Porcine circovirus 3 (PCV3) is a new species of the Circovirus genus, which has recently been associated with different clinical syndromes. Its presence has been reported in different countries of North and South America, Asia and recently also Europe (Poland). However, different from the other continents, no European PCV3 sequence is currently available in public databases. There is a strong need of epidemiological data and full-genome sequences from Europe because of its relevance in the understanding of PCV3 molecular epidemiology and control. To fill this lack of information, samples collected in Denmark, Italy and Spain in 2016 and 2017 were screened for PCV3. Of the Danish samples, 36 of 38 the lymph nodes, six of 20 serum samples and two of 20 lung samples tested positive. Similarly, 10 of 29 lungs, 20 of 29 organ pools, six of 33 sera and one of eight nasal swabs tested PCV3 positive in Italy. Fourteen of 94 serum pools from seven of 14 Spanish farms were also positive. Despite the convenience nature of the sampling prevents any precise prevalence estimation, the preliminary screening of the data from three European countries confirmed a rather wide PCV3 distribution in Europe. Furthermore, the analysis of the six obtained complete European PCV3 genomes and their comparison with the public available sequences seems to support a remarkable worldwide PCV3 circulation. These results underline once more the urgency of more extensive epidemiological studies to refine the current knowledge on PCV3 evolution, transmission, spreading patterns and impact on pig health.

Porcine circovirus 3 (PCV-3) is a recently described virus belonging to the family Circoviridae. It represents the third member of genus Circovirus able to infect swine, together with PCV-1, considered non-pathogenic, and PCV-2, one of the most economically relevant viruses for the swine worldwide industry. PCV-3 was originally found by metagenomics analyses in 2015 in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. The lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that PCV-3 might etiologically be involved in disease occurrence. Subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that PCV-3 was already present in pigs by early 1990s. In fact, current evidence suggests that PCV-3 is a rather widespread virus worldwide. Recently, the virus DNA has also been found in wild boar, expanding the scope of infection susceptibility among the Suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. Phylogenetic studies with available PCV-3 partial and complete sequences from around the world have revealed high nucleotide identity (>96%), although two main groups and several subclusters have been described as well. Moreover, it has been proposed the existence of a most common ancestor dated around 50 years ago. Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. Therefore, the present review will update the current knowledge and future trends of research on PCV-3.

Porcine circovirus 3 (PCV-3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV-3 infection dynamics. Finally, to elucidate PCV-3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV-3 DNA was extracted and amplified with a conventional PCR. For the PCV-3 PCR-positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV-3 PCR-positive samples from different years, different time-points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV-3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long-term infections, since 5 out of 10 PCV-3 PCR-positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV-3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV-3 PCR-positive samples was moderate to low. All partial and complete PCV-3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV-3 infection, showing high frequency of detection in this animal species. Furthermore, PCV-3 can be found in different tissues of wild boar and is apparently able to cause persistent infection.

SummaryPorcine circovirus 3 (PCV-3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. In this study, sera samples originating from 654 pigs of different production phases and clinical/pathological conditions, submitted for diagnostic purposes between 1996 and 2017, were randomly selected. Detection of PCV-3 genome in such samples was attempted with a previously described PCR method, and the par- Although the information obtained was limited, PCV-3 did not appear to be linked to any specific pathological condition or age group. K E Y W O R D Spolymerase chain reaction, Porcine circovirus 3, retrospective, sequencing, Spain

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Porcine circovirus type 3 (PCV-3) is a recently discovered virus in domestic pigs and wild boar. The virus has been described in pigs with different clinical/pathological presentations and healthy animals, but the dynamics of infection is currently unknown. The aim of this study was to longitudinally monitor PCV-3 infection in 152 pigs from four different healthy farms (A, B, C and D) by means of PCR in serum. The selected animals were sampled five (farm A) or six (farms B–D) times from weaning until the end of the fattening period. PCV-3 genome was found in pigs from all tested ages and farms; few animals had an apparent long-term infection (4–23 weeks). PCV-3 frequency of detection remained fairly uniform along tested ages within farms A and C, but was more variable among sampling times in farms B and D. Eight partial genome sequences were obtained from six different animals. Phylogenetic tree and pairwise distance analysis showed high similarity among sequences and with available genomes from different countries. This is the first study on PCV-3 infection dynamics in longitudinally sampled pigs. Most pigs got infection during their life, although PCV-3 did not appear to be linked with any specific age.

Porcine circovirus 3 (PCV-3) has been suggested as a putative causal agent of swine reproductive disease. A number of different studies have pointed out this association, but there is still a lack of information regarding the normal rates of PCV-3 infection in farms with normal reproductive parameters. The objective of the present study was to assess the frequency of PCV-3 detection in primiparous and multiparous sows and in tissues from their respective fetuses from farms with average reproductive parameters. Sera from 57 primiparous and 64 multiparous sows from 3 different farms were collected at two time points. Brain and lung tissues from 49 mummies and 206 stillborn were collected at farrowing. Samples were tested by PCR, and when positive, quantified by quantitative PCR. Thirty-nine complete genomes were obtained and phylogenetically analyzed. All sera from multiparous sows were negative, while 19/57 (33.3%) primiparous sows were PCV-3 PCR positive. From the 255 tested fetuses, 86 (33.7%) had at least one tissue positive to PCV-3. The frequency of detection in fetuses from primiparous sows (73/91, 80.2%) was significantly higher than those from multiparous ones (13/164, 7.9%). It can be concluded that PCV-3 is able to cause intrauterine infections in absence of overt reproductive disorders.

Molecular‐based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described ‘gold standard’ methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV‐3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real‐time qPCR (qPCR) for detection of PCV‐3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84–100) and qPCR (96%; PCI% 90–100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV‐3 in Italian swine population. Significance and Impact of the Study The continuous discovery of new pathogens poses a challenge in the development and evaluation of adequate diagnostic tools. In fact, since molecular‐based tools sometimes are the only available laboratory techniques, it is typically difficult to evaluate their diagnostic performances in the absence of a gold standard. The present study assesses this issue, demonstrating the excellent performances of two PCR‐based assays for porcine circovirus 3 (PCV‐3) detection using a Bayesian latent class analysis approach. Therefore, the molecular tests evaluated under this study constitute reliable tools for the routine diagnosis and surveillance programs of PCV‐3 circulating in swine populations.