Despite its recent discovery in 2014 in China, the PPV6 has been found in many countries around the world (Kim et al. 2021; Miłek et al. 2019; Ni et al. 2014; Schirtzinger et al. 2015). During the large-scale studies in Poland, PPV6 was identified on 16 farms out of 19 and was the second most detected virus in 10.7% (n = 150) of oral fluids pools, 10.0% (n = 254) of serum pools, and 3.6% (n = 252) of fecal pools (Miłek et al. 2019). A high detection rate of PPV6 (21.5%; n = 1000) was also demonstrated in South Korea by Kim S.C. et al. (2022). In addition, 16.3% (n = 171) of positive cases were reported in China (Ni et al. 2014). However, there is no data on the PPV6 circulation in Russia. Consequently, the purpose of our study was to investigate whether the virus was circulating in pig farms in Russia and study its prevalence in some regions.
The results of our studies showed that 9.4% (n = 521) of tested samples were positive for PPV6. Viral DNA was found in four regions out of seven: 18.2% (n = 154) in the Krasnoyarsk Krai, 14.3% (n = 91) in Tomsk, 11.9% (n = 59) in Kemerovo, and 2.4% (n = 42) in the Moscow Regions, respectively. Phylogenetic analysis of the capsid protein gene of the Kem-8 isolate showed complete nucleotide identity with the Spanish isolate 3456/2012 (MH558679), which is close to the Polish isolates. The 3456/2012 isolate from Spain was detected in pools of tissue from healthy animals, and the association between PPV6 infection and clinical manifestations in pigs has not been reported (Franzo et al. 2019). The clade also included the Canadian isolate SK CA (MH820262), the US isolates KSU4-NE (KR709265) and KSU7-SD (KR709268).
It is known that the application of contaminated semen could lead to rapid pathogen transmission and disease outbreaks among sows (Maes et al. 2016). During PPV1 infection, it has been confirmed that boars excrete the virus with semen and play a certain role in the virus dissemination (Maes et al. 2016; Mengeling and Cutlip 1976). For PPV6, these data have not been studied. The detection of PPV6 in boars imported for reproduction is of scientific interest and highlights the particular need to test them for various viral infections, including novel parvoviruses, before being used in insemination.
The serum samples from Krasnoyarsk Krai were obtained from pigs of three different age groups: weaned piglets, fattening piglets, and sows. Therefore, we decided to analyze the spread of PPV6 among them. We determined that the PPV6 prevalence in fattening piglets (aged 3–9 months) was 32.2% (n = 59) and in sows (aged 2–3 years) − 20.9% (n = 43), respectively. In weaned piglets (aged 20–60 days), the PPV6 DNA was not detected. Similar results of PPV6 distribution among fattening piglets were reported in the investigations of Miłek D. et al. (2019) and Ni J et al. (2014). A low prevalence of PPV6 among weaned piglets (3.6%) and sows (10.5%) and a high prevalence among fattening piglets (21.5–36.5%) were determined in the studies of Kim S.C. et al. (2022). It can be speculated that the significant prevalence of PPV6 among fattening piglets is due to the decrease in maternal antibodies level (Miłek et al. 2019). It is assumed, that a significantly higher virus detection rate in sows may indicate a chronic course of the infection (Miłek et al. 2019). However, the pathogenic role of PPV6 in the porcine organism has not been determined yet, as the virus has never been isolated in cell cultures and the experimental infection of pigs has not been carried out.
Ni J. et al. (2014) attempted to isolate PPV6 in PK-15, Vero, and Marc-145 cell cultures. However, the virus did not replicate in these cultures. In order to identify the cell system that provides the highest accumulation of the virus, we carried out PPV6 isolation in the continuous cell cultures (SPEV, SK and ST). As the concentration of viral DNA in the Kem-8 sample was the highest, we decided to use it for virus isolation. It is known that for the reproduction of parvoviruses in cell cultures, components of the DNA-synthesizing apparatus of the host cell, in particular alpha and gamma DNA polymerases, which are synthesized in the S-phase of the cell cycle, are needed (Mészáros et al. 2017; Orlyankin et al. 1984). Therefore, cell cultures with high mitotic activity are required for virus isolation. Consequently, the infection of cell cultures must be carried out at the time of the cells’ seeding or 20–24 h after it (Elias et al. 2004; Orlyankin et al. 1984). We introduced PPV6 to the cell suspension at the time of seeding in the plate without prior adsorption of the virus by cells. In each cell line, we conducted six serial passages. For each new passage of the virus, a cell lysate from the previous passage obtained by three cycles of freezing and thawing was used. During microscopic examination, we did not observe obvious differences between infected and mock-infected cell cultures. In the case of PPV1 infection, the cytopathogenic effect (CPE) in cell culture appeared when cells were infected with a high multiplicity of infection (MOI) At low MOI, CPE does not appear (Fernandes et al. 2017; Orlyankin et al. 1984).
It is known that cell apoptosis is a hallmark of all known parvoviruses (Jager et al. 2021; Mészáros et al. 2017; Streck and Truyen 2020; Zhang et al. 2015; Zhao et al. 2016). To identify, whether the apoptotic changes occurred in infected cell cultures, we applied acridine orange staining. This dye, when embedded in a DNA molecule, helps to detect apoptotic cells by the characteristic bright yellow-green glow of the nucleus. Viable cells exhibit diffuse green fluorescence (Bartzatt 2016; Zhao et al. 2016). We also observed a bright yellow-green DNA fluorescence in infected cell cultures, which indicated an increase in the amount of DNA in the nuclei of infected cell cultures compared to the mock-infected ones. In the ST cell culture, apoptosis was less manifest. By qPCR, we also determined that PPV6 did not accumulate in this culture as the level of Ct increased with each passage. So, further investigation of PPV6 in this culture was suspended. The qPCR results showed that the most promising cultures for PPV6 isolation are SPEV and SK. During virus cultivation, it is very important to pay attention to the cells’ concentration and to their division process, as in cell culture with a formed monolayer, PPV6 does not replicate. These results are consistent with the literature data reporting that parvoviruses practically do not replicate in cell cultures with a complete monolayer (Mészáros et al. 2017; Orlyankin et al. 1984; Streck et al. 2015; Zhang et al. 2015).
Staining of infected cells with Azure-Eosin allowed us to study in detail the morphological changes that occurred in PPV6-infected cells. We observed common signs of PPV6 infection in SPEV and SK cell cultures as swelling or enlargement of nuclei and nucleoli; pyknosis of nuclei; DNA fragmentation, chromatin margination, nuclear condensation; formation of symplasts containing 5 to 7 nuclei; and an increase in the number of various pathological mitoses. In some infected cells, we have also noted colchicine-like metaphases and metaphase lagging, which are characteristics of tumor cells. It is known that H-1 parvovirus has the ability to selectively infect and lyse cancer cells and is widely used in oncological therapy (Telerman et al. 1993). At the same time, there is no data on the oncotropism of PPV6, and its application in oncotherapy warrants further investigation.