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.

Coronavirus takeover of host cell translation and intracellular antiviral response: a molecular perspective.

Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion.

Molecular Characterization of Complete Genome Sequence of an Avian Coronavirus Identified in a Backyard Chicken from Tanzania.

A complete genome sequence of an avian coronavirus (AvCoV; 27,663 bp excluding 3' poly(A) tail) was determined using nontargeted next-generation sequencing (NGS) of an oropharyngeal swab from a backyard chicken in a live bird market in Arusha, Tanzania. The open reading frames (ORFs) of the Tanzanian strain TZ/CA127/19 are organized as typical of gammaCoVs (Coronaviridae family): 5'UTR-[ORFs 1a/1b encoding replicase complex (Rep1ab) non-structural peptides nsp2-16]-[spike (S) protein]-[ORFs 3a/3b]-[small envelop (E) protein]-[membrane (M) protein]-[ORFs 4a/4c]-[ORFs 5a/5b]-[nucleocapsid (N) protein]-[ORF6b]-3'UTR. The structural (S, E, M and N) and Rep1ab proteins of TZ/CA127/19 contain features typically conserved in AvCoVs, including the cleavage sites and functional motifs in Rep1ab and S. Its genome backbone (non-spike region) is closest to Asian GI-7 and GI-19 infectious bronchitis viruses (IBVs) with 87.2-89.7% nucleotide (nt) identities, but it has a S gene closest (98.9% nt identity) to the recombinant strain ck/CN/ahysx-1/16. Its 3a, 3b E and 4c sequences are closest to the duck CoV strain DK/GD/27/14 at 99.43%, 100%, 99.65% and 99.38% nt identities, respectively. Whereas its S gene phylogenetically cluster with North American TCoVs and French guineafowl COVs, all other viral genes group monophyletically with Eurasian GI-7/GI-19 IBVs and Chinese recombinant AvCoVs. Detection of a 4445 nt-long recombinant fragment with breakpoints at positions 19,961 and 24,405 (C- and N-terminus of nsp16 and E, respectively) strongly suggested that TZ/CA127/19 acquired its genome backbone from an LX4-type (GI-19) field strain via recombination with an unknown AvCoV. This is the first report of AvCoV in Tanzania and leaves unanswered the questions of its emergence and the biological significance.

Evaluating anti-viral effect of Ivermectin on porcine epidemic diarrhea virus and analyzing the related genes and signaling pathway by RNA-seq in vitro.

Porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there remains no effective drugs for PEDV infection. Ivermectin is an FDA-approved anthelmintic drug used to treat worm infections. In this study, we reported the broad-spectrum antiviral activity of Ivermectin in vitro. Ivermectin can inhibit PEDV infections of different genotypes. Avermectin derivatives can also inhibit PEDV infections. A time of addition assay showed that Ivermectin exhibited potent anti-PEDV activity when added simultaneously with or post virus infection. Furthermore, Ivermectin significantly inhibited the late stage of viral infection by affecting viral release. RNA sequencing indicates Ivermectin induces cell cycle arrest, which may be related to its ability to inhibit viral release. Interestingly, when combined with Niclosamide, Ivermectin demonstrated an enhanced anti-PEDV effect. These findings highlight Ivermectin as a novel antiviral agent with potential for the development of drugs against PEDV infection.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Genomic characterization of Alphacoronavirus from Mops condylurus bats in Nigeria.

Coronaviruses (CoVs) are responsible for sporadic, epidemic and pandemic respiratory diseases worldwide. Bats have been identified as the reservoir for CoVs. To increase the number of complete coronavirus genomes in Africa and to comprehend the molecular epidemiology of bat Alphacoronaviruses (AlphaCoVs), we used deep metagenomics shotgun sequencing to obtain three (3) near-complete genomes of AlphaCoVs from Mops condylurus (Angolan free-tailed) bat in Nigeria. Phylogenetic and pairwise identity analysis of open reading frame 1ab (ORF1ab), spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes of AlphaCoV in this study to previously described AlphaCoVs subgenera showed that the Nigerian AlphaCoVs may be members of potentially unique AlphaCoV subgenera circulating exclusively in bats in the Molossidae bat family. Recombination events were detected, suggesting the evolution of AlphaCoVs within the Molossidae family. The pairwise identity of the S gene in this study and previously published S gene sequences of other AlphaCoVs indicate that the Nigerian strains may have a genetically unique spike protein that is distantly related to other AlphaCoVs. Variations involving non-polar to polar amino acid substitution in both the Heptad Repeat (HR) regions 1 and 2 were observed. Further monitoring of bats to understand the host receptor use requirements of CoVs and interspecies CoV transmission in Africa is necessary to identify and prevent the potential danger that bat CoVs pose to public health.

