SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity.

Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.

The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.

DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.

Perfusion change in benign prostatic hyperplasia before and after castration in a canine model: Contrast enhanced ultrasonography and CT perfusion study.

Hormonal and vascular changes affecting the canine prostate after castration were investigated to identify the effects of hormones and perfusion on the development of benign prostatic hyperplasia (BPH). Concentrations of serum testosterone and estrogen and intraprostatic dihydrotestosterone (DHT) were compared between 5 normal dogs, 6 dogs with mild BPH, and 6 dogs with marked BPH. In addition, prostatic perfusion using contrast-enhanced ultrasonography (CEUS) and CT perfusion (CTP), as well as CT volumes of the prostates were compared. The changes in these values following castration were assessed in 6 dogs with marked BPH. CEUS revealed significantly slower prostatic arterial inflow and relatively faster venous outflow in BPH versus normal prostate. Permeability and blood volume were not significantly different between the groups via CTP. Intraprostatic DHT level was higher in BPH than in normal prostate and decreased significantly following castration, which was accompanied by a rapid decrease in prostatic volume. On CEUS, arterial inflow to the prostate significantly decreased following castration. Blood volume within the prostate decreased significantly by day 60 following castration. Permeability increased significantly during the early phase after castration; however, by day 60 post-castration, all perfusion parameters decreased significantly. Perfusion changes including venous parameters measured by CEUS and blood volume changes measured by CTP, however, did not support the backflow theory postulating that BPH is induced by vascular changes from congested testes. The major etiology for the development of BPH is attributed to be increased levels of DHT rather than vascular changes.

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate.1-3 Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies.4 Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis,5 these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate.1-3 Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies.4 Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis,5 these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor.IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Anti-rotaviral effects of Glycyrrhiza uralensis extract in piglets with rotavirus diarrhea.

BACKGROUND: Since rotavirus is one of the leading pathogens that cause severe gastroenteritis and represents a serious threat to human and animal health, researchers have been searching for cheap, safe, and effective anti-rotaviral drugs. There is a widespread of interest in using natural products as antiviral agents, and among them, licorice derived from Glycyrrhiza spp. has exerted antiviral properties against several viruses. In this study, anti-rotaviral efficacy of Glycyrrhiza uralensis extract (GUE) as an effective and cheaper remedy without side-effects was evaluated in colostrums-deprived piglets after induction of rotavirus diarrhea. METHODS: Colostrums-deprived piglets were inoculated with porcine rotavirus K85 (G5P[7]) strain. On the onset of diarrhea, piglets were treated with different concentration of GUE. To evaluate the antiviral efficacy of GUE, fecal consistency score, fecal virus shedding and histological changes of the small intestine, mRNA expression levels of inflammation-related cytokines (IL8, IL10, IFN-ß, IFN-γ and TNF-α), signaling molecules (p38 and JNK), and transcription factor (NFκB) in the small intestine and spleen were determined. RESULTS: Among the dosages (100-400 mg/ml) administrated to animals, 400 mg/ml of GUE cured diarrhea, and markedly improved small intestinal lesion score and fecal virus shedding. mRNA expression levels of inflammation-related cytokines (IL8, IL10, IFN-ß, IFN-γ and TNF-α), signaling molecules (p38 and JNK), and transcription factor (NFκB) in the small intestine and spleen were markedly increased in animals with RVA-induced diarrhea, but dose- dependently decreased in GUE treated animals after RVA-induced diarrhea. CONCLUSIONS: GUE cures rotaviral enteritis by coordinating antiviral and anti-inflammatory effects. Therapy of this herbal medicine can be a viable medication for curing rotaviral enteritis in animals and humans.

Azuki bean (Vigna angularis) extract inhibits the development of experimentally induced atopic dermatitis-like skin lesions in NC/Nga mice.

The present study investigated the effects of azuki bean (Vigna angularis) extract (VAE) on the progress of atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by 1-chloro-2,4-dinitrobenzene. The efficacy of VAE in NC/Nga mice was determined by measuring gross and histological skin lesions, serum IgE levels, eosinophil ratio in peripheral leucocytes, and mRNA expression levels of interleukin (IL)-4, tumour necrosis factor (TNF)-α and interferon (IFN)-γ in splenocytes. Continuous ingestion of VAE inhibited the development of the AD-like skin lesions in a dose-dependent manner. In the VAE-treated mice, the numbers of mast cells in the skin, eosinophil ratio in peripheral leucocytes, relative mRNA expression of inflammatory cytokines in the spleen, and serum IgE levels were significantly reduced. Results suggest that VAE can inhibit the development of AD-like skin lesions in NC/Nga mice by regulating immune mediators and cells, and may be an effective alternative therapy for AD.