Newly identified lineages of porcine hemagglutinating encephalomyelitis virus exhibit respiratory phenotype.

Swine pathogens have a long history of zoonotic transmission to humans, occasionally leading to sustained outbreaks or pandemics. Through a retrospective epidemiological study of swine populations in China, we describe novel lineages of porcine hemagglutinating encephalomyelitis virus (PHEV) complex coronaviruses (CoVs) that cause exclusively respiratory symptoms with no signs of the neurological symptoms typically associated with classical PHEV infection. Through large-scale epidemiological surveillance, we show that these novel lineages have circulated in at least eight provinces in southeastern China. Phylogenetic and recombination analyses of twenty-four genomes identified two major viral lineages causing respiratory symptoms with extensive recombination within them, between them, and between classical PHEV and the novel respiratory variant PHEV (rvPHEV) lineages. Divergence times among the sampled lineages in the PHEV virus complex date back to 1886-1958 (mean estimate 1928), with the two major rvPHEV lineages separating approximately 20 years later. Many rvPHEV viruses show amino acid substitutions at the carbohydrate-binding site of hemagglutinin esterase (HE) and/or have lost the cysteine required for HE dimerization. This resembles the early adaptation of human CoVs, where HE lost its hemagglutination ability to adapt to growth in the human respiratory tract. Our study represents the first report of the evolutionary history of rvPHEV circulating in swine and highlights the importance of characterizing CoV diversity and recombination in swine to identify pathogens with outbreak potential that could threaten swine farming.

Risk Assessment of the Possible Intermediate Host Role of Pigs for Coronaviruses with a Deep Learning Predictor.

Swine coronaviruses (CoVs) have been found to cause infection in humans, suggesting that Suiformes might be potential intermediate hosts in CoV transmission from their natural hosts to humans. The present study aims to establish convolutional neural network (CNN) models to predict host adaptation of swine CoVs. Decomposing of each ORF1ab and Spike sequence was performed with dinucleotide composition representation (DCR) and other traits. The relationship between CoVs from different adaptive hosts was analyzed by unsupervised learning, and CNN models based on DCR of ORF1ab and Spike were built to predict the host adaptation of swine CoVs. The rationality of the models was verified with phylogenetic analysis. Unsupervised learning showed that there is a multiple host adaptation of different swine CoVs. According to the adaptation prediction of CNN models, swine acute diarrhea syndrome CoV (SADS-CoV) and porcine epidemic diarrhea virus (PEDV) are adapted to Chiroptera, swine transmissible gastroenteritis virus (TGEV) is adapted to Carnivora, porcine hemagglutinating encephalomyelitis (PHEV) might be adapted to Primate, Rodent, and Lagomorpha, and porcine deltacoronavirus (PDCoV) might be adapted to Chiroptera, Artiodactyla, and Carnivora. In summary, the DCR trait has been confirmed to be representative for the CoV genome, and the DCR-based deep learning model works well to assess the adaptation of swine CoVs to other mammals. Suiformes might be intermediate hosts for human CoVs and other mammalian CoVs. The present study provides a novel approach to assess the risk of adaptation and transmission to humans and other mammals of swine CoVs.

A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered emerging alphacoronavirus. SADS-CoV shares over 90% genome sequence identity with bat alphacoronavirus HKU2. SADS-CoV was associated with severe diarrhea and high mortality rates in piglets. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. Here, monoclonal antibody (MAb) 6E8 against SADS-CoV N protein accurately recognized SADS-CoV infection. Then, MAb 6E8 was utilized as a blocking antibody to develop blocking ELISA (bELISA). We customized the rN coating antigen with concentration 0.25 µg/mL. According to receiver operator characteristic curve analysis, the cutoff value of the bELISA was determined as 38.19% when the max Youden index was 0.955, and specificity was 100%, and sensitivity was 95.5%. Specificity testing showed that there was no cross-reactivity with other serum positive swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV). In conclusion, we customized a novel and high-quality blocking ELISA for detection of SADS-CoV infection, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection. IMPORTANCE SADS-CoV was reported to be of high potential for dissemination among various of host species. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. We customed a novel and high-quality bELISA assay for detection of SADS-CoV N protein antibodies, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection.

Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Is Lethal to Neonatal Mice.

Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. We rescued synthetic wild-type SADS-CoV using one-step assembly of a viral cDNA clone by homologous recombination in yeast. Furthermore, we characterized SADS-CoV replication in vitro and in neonatal mice. We found that SADS-CoV caused severe watery diarrhea, weight loss, and a 100% fatality rate in 7- and 14-day-old mice after intracerebral infection. We also detected SADS-CoV-specific N protein in the brain, lungs, spleen, and intestines of infected mice. Furthermore, SADS-CoV infection triggers excessive cytokine expression that encompasses a broad array of proinflammatory mediators, including interleukin 1ß (IL-1ß), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), C-X-C motif chemokine ligand 10 (CXCL10), interferon beta (IFN-ß), IFN-γ, and IFN-λ3. This study highlights the importance of identifying neonatal mice as a model for developing vaccines or antiviral drugs against SADS-CoV infection. IMPORTANCE SADS-CoV is the documented spillover of a bat coronavirus that causes severe disease in pigs. Pigs are in frequent contact with both humans and other animals and theoretically possess a greater chance, compared to many other species, of promoting cross-species viral transmission. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Animal models are an essential feature of the vaccine design toolkit. Compared with neonatal piglets, the mouse is small, making it an economical choice for animal models for SADS-CoV vaccine design. This study showed the pathology of neonatal mice infected with SADS-CoV, which should be very useful for vaccine and antiviral studies.

Characterization and cross-protection of experimental infections with SeCoV and two PEDV variants.

The aim of this study was to characterize the infection of weaned pigs with swine enteric coronavirus (SeCoV) - a chimeric virus most likely originated from a recombination event between porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus, or its mutant porcine respiratory coronavirus - and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals. Piglets within each group were primary inoculated with one of the investigated viral strains (B: PEDV; C: SeCoV and D: rPEDV-SeCoV) or mock-inoculated (A), and exposed to rPEDV-SeCOV at day 20 post-infection; thus, group A was primary challenged (-/rPEDV-SeCoV), groups B and C were subjected to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively), and group D to a homologous re-challenge (rPEDV-SeCoV/rPEDV-SeCoV), Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection, all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhoea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposure to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in faeces.

Detection and Genetic Diversity of Porcine Coronavirus Involved in Diarrhea Outbreaks in Spain.

Porcine enteric coronaviruses include some of the most relevant viral pathogens to the swine industry such as porcine epidemic diarrhea virus (PEDV) or porcine transmissible gastroenteritis virus (TGEV) as well as several recently identified virus such as swine enteric coronavirus (SeCoV), porcine deltacoronavirus (PDCoV) or swine enteric alphacoronavirus (SeACoV). The aim of this study is the identification and characterization of enteric coronaviruses on Spanish pig farms between 2017 and 2019. The study was carried out on 106 swine farms with diarrhea outbreaks where a viral etiology was suspected by using two duplex RT-PCRs developed for the detection of porcine enteric coronaviruses. PEDV was the only coronavirus detected in our research (38.7% positive outbreaks, 41 out of 106) and neither TGEV, SeCoV, PDCoV nor SeACoV were detected in any of the samples. The complete S-gene of all the PEDV isolates recovered were obtained and compared to PEDV and SeCoV sequences available in GenBank. The phylogenetic tree showed that only PEDV of the INDEL 2 or G1b genogroup has circulated in Spain between 2017 and 2019. Three different variants were detected, the recombinant PEDV-SeCoV being the most widespread. These results show that PEDV is a relevant cause of enteric disorders in pigs in Spain while new emerging coronavirus have not been detected so far. However, the monitoring of these virus is advisable to curtail their emergence and spread.

Nucleocapsid proteins from other swine enteric coronaviruses differentially modulate PEDV replication.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

First outbreak with chimeric swine enteric coronavirus (SeCoV) on pig farms in Slovakia - lessons to learn.

This report describes the first disease outbreak caused by chimeric swine enteric coronavirus (SeCoV) on two pig farms in Slovakia in early 2015. The infection was introduced by import of two breeding boars which were placed in provisional quarantine in a unit not strictly separated from other healthy pigs in the same building. Subsequently, loss of appetite and diarrhoea were observed in both boars during the first three days in the isolation unit. The infection gradually spread to the farrowing area and throughout the farm in two weeks and later to another nearby farm. Yellow watery diarrhoea accompanied by dehydration and death was observed in piglets with a mortality ranging from 30 to 35%. In the absence of an available vaccine, the pregnant sows were dosed by mouth with a 10% suspension prepared from the intestine and faeces of infected piglets in warm water. Three weeks after dosing, new litters of piglets were born which remained healthy with no development of diarrhoea.

Spillover of Swine Coronaviruses, United States.

Porcine epidemic diarrhea virus, a pathogen first detected in US domestic swine in 2013, has rapidly spilled over into feral swine populations. A better understanding of the factors associated with pathogen emergence is needed to better manage, and ultimately prevent, future spillover events from domestic to nondomestic animals.