Transcriptome Profiling of Vero E6 Cells during Original Parental or Cell-Attenuated Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines.

Strong association between angiotensin-converting enzyme gene InDel polymorphism and COVID-19 diseases / Fuerte asociación entre el polimorfismo InDel del gen de la enzima convesiva de angiotensina y las enfermedades por COVID-19

Background and ObjectivesThe COVID-19 pandemic that emerged in China in late 2019 and spread rapidly around the world. There is evidence that COVID-19 infection can be influenced by genetic variations in the host. The aim of this study was to investigate the association between ACE InDel polymorphism and COVID-19 in Northern Cyprus.Patients and methodsThis study included 250 patients diagnosed with COVID-19 and 371 healthy controls. Genotyping for the ACE InDel gene polymorphism was performed by polymerase chain reaction.ResultsThe frequency of ACE DD homozygotes was significantly increased in COVID-19 patients compared to the control group (p=0.022). The difference in the presence of the D allele between the patient and control groups was statistically significant (57.2% and 50.67%, respectively, p<0.05). Individuals with the genotype II were found to have a higher risk of symptomatic COVID-19 (p=0.011). In addition, chest radiographic findings were observed more frequently in individuals with the genotype DD compared to individuals with the genotypes ID and II (p=0.005). A statistically significant difference was found when the time of onset of symptoms for COVID-19 and duration of treatment were compared with participants’ genotypes (p=0.016 and p=0.014, respectively). The time of onset of COVID-19 was shorter in individuals with the genotype DD than in individuals with the genotype II, while the duration of treatment was longer.ConclusionIn conclusion, the ACE I/D polymorphism has the potential to predict the severity of COVID-19. (AU)

Antecedentes y objetivosLa pandemia de COVID-19 surgió en China a fines de 2019 y se extendió rápidamente por todo el mundo. Existe evidencia de que la infección por COVID-19 puede verse influenciada por variaciones genéticas en el huésped. El objetivo de este estudio fue investigar la asociación entre el polimorfismo ACE InDel y COVID-19 en el norte de Chipre.Pacientes y métodosSe incluyeron 250 pacientes diagnosticados de COVID-19 y 371 controles sanos. El genotipado del polimorfismo del gen ACE InDel se realizó mediante reacción en cadena de la polimerasa.ResultadosLa frecuencia de homocigotos ACE DD aumentó significativamente en pacientes con COVID-19 en comparación con el grupo de control (p=0,022). La diferencia en la presencia del alelo D entre los grupos de pacientes y control fue estadísticamente significativa (57,2% y 50,67%, respectivamente, p<0,05). Las personas con el genotipo II tenían un mayor riesgo de COVID-19 sintomático (p=0,011). Además, los hallazgos radiográficos de tórax se observaron con mayor frecuencia en individuos con el genotipo DD en comparación con los individuos con los genotipos ID y II (p=0,005). Se encontró una diferencia estadísticamente significativa cuando se comparó el tiempo de aparición de los síntomas de COVID-19 y la duración del tratamiento con los genotipos de los participantes (p=0,016 y p=0,014, respectivamente). El tiempo de aparición de COVID-19 fue más corto en individuos con genotipoDD que en individuos con genotipoII, mientras que la duración del tratamiento fue más prolongada.ConclusionesEl polimorfismo ACE I/D podría predecir la gravedad de la COVID-19. (AU)

Acute coronavirus infection triggers a TNF-dependent osteoporotic phenotype in mice.

