Investigating porcine parvovirus 4 infection using in situ polymerase chain reaction

Porcine parvovirus 4 (PPV4) is a representative of the Copiparvovirus genus classified as Ungulate copiparvovirus 2. PPV4 was detected recently in the lung lavage samples of diseased pigs co-infected with porcine circovirus type 2 (PCV2). PPV4 is unique in that its genomic sequence is most closely related to bovine parvovirus 2 classified as Ungulate copiparvovirus 1. The pathogenic nature of PPV4 remains to be determined while it seems that PPV4 is widespread in pig populations in Central and Eastern Europe. The aim of this study was to detect novel PPV4, using two in situ methods that target nucleic acids, like in situ hybridization (ISH) and in situ polymerase chain reaction (IS-PCR).

In order to investigate its role in pathological conditions samples from 46 and 22 dead animals from two farms were examined – necropsied and checked for PPV4 and PCV2 by PCR; tissues embedded in paraffin blocks were submitted to H&E and Brown and Brenn staining, immunohistochemistry (IHC) to detect CD3, CD79α, swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, PRRSV, swine influenza, Mycoplasma hyopneumoniae and in situ hybridization to detect PPV4, ss and dsDNA of PCV2. PPV4 positive samples were submitted to IS-PCR including double staining method to detect PPV4 and cell marker.

Out of the 46 lung and lymph node samples tested 6 were positive for the presence of PPV4. PPV4 enhanced necrosis in lymph nodes and was associated with the decreased replication of PCV2 in lymphoid follicles. In lymph nodes PPV4 caused a statistically significant increase in the number of T lymphocytes and was linked to a decreased level of most immune cells including macrophages. PPV4-positive cells were observed in lymph nodes, mostly in the cortex and to some extent in the medulla. Positive cells displayed lymphocyte-like or macrophage-like morphology in haemorrhagic areas where inflammatory cells were mixed with erythrocytes. Only limited number of PPV4-infected foci displayed CD3 antigen in contrast, while in lungs, PPV4- positive cells expressed poorly SLAIIDQ antigen and no CD3 or lysozyme antigens at all. Very discrete microscopic lesions were observed in IS-PCRpositive lungs consisting of lung oedema, congestion and very weak proliferation of alveolar walls.

We found that PPV4 viruses were localized mostly in lymphocytic cells in lungs, lymph nodes and liver. PPV4 could play a role in necrotizing lymphadenitis in the background of PCV2 infection with unclear pathogenesis. Low level of SLAIIDQ and CD3 was expressed by infected cells suggesting that PPV4 may have a specific tropism for immature lymphocytes and/or NK cells.

Disclosure of Interest: None Declared.