Identification of two novel B-cell epitopes located on the spike protein of swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging alpha-coronavirus that causes diarrhea in piglets and results in serious economic losses. During SADS-CoV infection, the spike protein (S) serves as a crucial structural component of the virion, interacting with receptors and eliciting the production of neutralizing antibodies. Due to the potential risk of zoonotic transmission of SADS-CoV, the identification and screening of epitopes on the S glycoproteins will be crucial for development of sensitive and specific diagnostic tools. In this study, we immunized BALB/c mice with recombinant SADS-CoV S trimer protein and generated two S1-specific monoclonal antibodies (mAbs): 8D6 and 6E9, which recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 8D6 was mapped to 311NPDQRD316, the minimal fragment recognized by mAb 6E9 was mapped to 492ARFVDRL498. Homology analysis of the regions corresponding to 13 typical strains of different SADS-CoV subtypes showed high conservation of these two epitopes. These findings contribute to a deeper understanding of the structure of the SADS-CoV S protein, which is valuable for vaccine design and holds potential for developing diagnostic methods to detect SADS-CoV.

Five-year longitudinal surveillance reveals the continual circulation of both alpha- and beta-coronaviruses in Plateau and Gansu pikas (Ochotona spp.) at Qinghai Lake, China1.

The discovery of alphacoronaviruses and betacoronaviruses in plateau pikas (Ochotona curzoniae) expanded the host range of mammalian coronavirus (CoV) to a new order - Lagomorpha. However, the diversity and evolutionary relationships of CoVs in these plateau-region-specific animal population remains uncertain. We conducted a five-year longitudinal surveillance of CoVs harboured by pikas around Qinghai Lake, China. CoVs were identified in 33 of 236 plateau pikas and 2 of 6 Gansu pikas (Ochotona cansus), with a total positivity rate of 14.5%, and exhibiting a wide spatiotemporal distribution across seven sampling sites and six time points. Through meta-transcriptomic sequencing and RT-PCR, we recovered 16 near-complete viral genome sequences. Phylogenetic analyses classified the viruses as variants of either pika alphacoronaviruses or betacoronaviruses endemic to plateau pikas from the Qinghai-Tibet Plateau region. Of particular note, the pika-associated betacoronaviruses may represent a novel subgenus within the genus Betacoronavirus. Tissue tropism, evaluated using quantitative real-time PCR, revealed the presence of CoV in the rectal and/or lung tissues, with the highest viral loads at 103.55 or 102.80 RNA copies/µL. Surface plasmon resonance (SPR) assays indicated that the newly identified betacoronavirus did not bind to human or pika Angiotensin-converting enzyme 2 (ACE2) or Dipeptidyl peptidase 4 (DPP4). The findings highlight the ongoing circulation and broadening host spectrum of CoVs among pikas, emphasizing the necessity for further investigation to evaluate their potential public health risks.

Recombinant porcine interferon delta 8 inhibits swine acute diarrhoea syndrome coronavirus infection in vitro and in vivo.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which originates from zoonotic transmission of bat coronaviruses in the HKU2 lineage, causes severe illness in pigs and carries a high risk of spreading to humans. At present, there are no licenced therapeutics for the treatment of SADS-CoV. In this study, we examined the effectiveness of recombinant porcine interferon delta 8 (IFN-δ8) against SADS-CoV both in vitro and in vivo. In vitro experiments showed that IFN-δ8 inhibited SADS-CoV proliferation in a concentration-dependent manner, with complete inhibition occurring at a concentration of 5 µg/mL. In vivo experiments demonstrated that two 50 µg/kg doses of IFN-δ8 injected intraperitoneally protected piglets against lethal challenge, blocked viral shedding, attenuated intestinal damage, and decreased the viral load in the jejunum and ileum. Further findings suggested that IFN-δ8 inhibited SADS-CoV infection by increasing the expression of IFN-stimulated genes. These results indicate that IFN-δ8 shows promise as a biological macromolecule drug against SADS-CoV infection.

Bat Rhinacoviruses Related to Swine Acute Diarrhoea Syndrome Coronavirus Evolve under Strong Host and Geographic Constraints in China and Vietnam.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV; Coronaviridae, Rhinacovirus) was detected in 2017 in Guangdong Province (China), where it caused high mortality rates in piglets. According to previous studies, SADS-CoV evolved from horseshoe bat reservoirs. Here, we report the first five Rhinacovirus genomes sequenced in horseshoe bats from Vietnam and their comparisons with data published in China. Our phylogenetic analyses provided evidence for four groups: rhinacoviruses from Rhinolphus pusillus bats, including one from Vietnam; bat rhinacoviruses from Hainan; bat rhinacoviruses from Yunnan showing a divergent synonymous nucleotide composition; and SADS-CoV and related bat viruses, including four rhinacoviruses from Vietnam sampled in Rhinolophus affinis and Rhinolophus thomasi. Our phylogeographic analyses showed that bat rhinacoviruses from Dien Bien (Vietnam) share more affinities with those from Yunnan (China) and that the ancestor of SADS-CoVs arose in Rhinolophus affinis circulating in Guangdong. We detected sequencing errors and artificial chimeric genomes in published data. The two SADS-CoV genomes previously identified as recombinant could also be problematic. The reliable data currently available, therefore, suggests that all SADS-CoV strains originate from a single bat source and that the virus has been spreading in pig farms in several provinces of China for at least seven years since the first outbreak in August 2016.

