Franzo and Segalés Virology Journal (2023) 20:26 Virology Journal https://doi.org/10.1186/s12985-023-01984-6 COMMENT Open Access Porcine circovirus 3 (PCV‑3) variability: Is it in the virus or in the classification criteria? Giovanni Franzo1* and Joaquim Segalés2,3,4* Abstract The continuous discovery of new viruses during the last decades has increased the need for new classification approaches and rules. Currently, the International Committee on Taxonomy of Viruses classifies viruses up to the species level. However, because of the higher variability of most of these infectious agents, a below-species cat- egorization is often required for proper epidemiological investigations. Unfortunately, variable criteria are typically proposed by different research groups, leading to misleading and poorly reproducible results. This scenario occurred for the recently identified Porcine circovirus 3. Although genotype definition standards had been defined by a group of experts in the field, recent articles have been published introducing new genotypes, whose classification rules are not reported. We therefore would like to stress the usefulness of defining and maintaining a common language to allow proper results comparison among groups. We consider the consensus opinion of a heterogeneous expert team as the most valuable approach. Nevertheless, if other approaches are proposed, the disclosure of the criteria and the comparison with previous literature should be deemed mandatory to allow effective results reproducibility, interpre- tation and sharing. Keywords Porcine circovirus 3 (PCV-3), Genotyping, Classification, Standardized criteria, Variability Background which are usually based on the increasing knowledge on The last decades have been featured by the discovery of genome sequences and their similarity rather than on an astonishing number of new viruses, often through phenotypic or clinical features. molecular techniques only. The need for organizing such According to the International Committee on Taxon- an amazing amount of new data has prompted an inten- omy of Viruses (ICTV, https://​ictv.​global/), viruses are sive classification or re-classification and naming efforts, classified up to the species level. However, because of the remarkable variability and high evolutionary rates of sev- eral viral species, the “species-level” resolution often fails *Correspondence: Giovanni Franzo to properly describe the epidemiological and biological giovanni.franzo@unipd.it features of most viruses infecting humans, animals or Joaquim Segalés plants. For this reason, different proposals of sub-species joaquim.segales@uab.cat 1 Department of Animal Medicine, Production and Health (MAPS), Padua classification have been suggested for most viruses, and University, 35020 Legnaro, Italy porcine circoviruses (PCV) are not an exception. 2 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, The most well-known PCV, Porcine circovirus 2 (PCV- Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Unitat Mixta d’Investigació IRTA‑UAB en Sanitat Animal, Centre de 2), was subjected to further subclassifications (clades, Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma genogroups, genotypes) since the first years after its iden- de Barcelona (UAB), 08193 Bellaterra, Spain 4 tification [1]. Although conflicting criteria and nomen- OIE Collaborating Centre for the Research and Control of Emerging and Re-Emerging Swine Diseases in Europe (IRTA-CReSA), clature were initially published [2–5], a consensus on 08193 Bellaterra, Spain sub-species (genotypes) classification criteria and names was reached among researchers over time [4]. Moreover, © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Franzo and Segalés Virology Journal (2023) 20:26 Page 2 of 3 the global convergence at the international level to spe- 3c) sequences are aligned, all their strains were closely cific criteria, including regular updates [6, 7], allowed related to the PCV-3a ones previously reported [9]. the establishment of a common language among people Moreover, the maximum genetic distance among these working in the field. reported ORF2 sequences was 0.0388 (3.8%), lower than A comparable scenario emerged with the identifica- the threshold suggested for a new genotype definition. tion of Porcine circovirus 3 (PCV-3) [8]. Based on the Therefore, based on the previously proposed criteria, all positive PCV-2 experience, sub-species classification cri- their sequences would be PCV-3a. Noteworthy, these teria and names were proposed by a team of circovirus authors [10] did not provide a comprehensive defini- experts from several research groups located in differ- tion of genotyping criteria in their article, which further ent parts of the world [9]. Briefly, the following criteria confuses the nomenclature and makes it difficult to com- were