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

Abstract

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.

Fig 1. Intra and inter-cluster pairwise p-distances among ORF2 sequences.

Density plot reporting the pairwise p-distance distribution within and between clusters. Different clusters have been color-coded.

Fig 2. PCV-2 Neighbor-Joining phylogenetic tree.

Neighbor-Joining phylogenetic tree reconstructed based on row genetic distances (i.e. pairwise p-distance) calculated on a collection of strains representative of the proposed PCV-2 genotypes. Both the cluster and genotype nomenclature are reported. Bootstrap support is displayed near the corresponding node. A tree reconstructed based on a more extensive sequence collection (n = 2610) is provided as S1 Fig.

Fig 3. Count of sequences for each collection year.

Different clusters have been color-coded.

Fig 4. Geographic distribution of major PCV-2 clusters.

The PCV-2 cluster distribution has been summarized using continent-specific pie-charts whose slide size is proportional to the cluster frequency (color-coded).