TMPRSS13 promotes the cell entry of swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused severe intestinal diseases in pigs. It originates from bat coronaviruses HKU2 and has a potential risk of cross-species transmission, raising concerns about its zoonotic potential. Viral entry-related host factors are critical determinants of susceptibility to cells, tissues, or species, and remain to be elucidated for SADS-CoV. Type II transmembrane serine proteases (TTSPs) family is involved in many coronavirus infections and has trypsin-like catalytic activity. Here we examine all 18 members of the TTSPs family through CRISPR-based activation of endogenous protein expression in cells, and find that, in addition to TMPRSS2 and TMPRSS4, TMPRSS13 significantly facilitates SADS-CoV infection. This is confirmed by ectopic expression of TMPRSS13, and specific to trypsin-dependent SADS-CoV. Infection with pseudovirus bearing SADS-CoV spike protein indicates that TMPRSS13 acts at the entry step and is sensitive to serine protease inhibitor Camostat. Moreover, both human and pig TMPRSS13 are able to enhance the cell-cell membrane fusion and cleavage of spike protein. Overall, we demonstrate that TMPRSS13 is another host serine protease promoting the membrane-fusion entry of SADS-CoV, which may expand its host tropism by using diverse TTSPs.

Seroprevalence of the novel swine acute diarrhea syndrome coronavirus in China assessed by enzyme-linked immunosorbent assay.

The endemic outbreak of SADS-CoV has resulted in economic losses and potentially threatened the safety of China's pig industry. The molecular epidemiology of SADS-CoV in pig herds has been investigated in many provinces in China. However, there are no data over a long-time span, and there is a lack of extensive serological surveys to assess the prevalence of SADS-CoV in Chinese swine herds since the discovery of SADS-CoV. In this study, an indirect anti-SADS-CoV IgG enzyme-linked immunosorbent assay (ELISA) based on the SADS-CoV S1 protein was established to investigate the seroprevalence of SADS-CoV in Chinese swine herds. Cross-reactivity assays, indirect immunofluorescence, and western blotting assays showed that the developed ELISA had excellent SADS-CoV specificity. In total, 12,978 pig serum samples from 29 provinces/municipalities/autonomous regions in China were tested from 2022 to 2023. The results showed that the general seroprevalence of SADS-CoV in China was 59.97%, with seroprevalence ranging from 16.7% to 77.12% in different provinces and from 42.61% to 68.45% in different months. SADS-CoV is widely prevalent in China, and its seroprevalence was higher in Northeast China, North China, and Central China than in other regions. Among the four seasons, the prevalence of SADS-CoV was the highest in spring and the lowest in autumn. The results of this study provide the general seroprevalence profile of SADS-CoV in China, facilitating the understanding of the prevalence of SADS-CoV in pigs. More importantly, this study is beneficial in formulating preventive and control measures for SADS-CoV and may provide directions for vaccine development.

NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Quercetin inhibition of porcine intestinal alpha coronavirus in vitro and in vivo.

BACKGROUND: Porcine acute diarrhea syndrome coronavirus (SADS-CoV) is one of the novel pathogens responsible for piglet diarrhea, contributing to substantial economic losses in the farming sector. The broad host range of SADS-CoV raises concerns regarding its potential for cross-species transmission. Currently, there are no effective means of preventing or treating SADS-CoV infection, underscoring the urgent need for identifying efficient antiviral drugs. This study focuses on evaluating quercetin as an antiviral agent against SADS-CoV. RESULTS: In vitro experiments showed that quercetin inhibited SADS-CoV proliferation in a concentration-dependent manner, targeting the adsorption and replication stages of the viral life cycle. Furthermore, quercetin disrupts the regulation of the P53 gene by the virus and inhibits host cell cycle progression induced by SADS-CoV infection. In vivo experiments revealed that quercetin effectively alleviated the clinical symptoms and intestinal pathological damage caused by SADS-CoV-infected piglets, leading to reduced expression levels of inflammatory factors such as TLR3, IL-6, IL-8, and TNF-α. CONCLUSIONS: Therefore, this study provides compelling evidence that quercetin has great potential and promising applications for anti- SADS-CoV action.

Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Swine acute diarrhea syndrome coronavirus Nsp1 suppresses IFN-λ1 production by degrading IRF1 via ubiquitin-proteasome pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

Presence of Alphacoronavirus in Tree- and Crevice-Dwelling Bats from Portugal.