AIMS: Millions of people died during the COVID-19 pandemic, but the vast majority of infected individuals survived. Now, some consequences of the disease, known as long COVID, are been revealed. Although the respiratory system is the target of Sars-CoV-2, COVID-19 can influence other parts of the body, including bone. The aim of this work was to investigate the impact of acute coronavirus infection in bone metabolism. MAIN METHODS: We evaluated RANKL/OPG levels in serum samples of patients with and without acute COVID-19. In vitro, the effects of coronavirus in osteoclasts and osteoblasts were investigated. In vivo, we evaluated the bone phenotype in a BSL2 mouse model of SARS-like disease induced by murine coronavirus (MHV-3). KEY FINDINGS: Patients with acute COVID-19 presented decreased OPG and increased RANKL/OPG ratio in the serum versus healthy individuals. In vitro, MHV-3 infected macrophages and osteoclasts, increasing their differentiation and TNF release. Oppositely, osteoblasts were not infected. In vivo, MHV-3 lung infection triggered bone resorption in the femur of mice, increasing the number of osteoclasts at 3dpi and decreasing at 5dpi. Indeed, apoptotic-caspase-3+ cells have been detected in the femur after infection as well as viral RNA. RANKL/OPG ratio and TNF levels also increased in the femur after infection. Accordingly, the bone phenotype of TNFRp55-/- mice infected with MHV-3 showed no signs of bone resorption or increase in the number of osteoclasts. SIGNIFICANCE: Coronavirus induces an osteoporotic phenotype in mice dependent on TNF and on macrophage/osteoclast infection.

Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability.

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed -1 ribosomal frameshift (-1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.

In silico and in vitro evaluation of antiviral activity of wogonin against main protease of porcine epidemic diarrhea virus.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen's genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`.

MicroRNA-155 is a main part of proinflammatory puzzle during severe coronavirus disease 2019 (COVID-19)

Genetic and epigenetic parameters play critical roles in determining the outcomes of the severe acute respiratory syndrome coronavirus type 19 (SARS-CoV-2) infection. MicroRNAs (miRNAs) are an important part of the epigenetic factors that regulate several functions of the immune cells and also viruses. Accordingly, the molecules can regulate expression of the immune cell proteins and virus in the host cells. Among the miRNAs, miRNA-155 (miR-155) is well-studied in patients suffering from severe coronavirus disease 2019 (COVID-19). It has been reported that the SARS-CoV-2 infected patients may be directed to induce a cytokine storm or severe proinflammatory responses. This review article discusses the pathological roles of miR-155 during COVID-19 infection (AU)

Detection of coronaviruses in insectivorous bats of Fore-Caucasus, 2021.

Coronaviruses (CoVs) pose a huge threat to public health as emerging viruses. Bat-borne CoVs are especially unpredictable in their evolution due to some unique features of bat physiology boosting the rate of mutations in CoVs, which is already high by itself compared to other viruses. Among bats, a meta-analysis of overall CoVs epizootiology identified a nucleic acid observed prevalence of 9.8% (95% CI 8.7-10.9%). The main objectives of our study were to conduct a qPCR screening of CoVs' prevalence in the insectivorous bat population of Fore-Caucasus and perform their characterization based on the metagenomic NGS of samples with detected CoV RNA. According to the qPCR screening, CoV RNA was detected in 5 samples, resulting in a 3.33% (95% CI 1.1-7.6%) prevalence of CoVs in bats from these studied locations. BetaCoVs reads were identified in raw metagenomic NGS data, however, detailed characterization was not possible due to relatively low RNA concentration in samples. Our results correspond to other studies, although a lower prevalence in qPCR studies was observed compared to other regions and countries. Further studies should require deeper metagenomic NGS investigation, as a supplementary method, which will allow detailed CoV characterization.

Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy.

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.

Three Amino Acid Substitutions in the Spike Protein Enable the Coronavirus Porcine Epidemic Diarrhea Virus To Infect Vero Cells.

