Claudia Maria Tucciarone

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.

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.

Effect of different vaccination strategies on IBV QX population dynamics and clinical outbreaks.

Abstract The extreme variability and rapid evolution of Infectious bronchitis virus (IBV) has always represented the key challenge for its control because of the limited cross-protection among different strains. Several experimental trials have proven a broadening of the protection spectrum when animals are vaccinated with multiple genotypes. Nevertheless, the conditions of vaccine administration in field are so different that the generalization of experimental results is, at least, questionable. In the present study a large scale epidemiological-phylodynamic approach was used to reconstruct the demographic history of the major field genotype (i.e. the QX one) circulating in Italy and Spain. These two countries were selected because, even if they share a comparable epidemiological scenario, the implemented vaccination protocols did not vary in Spain while changed dramatically in Italy over the time period considered. One hundred and ninety-five Italian and 98 Spanish non-recombinant sequences of the hyper-variable region of the S1 gene obtained between 2012 and 2016 were analyzed using a serial coalescent-based approach to reconstruct viral population history over time. While the IBV QX population dynamics remained constant in Spain, a much more complex pattern was evidenced in Italy; both in terms of viral population size and clinical outbreak frequency. Remarkably, a strong association with changes in vaccination strategies was recognized. This allowed demonstrating, by accomplishing all Hill’s criteria for causation, the cause-effect relationship between the vaccine administration/withdrawal and the variation in viral population dynamics and, above all, IBV related outbreaks. Thus, a robust confirmation about the efficacy of IBV vaccination in field conditions was provided. Additionally, the history herein reported testifies the primary importance of rigorously planning not only the intervention strategies but also their monitoring and evaluation.

Porcine circovirus type 3: a threat to the pig industry?

The recent discovery of porcine circovirus 3 (PCV3) has caused some concern among field veterinarians and brought the attention of the scientific world, likely because of some similarities with porcine circovirus 2 (PCV2). Several manuscripts reporting the detection of PCV3 and the presence of different clinical conditions have been published to date.1–5 A priori emphasis has been attributed to the association between PCV3 infections and clinical pathological conditions, even if little evidence is currently available to sustain this association. We conducted a brief exploratory study in northern Italy: 116 samples (from 17 gilts, …

Canine parvovirus type 2 (CPV-2) and Feline panleukopenia virus (FPV) codon bias analysis reveals a progressive adaptation to the new niche after the host jump

Based on virus dependence from host cell machinery, their codon usage is expected to show a strong relation with the host one. Even if this association has been stated, especially for bacteria viruses, the linkage is considered to be less consistent for more complex organisms and a codon bias adaptation after host jump has never been proven. Canine parvovirus type 2 (CPV-2) was selected as a model because it represents a well characterized case of host jump, originating from Feline panleukopenia virus (FPV). The current study demonstrates that the adaptation to specific tissue and host codon bias affected CPV-2 evolution. Remarkably, FPV and CPV-2 showed a higher closeness toward the codon bias of the tissues they display the higher tropism for. Moreover, after the host jump, a clear and significant trend was evidenced toward a reduction in the distance between CPV-2 and the dog codon bias over time. This evidence was not confirmed for FPV, suggesting that an equilibrium has been reached during the prolonged virus-host co-evolution. Additionally, the presence of an intermediate pattern displayed by some strains infecting wild species suggests that these could have facilitated the host switch also by acting on codon bias.

Development and validation of direct PCR and quantitative PCR assays for the rapid, sensitive, and economical detection of porcine circovirus 3:

Since the identification of species Porcine circovirus 2, the relevance of genus Circovirus has increased given its impact on the swine industry. A new species (Porcine circovirus 3, PCV-3) has been detected in association with various clinical conditions. Consequently, there is an urgent need for reliable and widely accessible tests for both routine diagnostic and research purposes. We developed a direct PCR (requiring no DNA extraction) and a quantitative (q)PCR targeting the conserved rep gene to detect the PCV-3 genome. Test performance was assessed by testing 120 field samples within different matrices. Both methods were sensitive (detection of 10 viral genome/µL), specific, and repeatable. The substantially perfect agreement between the 2 assays strongly supports their high sensitivity and specificity. The low cost and short processing time of the direct PCR protocol, together with the reliable quantitative results provided by qPCR, support the establishment of common testing guidelines.

Think globally, act locally: Phylodynamic reconstruction of infectious bronchitis virus (IBV) QX genotype (GI-19 lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales.

