Porcine epidemic diarrhea virus (PEDV) belongs to the alphacoronavirus of the Coronaviridae. It is the major etiological agent of the recent outbreaks of piglet diarrhea and death in the US. Limited knowledge is currently available regarding the role of dendritic cells in PEDV infection. Here, we observed that PEDV did not replicate in monocyte-derived dendritic cells as evidenced by the decrease of viral gene transcript copies in infected cells by qRT-PCR and the absence of viral proteins by immunofluorescence staining as well as the absence of virus particles in infected cells by transmission electron microscopy. In addition, PEDV did not compromise cell viability at 48, 72, and 96h after infection at either a MOI of 2.5 or 5. Interestingly, an increased transcription of type I interferon including interferon-α and β was observed in infected cells compared to mock infected cells. Surprisingly, we did not detect any interferon-β in the supernatants of infected cells. A slight increase in interferon-α protein production in the supernatants of PEDV-infected cells was observed compared to mock infected cells. We also observed a markedly increased transcription of interferon inducible protein -10 (IP-10). Overall, PEDV does not replicate in porcine Mo-DC, but activates the transcription of type I interferon and chemokine IP-10.

Porcine respiratory disease complex (PRDC) is a significant source of morbidity and mortality, manifested by pneumonia of multiple etiologies, where a variety of pathogens and environment and management practices play a role in the disease. Porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), and porcine circovirus 2 (PCV2) are well-established pathogens in PRDC. Porcine parvovirus 2 (PPV2) has been identified in both healthy and clinically diseased pigs at a high prevalence worldwide. Despite widespread circulation, the significance of PPV2 infection in PRDC and its association with other co-infections are unclear. Here, PPV2 was detected in the lung tissue in 39 of 100 (39%) PRDC-affected pigs by quantitative polymerase chain reaction (qPCR). Using in situ hybridization (ISH) in conjunction with tissue microarrays (TMA), PPV2 infection was localized in alveolar macrophages and other cells in the lungs with interstitial pneumonia in 28 of 99 (28.2%) samples. Viral load tended to correlate with the number of macrophages in the lungs. Assessment of the frequency, viral titers, and tissue distributions showed no association between infection of PPV2 and other major viral respiratory pathogens. In one-third of the PPV2-positive samples by qPCR, no other known viruses were identified by metagenomic sequencing. The genome sequences of PPV2 were 99.7% identical to the reference genomes. Although intensive intranuclear and intracytoplasmic signals of PPV2 were mainly detected in alveolar macrophages by ISH, no obvious virus replication was noted in in vitro cell culture. Together, these results suggest that PPV2 is associated, but may not be the sole causative agent, with PRDC, warranting the control and prevention of this underdiagnosed virus.

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in pregnant sows and acute respiratory disease in young pigs. It is a leading infectious agent of swine respiratory complex, which has significant negative economic impact on the swine industry. Commercial markets currently offer both live attenuated and killed vaccines; however, increasing controversy exists about their efficacy providing complete protection. Virus-like particles (VLPs) possess many desirable features of a potent vaccine candidate and have been proven to be highly immunogenic and protective against virus infections. Here we explored the efficacy of PRRSV VLPs together with the use of a novel 2′, 3′-cGAMP VacciGrade™ adjuvant.MethodsAnimals were immunized twice intranasally with phosphate buffered saline (PBS), PRRSV VLPs, or PRRSV VLPs plus 2′, 3′-cGAMP VacciGrade™ at 2 weeks apart. Animals were challenged with PRRSV-23983 at 2 weeks post the second immunization. PRRSV specific antibody response and cytokines were measured. Viremia, clinical signs, and histological lesions were evaluated.ResultsPRRSV N protein specific antibody was detected in all animals at day 10 after challenge, but no significant difference was observed among the vaccinated and control groups. Surprisingly, a significantly higher viremia was observed in the VLPs and VLPs plus the adjuvant groups compared to the control group. The increased viremia is correlated with a higher interferon-α induction in the serum of the VLPs and the VLPs plus the adjuvant groups.ConclusionsIntranasal immunizations of pigs with PRRSV VLPs and VLPs plus the 2′, 3′-cGAMP VacciGrade™ adjuvant exacerbates viremia. A higher level of interferon-α production, but not interferon-γ and IL-10, is correlated with enhanced virus replication. Overall, PRRSV VLPs and PRRSV VLPs plus the adjuvant fail to provide protection against PRRSV challenge. Different dose of VLPs and alternative route of vaccination such as intramuscular injection should be explored in the future studies to fully assess the feasibility of such a vaccine platform for PRRSV control and prevention.

While rotavirus (RV) is primarily known to cause gastroenteritis in many animals, several epidemiological studies have shown concurrent respiratory symptoms with fecal and nasal virus shedding. However, respiratory RV infections have rarely been investigated. By screening clinical samples submitted for diagnostic testing, porcine rotavirus A (RVA) was detected by quantitative reverse transcription PCR (qRT-PCR) in 28 out of 91 (30.8%) lungs obtained from conventionally reared pigs with respiratory signs. Among the positive cases, intensive RVA signals were mainly localized in alveolar macrophages (n = 3) and bronchiolar epithelial cells (n = 1) by RNAscope®in situ hybridization (ISH). The signals of RVA in bronchiolar epithelial cells were verified by ISH with different probes, immunohistochemistry, and transmission electron microscopy. Furthermore, additional cases with RVA ISH-positive signals in alveolar macrophages (n = 9) and bronchial epithelial cells (n = 1) were identified by screening 120 archived formalin-fixed and paraffin-embedded lung samples using tissue microarrays. Overall, our study showed a high frequency of RVA detection in lungs from conventional pigs with respiratory disease. Further research is needed to determine if RVA infection in the respiratory epithelium correlates with nasal shedding of rotavirus and its contribution to respiratory disease.

Astroviruses (AstVs) cause gastrointestinal disease in mammals and avians. Emerging evidence suggests that some AstVs have extraintestinal tissue tropism, with AstVs detected in the liver, kidney, central nervous system, and the respiratory tract variably associated with disease. In cattle, AstV infection has been linked to gastroenteric or neurologic disease. Here, metagenomic sequencing of a lung from a bovine with respiratory disease identified a novel AstV with a predicted capsid-encoding ORF2 amino acid sequence with 66% identity to caprine astrovirus (CAstV G2.1). A quantitative reverse transcription PCR (qRT-PCR) targeting ORF2 found four out of 49 (8%) lungs and one out of 48 (2%) enteric samples obtained from bovine diagnostic submissions positive for the novel bovine astrovirus (BAstV). In two strongly qRT-PCR-positive lung samples, intense novel BAstV nucleic acid signals were mainly localized in the cytoplasm of alveolar macrophages and mononuclear cells using RNAscope® in situ hybridization (ISH). Genetic analysis of two novel BAstV genomes determined from qRT-PCR positive samples found high similarity for ORF1ab nucleotide sequence (92.1% and 93.9%) to BAstV strain BSRI-1, while ORF2 nucleotide sequence was most similar to CAstV G2.1 (74.6% and 77.6%). Phylogenetic analysis of the novel BAstV sequences found a close genetic relationship to the single BAstV (BSRI-1) previously identified from a bovine respiratory sample as well as bovine and caprine AstVs identified from various tissues. Further research is needed to determine the clinical significance of BAstV in respiratory diseases.