Characterization of the Humoral Immune Response to Porcine Epidemic Diarrhea Virus Infection under Experimental and Field Conditions Using an AlphaLISA Platform.

Coronavirus infections are a continuous threat raised time and again. With the recent emergence of novel virulent strains, these viruses can have a large impact on human and animal health. Porcine epidemic diarrhea (PED) is considered to be a reemerging pig disease caused by the enteropathogenic alphacoronavirus PED virus (PEDV). In the absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine are critical. Early detection and differential diagnosis of PEDV infections increase the chance of successful control of the disease. Therefore, there is a continuous need for development of reduced assay-step protocols, no-wash, high-throughput immunoassays. This study described the characterization of the humoral immune response against PEDV under experimental and field conditions using a rapid, sensitive, luminescent proximity homogenous assay (AlphaLISA). PEDV IgG and IgA antibodies were developed toward the beginning of the second week of infection. PEDV IgG antibodies were detected for at least 16 weeks post-exposure. Remarkably, the serum IgA levels remained high and relatively stable throughout the study, lasting longer than the serum IgG response. Overall, AlphaLISA allows the detection and characterization of pathogen-specific antibodies with new speed, sensitivity, and simplicity of use. Particularly, the bridge assay constitutes a rapid diagnostic that substantially improves upon the "time to result" metric of currently available immunoassays.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Molecular mechanisms of human coronavirus NL63 infection and replication.

Human coronavirus NL63 (HCoV-NL63) is spread globally, causing upper and lower respiratory tract infections mainly in young children. HCoV-NL63 shares a host receptor (ACE2) with severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 but, unlike them, HCoV-NL63 primarily develops into self-limiting mild to moderate respiratory disease. Although with different efficiency, both HCoV-NL63 and SARS-like CoVs infect ciliated respiratory cells using ACE2 as receptor for binding and cell entry. Working with SARS-like CoVs require access to BSL-3 facilities, while HCoV-NL63 research can be performed at BSL-2 laboratories. Thus, HCoV-NL63 could be used as a safer surrogate for comparative studies on receptor dynamics, infectivity and virus replication, disease mechanism, and potential therapeutic interventions against SARS-like CoVs. This prompted us to review the current knowledge on the infection mechanism and replication of HCoV-NL63. Specifically, after a brief overview on the taxonomy, genomic organization and virus structure, this review compiles the current HCoV-NL63-related research in virus entry and replication mechanism, including virus attachment, endocytosis, genome translation, and replication and transcription. Furthermore, we reviewed cumulative knowledge on the susceptibility of different cells to HCoV-NL63 infection in vitro, which is essential for successful virus isolation and propagation, and contribute to address different scientific questions from basic science to the development and assessment of diagnostic tools, and antiviral therapies. Finally, we discussed different antiviral strategies that have been explored to suppress replication of HCoV-NL63, and other related human coronaviruses, by either targeting the virus or enhancing host antiviral mechanisms.

Characterization of the Subclinical Infection of Porcine Deltacoronavirus in Grower Pigs under Experimental Conditions.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at -7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

The N-terminal Subunit of the Porcine Deltacoronavirus Spike Recombinant Protein (S1) Does Not Serologically Cross-react with Other Porcine Coronaviruses.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) -7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.

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.