Coronaviruses (CoVs) are RNA viruses capable of infecting a wide range of hosts, including mammals and birds, and have caused significant epidemics such as the ongoing COVID-19 pandemic. Bats, the second most diverse mammalian order, are hosts for various CoVs due to their unique immune responses and ecological traits. This study investigates CoV prevalence in crevice- and tree-dwelling bats in Portugal, a country with limited prior research on bat CoVs. Using nested RT-PCR and sequencing, we screened 87 stool samples from bats, identifying one sample (1.15%) that was positive for Alphacoronavirus, belonging to Pipistrellus pipistrellus. Phylogenetic analysis revealed close genetic relationships with Alphacoronavirus strains from the same bat species in Europe. The low prevalence suggests habitat-specific differences in viral transmission, with cave-dwelling bats exhibiting higher CoV prevalence due to population density and behaviour. These findings underscore the necessity for sustained surveillance efforts aimed at comprehending CoV dynamics within bat populations, especially concerning the risk of spillover events and viral evolution. Vital to this understanding is the monitoring of bat migration patterns, which serves as a crucial tool for elucidating CoV ecology and epidemiology. Such efforts are essential for ongoing research endeavours aimed at mitigating the potential for future zoonotic disease outbreaks.

Coinfection and nonrandom recombination drive the evolution of swine enteric coronaviruses.

Coinfection with multiple viruses is a common phenomenon in clinical settings and is a crucial driver of viral evolution. Although numerous studies have demonstrated viral recombination arising from coinfections of different strains of a specific species, the role of coinfections of different species or genera during viral evolution is rarely investigated. Here, we analyzed coinfections of and recombination events between four different swine enteric coronaviruses that infect the jejunum and ileum in pigs, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), and a deltacoronavirus, porcine deltacoronavirus (PDCoV). Various coinfection patterns were observed in 4,468 fecal and intestinal tissue samples collected from pigs in a 4-year survey. PEDV/PDCoV was the most frequent coinfection. However, recombination analyses have only detected events involving PEDV/TGEV and SADS-CoV/TGEV, indicating that inter-species recombination among coronaviruses is most likely to occur within the same genus. We also analyzed recombination events within the newly identified genus Deltacoronavirus and found that sparrows have played a unique host role in the recombination history of the deltacoronaviruses. The emerging virus PDCoV, which can infect humans, has a different recombination history. In summary, our study demonstrates that swine enteric coronaviruses are a valuable model for investigating the relationship between viral coinfection and recombination, which provide new insights into both inter- and intraspecies recombination events among swine enteric coronaviruses, and extend our understanding of the relationship between coronavirus coinfection and recombination.

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.

Re-emergence of severe acute diarrhea syndrome coronavirus (SADS-CoV) in Henan, central China, 2023.

Severe acute diarrhea syndrome coronavirus (SADS-CoV) was first detected in Guangdong province of China in 2017. And yet from May 2021 to Jun 2023, there were no SADS-CoV outbreaks. In this study, we reported the recent outbreak of SADS-CoV in China on Jun 2023. Phylogenetic analysis showed the novel strain was derived from the ongoing transmission and evolution of SADS-CoV in China, rather than a separate cross-species transmission from bats. Also, the novel strain was found to participate in a recombant event as a minor parent and a missing base in the genome was discovered indicating an novel evolutionary pathway. Through virulence assays in piglets, we further determined that novel strain (SADS-CoV/HNNY/2023) was a highly virulent SADS-CoV strain with typical clinical symptoms: acute diarrhea, vomiting, rapid weight loss. Therefore, the re-emergence of SADS-CoV strains should be brought to people's attention.

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

Crystal Structures of Fusion Cores from CCoV-HuPn-2018 and SADS-CoV.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Cepharanthine analogs mining and genomes of Stephania accelerate anti-coronavirus drug discovery.

Cepharanthine is a secondary metabolite isolated from Stephania. It has been reported that it has anti-conronaviruses activities including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we assemble three Stephania genomes (S. japonica, S. yunnanensis, and S. cepharantha), propose the cepharanthine biosynthetic pathway, and assess the antiviral potential of compounds involved in the pathway. Among the three genomes, S. japonica has a near telomere-to-telomere assembly with one remaining gap, and S. cepharantha and S. yunnanensis have chromosome-level assemblies. Following by biosynthetic gene mining and metabolomics analysis, we identify seven cepharanthine analogs that have broad-spectrum anti-coronavirus activities, including SARS-CoV-2, Guangxi pangolin-CoV (GX_P2V), swine acute diarrhoea syndrome coronavirus (SADS-CoV), and porcine epidemic diarrhea virus (PEDV). We also show that two other genera, Nelumbo and Thalictrum, can produce cepharanthine analogs, and thus have the potential for antiviral compound discovery. Results generated from this study could accelerate broad-spectrum anti-coronavirus drug discovery.