Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2␣ (eIF2␣) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316 -323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca 2ϩ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca 2ϩ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca 2ϩ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca 2ϩ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca 2ϩ homeostasis and accumulation of ROS. IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca 2ϩ and ROS levels.Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca 2ϩ levels and cellular ROS levels as a result of activation of the PERK pathway.

Background Porcine circovirus type 3 (PCV3) was first reported in US in 2016. The virus was also identified later in China. Prevalence of PCV3 in Zhejiang province in southeastern China is not clear though it has been reported in many parts of China. Results PCV3 infection and its co-infection with other swine viral pathogens in pig herds of Zhejiang province were retrospectively investigated by quantitative PCR (qPCR) and its sero-prevalence by indirect ELISA. PCV3 was found positive in 67.1% of the 283 clinical samples taken from 2014 to 2017 as shown by qPCR. Single infection with PCV3 accounted for only one-third of the samples, and majority were of co-infections, predominantly with PEDV (41.6%) but generally low with other swine viruses. Indirect ELISA using the PCV3 capsid protein as the coating antigen revealed an average sero-positive rate of 52.6% (40.8 to 60.8%) in 2345 serum samples from 2011 to 2017, with earliest yet high positive findings in samples taken in 2012. Of 203 serum samples, the qPCR method showed more positive findings than ELISA (81.3% vs 56.2%). With 89 serum samples negative by ELISA, vast majority ( n = 81) were found positive by qPCR. There was negative correlation in levels of PCV3 DNA and anti-capsid antibody response. ORF2-based phylogenetic analysis revealed three major groups (PCV3a, PCV3b and PCV3c) of the 200 strains, 38 from this study and 162 reference strains from GenBank. Most of the strains from this study were clustered into PCV3c. Of the putative signature residues of the capsid protein (aa 24, 27, 77 and 150) relative to the three groups, only the PCV3a group strains showed a distinct pattern of residues VKSI (95% of the strains), while the other two groups did not have such a ‘signature’ pattern. Conclusions Results from this study provided further evidence that the novel virus PCV3 was widely distributed in China and might have emerged in Zhejiang province before 2014, most probably back in 2012 when there was high PCV3 sero-prevalence. PCV3 might be viremic in pigs and could spread by fecal shedding. Electronic supplementary material The online version of this article (10.1186/s12917-019-1977-7) contains supplementary material, which is available to authorized users.

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