suggested and validated to define a PCV-3 geno- pare different studies. This scenario clearly complicates type: maximum within-genotype genetic distance of 3% the understanding of PCV-3 epidemiology and can be at the complete genome and 6% at the ORF2 levels and extremely misleading for researchers and, especially, for bootstrap support or posterior probability in the phylo- field veterinarians, farmers and commercial companies genetic tree higher than 90%. Finally, it was suggested to also. formally accept a genotype only if at least five sequences While we assume that, being not officially recognized, are available in gene databases, to avoid the risk of defin- every sub-species classification scheme should be scien- ing poor-quality sequences or extremely low-fitness tifically scrutinized, we consider counterproductive to strains as separate genotypes. According to these criteria, introduce new schemes in absence of reasonable biologi- consistent results were obtained using different analysis cal or epidemiological reasons to update or replace pre- methods and datasets, and a single genotype was pro- existing criteria. Additionally, when new genotypes are posed (named as PCV-3a). Although sub-clusters could reported, the criteria for such classification are typically be identified within PCV-3a genotype, the bootstrap sup- absent and no comparison with previous literature is per- port was often low and inconsistencies among different formed, making these approaches arbitrary and poorly datasets and analysis methods were observed, suggest- reproducible. ing caution in the recognition of additional genotypes. To facilitate and standardize further studies, a reference Conclusion dataset was provided in the original publication [9]. Based on these considerations, we believed it opportune to stress the need to comply with shared standards for Main text PCV-3 sub-species classification criteria, to allow the The efforts for unifying a classification at a viral sub- creation of a common language among researchers and species level are important to offer a common language people working in the field. Although not formally rec- and facilitate comparisons among epidemiological stud- ognized by the ICTV, the below-species level classifica- ies at regional, national and worldwide levels. Therefore, tion represents an invaluable tool for the understanding the objective of this Comment is to get the attention of of viral infection epidemiology and, potentially, for the international scientific community regarding the con- their control. Since no universal rules for such sub-spe- venience of keeping clear-cut classification criteria and cies level classification have been established for viruses, using them in subsequent studies, at least until potential we consider the consensus opinion of a heterogeneous new well-substantiated ones may replace the original cri- expert team the most valuable approach. Nevertheless, if teria. More specifically, this Comment wants to address other approaches are proposed, the disclosure of the cri- the PCV-3 classification used in some recently published teria and the comparison with previous literature should studies [10–12]. be deemed mandatory to allow results comparison and These studies [10–12] report a variable number of reproducibility. PCV-3 genotypes, with different names from those that have been previously proposed [9]. These works men- Author contributions tion up to three different genotypes (PCV-3a, 3b and 3c). GF performed the phylogenetic analysis; GF and JS interpreted the data and Since the existing data to date pointed out to one sin- wrote the manuscript. Both authors read and approved the final version of the gle genotype for PCV-3 (PCV-3a) [9], the description of manuscript. novel genotypes is of worldwide interest. As an example, Funding we would like to comment on the case of Epidemiologi- No funding was received for the present work. cal and genetic characteristics of Porcine circovirus 3 in 15 provinces and municipalities of China between 2016 and 2020 [10]. When the so-called genotype (PCV-3a, 3b and Franzo and Segalés Virology Journal (2023) 20:26 Page 3 of 3 Declarations Competing interests The authors declare that they have no competing interests. Received: 26 November 2022 Accepted: 5 February 2023 References 1. Segalés J, Kekarainen T, Cortey M. The natural history of Porcine circovirus type 2: From an inoffensive virus to a devastating swine disease? Vet Microbiol. 2013;165:13–20. https://​doi.​org/​10.​1016/j.​vetmic.​2012.​12.​033. 2. Gagnon CA, Tremblay D, Tijssen P, Venne MH, Houde A, Elahi SM. The emergence of Porcine circovirus 2b genotype (PCV-2b) in swine in Canada. Can Vet J. 2007;48:811–9. 3. Grau-Roma L, Crisci E, Sibila M, López-Soria S, Nofrarias M, Cortey M, et al. 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