A Serological Investigation of Porcine Reproductive and Respiratory Syndrome and Three Coronaviruses in the Campania Region, Southern Italy.

Porcine coronaviruses and reproductive and respiratory syndrome (PRRS) are responsible for severe outbreaks that cause huge economic losses worldwide. In Italy, three coronaviruses have been reported historically: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Although repeated outbreaks have been described, especially in northern Italy, where intensive pig farming is common, there is a worrying lack of information on the spread of these pathogens in Europe. In this work, we determined the seroprevalence of three porcine coronaviruses and PRRSV in the Campania region, southern Italy. A total of 443 samples were tested for the presence of antibodies against porcine coronaviruses and PRRSV using four different commercial ELISAs. Our results indicated that PEDV is the most prevalent among porcine coronaviruses, followed by TGEV, and finally PRCV. PRRSV appeared to be the most prevalent virus (16.7%). For coronaviruses, seroprevalence was higher in pigs raised in intensive farming systems. In terms of distribution, TGEV is more widespread in the province of Avellino, while PEDV and PRRSV are more prevalent in the province of Naples, emphasizing the epidemic nature of both infections. Interestingly, TGEV-positive animals are more common among growers, while seropositivity for PEDV and PRRSV was higher in adults. Our research provides new insights into the spread of swine coronaviruses and PRRSV in southern Italy, as well as a warning about the need for viral surveillance.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

Detecting and Monitoring Porcine Hemagglutinating Encephalomyelitis Virus, an Underresearched Betacoronavirus.

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans.

Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.