conditions linked to the novel virus is unknown, and the most frequent outcome of PCV-3 infection is likely subclinical based on its worlwide distribution