Infectious bronchitis virus (IBV) represents one of the poultry industry major threats, particularly in high density producing countries. The emergence and spread of new IBV genotypes have frustrated the various disease control efforts implemented over time. Despite that, few comprehensive and large scale studies have been performed to understand the international and local spreading dynamics of this virus. In the present work, these phenomena were evaluated by implementing a Bayesian phylodynamic approach to reconstruct the epidemiological patterns and population history of the QX genotype (currently renamed GI-19 lineage), the most relevant IBV lineage of the Old-World. Our analysis, based on 807 partial S1 sequences of strains collected from 18 countries between 1993 and 2015, demonstrates that this genotype originated in China well before its first identification. After a prolonged local circulation, it started spreading to other European, Asian and Middle East countries in successive waves, which were mirrored by concomitant fluctuations in viral population size. Interestingly, the within-Europe spread was characterized by a higher estimated migration rate compared with the inter-continental one, potentially reflecting the closer geographic and economic relationships among these countries. Nevertheless, the colonization of new states by the GI-19 lineage appeared to occur mostly by single introduction events in both intra and inter-continental spread, likely because of epidemiological factor and health policy combination which seems to prevent the frequent introduction and mixing of different strains. On the other hand, the within Italy QX circulation reconstruction showed a much more intricate connection network among different locations, evidencing the difficulty in controlling IBV spread especially in highly densely poultry populated areas. The presence of several well supported epidemiological links among distantly related Italian regions testifies that animal transportation and indirect transmission routes rather than local airborne diffusion contribute to the QX success and persistence at local scale. Globally, the spreading dynamics and evolution of the QX genotype were reconstructed from its very origin to nowadays, demonstrating the need of more effective direct control measures, particularly within each country. Unfortunately, the incompleteness of available molecular epidemiology data represents an insurmountable limit which leaves many questions currently unsolved, thus highlighting the compulsoriness of a structured monitoring and data sharing system implementation.

Reconciling individual and population levels of porcine reproductive and respiratory syndrome virus evolution

In summary,the present study demonstrates that the forces favoring intra-animal diversification can cause the emergence of variants that,benefiting of their diversity at the population level,are able to succeed and spread in an often highly homogeneous environment,and to be further selected later.

Infectious bronchitis virus in Romanian broiler farms

genotypes and the percentage sequence identity between obtained samples and vaccine strains was 100 per cent, suggesting the detection of the vaccine strain (Franzo and others 2014). However, four strains were classified as variant 2 (GI-23); a genotype initially identified in Israel in 1998 and then progressively detected wider in the Middle East. Recent reports suggest it has reached Eastern Europe (Domanska-Blicharz and others 2016). Due to the clinical relevance of this genotype, different homologous vaccines have been developed; however, none is currently licensed in Romania. Full S1 gene sequencing (accession numbers: MF101744, MF101745, MF101746, MF101747) and comparison with the reference dataset suggested by Valastro and others (2016) and with other sequences available from Genbank confirmed the clustering with GI-23 strains (Supplement 1). Phylogenetic analysis and pairwise genetic and amino acid (AA) distance analysis revealed a high similarity between the detected strains with those recently reported in Poland (Domanska-Blicharz and others 2016) (p-distance of 0.001 and 0.000 for RNA and AA sequences, respectively). Comparison with the most closely related commercial vaccine (TAbic IB VAR206; Phibro Vaccines) revealed a p-distance of 0.006 and 0.01 at RNA and AA level, respectively, which was higher than the distance from the progenitor of the same vaccine (RNA p-distance of 0.005, AA p-distance of 0.006). These data, coupled with the knowledge of the applied vaccine protocols, support the field origin of the detected strains. In most instances, the variant 2-infected flocks showed clinical signs, confirming the virulence of this IBV lineage. The results of our monitoring activity, which detected and characterised Romanian variant 2 (GI-23) strains, corroborate the rising evidence of variant 2 genotype spreading westward through Europe.

First Report of Avian Metapneumovirus Subtype B Field Strain in a Romanian Broiler Flock during an Outbreak of Respiratory Disease

SUMMARY Avian metapneumovirus (aMPV) represents one of the most prevalent diseases of turkey, especially in combination with other pathogens, and its frequency is also increasing among chickens. Despite this evidence, epidemiologic data are poor and scattered, severely preventing control of the disease even in highly developed areas such as Europe. In the present study, the detection and characterization of an aMPV subtype B strain circulating in a vaccinated but symptomatic Romanian broiler flock is reported for the first time. The phylogenetic analysis based on the partial G gene sequence demonstrates the close relationship of the Romanian virus with a group of recently emerged Italian field strains for which vaccine-induced protection was experimentally proven to be partial. These preliminary results allow us to hypothesize the spreading of vaccine-escaping aMPV subtype B strains through Europe and, consequently, dictate the carrying out of a more systematic survey to confirm this theory and enforce adequate countermeasures.