Porcine epidemic diarrhea virus (PEDV), a continuously evolving pathogen, causes severe diarrhea in piglets, with high mortality rates. To prevent or mitigate the disease, it is common practice to develop live or inactivated PEDV vaccines based on cell-adapted viral variants. Propagating wild-type PEDV in cultured cells is, however, often challenging due to the lack of knowledge about the requirements for the cell adaptation of PEDV. In the present study, by using the RNA-targeted reverse genetic system for PEDV to apply S protein swapping followed by the rescue of the recombinant viruses, three key amino acid mutations in the S protein, A605E, E633Q, and R891G, were identified, which enable attenuated PEDV strain DR13 (DR13att) to efficiently and productively infect Vero cells, in contrast to the parental DR13 strain (DR13par). The former two key mutations reside inside and in the vicinity of the receptor binding domain (RBD), respectively, while the latter occurs at the N-terminal end of the fusion peptide (FP). Besides the three key mutations, other mutations in the S protein further enhanced the infection efficiency of the recombinant viruses. We hypothesize that the three mutations changed PEDV tropism by altering the S2' cleavage site and the RBD structure. This study provides basic molecular insight into cell adaptation by PEDV, which is also relevant for vaccine design. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a lethal pathogen for newborn piglets, and an efficient vaccine is needed urgently. However, propagating wild-type PEDV in cultured cells for vaccine development is still challenging due to the lack of knowledge about the mechanism of the cell adaptation of PEDV. In this study, we found that three amino acid mutations, A605E, E633Q, and R891G, in the spike protein of the Vero cell-adapted PEDV strain DR13att were critical for its cell adaptation. After analyzing the mutation sites in the spike protein, we hypothesize that the cell adaptation of DR13att was achieved by altering the S2' cleavage site and the RBD structure. This study provides new molecular insight into the mechanism of PEDV culture adaptation and new strategies for PEDV vaccine design.

Features of Distribution of the Allelic Variant of the OAS1 Gene Associated with Severe Form of the Coronavirus Infection in the Russian and Global Populations.

The study of the geographic distribution of the allelic variant of the OAS1 gene associated with severe form of the infections caused by RNA viruses was carried out using the rs10774671 polymorphic locus. The mutant allele encoding the p42 protein isoform was most prevalent in the Russian populations. A comparative analysis of the prevalence of the mutant allele in world populations showed that its frequency is 0.9 among the inhabitants of Northern Eurasia, while the allele encoding the p46 protein isoform is widespread among the population of West Central Africa. A cartographic analysis of the relationship between the population-frequency characteristics of the marker alleles and the geographical remoteness of the populations showed that the mutant allele is most often observed in the indigenous populations of the Far East, which suggests its East Asian origin.

Differential transcriptomic changes in the central nervous system and urinary bladders of mice infected with a coronavirus.

Multiple sclerosis (MS) often leads to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The aim of the study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder (UB) dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. Adult C57BL/6 male mice (N = 12) received either an intracranial injection of MHV (coronavirus-induced encephalomyelitis, CIE group), or sterile saline (control group). Spinal cord (SC) and urinary bladders (UB) were collected from CIE mice at 1 wk and 4 wks, followed by RNA isolation and NanoString nCounter Neuroinflammation assay. Transcriptome analysis of SC identified a significantly changed expression of >150 genes in CIE mice known to regulate astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Two genes were significantly upregulated (Ttr and Ms4a4a), and two were downregulated (Asb2 and Myct1) only in the UB of CIE mice. Siglec1 and Zbp1 were the only genes significantly upregulated in both tissues, suggesting a common transcriptomic link between neuroinflammation in the CNS and neurogenic changes in the UB of CIE mice.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Visual detection and differentiation of porcine epidemic diarrhea virus wild-type strains and attenuated vaccine strains using CRISPR/Cas13a-based lateral flow strip.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets. Infections result in high mortality and serious economic losses to the swine industry. PEDV attenuated vaccine does not completely protect against all mutant wild-type strains, and PEDV infection can periodically occur. A sensitive, accurate, and simple detection method for PEDV is needed to reduce the occurrence of the disease. In this study, the CRISPR/Cas13a system was combined with recombinase aided amplification to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The method is based on isothermal detection at 37°C. The results are used for visual readout. The assay had high sensitivity and specificity, with a detection limit of 101 copies/µL for the gene of interest, and no cross-reactivity with other pathogens. The Cas13a detection worked well with clinical samples. This visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a should be a powerful tool for detecting PEDV.