Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction.

BACKGROUND: Porcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®). RESULTS: We found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes. CONCLUSION: The overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia.

Co-infection status of novel parvovirus's (PPV2 to 4) with porcine circovirus 2 in porcine respiratory disease complex and porcine circovirus-associated disease from 1997 to 2012.

As global pig health diseases, porcine respiratory disease complex (PRDC) and porcine circovirus-associated disease (PCVAD) generate substantial economic losses despite pigs been vaccinated against the primary causative virus, highlighting the importance of understanding virome interactions and specifically co-factor infections. Established primary endemic pathogens for PRDC include porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSv) and swine influenza virus (SIV), and PCV2 aetiology in interaction with other co-infecting viruses can result in PCVAD. Porcine parvovirus (PPV) 1 is a well-characterized virus with an available vaccine preventing reproductive failure in sows. However, whilst novel PPV 2 to 7 viruses have been identified since 2001, their viral pathogenic potential in clinical and subclinical disease remains to be determined. Therefore, this study has sought to develop a better understanding of their potential role as associated co-infections in PRDC and PCVAD by examining archival samples for the presence of PCV2 and the novel parvoviruses PPV2-4 from clinically diseased pigs across production age stages. Epidemiologically, the novel PPV2 was found to be the most prevalent within the fattener age group with PPV2-4 statistically associated with pig respiratory disease and enteric ulcers. Additionally, statistical modelling by latent class analysis (LCA) on veterinary pathology scored pigs found a clustering co-factor association between PPV2 and PCV2, suggesting the novel PPV may be involved in PRDC and PCVAD. Phylogenetic analysis of novel PPVs revealed the PPV2 capsid evolution to be diverged from the original strains with a low nucleotide homology of 88%-96% between two distinct clades. These findings determine that novel PPV 2-4 viruses are statistically associated as co-infectors in a diseased pig population, and significantly detected PPV2 clustering co-infection frequency with PCV2 in PRDC and PCVAD diseased pigs through LCA analysis.

Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge.

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge.

Next-Generation Sequencing Coupled With in situ Hybridization: A Novel Diagnostic Platform to Investigate Swine Emerging Pathogens and New Variants of Endemic Viruses.

Next generation sequencing (NGS) can be applied to identify and characterize the entire set of microbes within a sample. However, this platform does not provide a morphological context or specific association between the viral or bacterial sequences detected and the histological lesions. This limitation has generated uncertainty whether the sequences identified by NGS are actually contributing or not for the clinical outcome. Although in situ hybridization (ISH) and immunohistochemistry (IHC) can be used to detect pathogens in tissue samples, only ISH has the advantage of being rapidly developed in a context of an emerging disease, especially because it does not require development of specific primary antibodies against the target pathogen. Based on the sequence information provided by NGS, ISH is able to check the presence of a certain pathogen within histological lesions, by targeting its specific messenger RNA, helping to build the relationship between the pathogen and the clinical outcome. In this mini review we have compiled results of the application of NGS-ISH to the investigation of challenging diagnostic cases or emerging pathogens in pigs, that resulted in the detection of porcine circovirus type 3, porcine parvovirus type 2, Senecavirus A, and Mycoplasma hyorhinis.