NLRP1 restricts porcine deltacoronavirus infection via IL-11 inhibiting the phosphorylation of the ERK signaling pathway.

Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes large-scale outbreaks of severe diarrhea disease in piglets. Unlike other porcine coronaviruses, PDCoV has a wide range of species tissue tropism, including primary human cells, which poses a significant risk of cross-species transmission. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) has a key role in linking host innate immunity to microbes and the regulation of inflammatory pathways. We now report a role for NLRP1 in the control of PDCoV infection. Overexpression of NLRP1 remarkably suppressed PDCoV infection, whereas knockout of NLRP1 led to a significant increase in PDCoV replication. A mechanistic study revealed that NLRP1 suppressed PDCoV replication in cells by upregulating IL-11 expression, which in turn inhibited the phosphorylation of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor U0126 effectively hindered PDCoV replication in pigs. Together, our results demonstrated that NLRP1 exerted an anti-PDCoV effect by IL-11-mediated inhibition of the phosphorylation of the ERK signaling pathway, providing a novel antiviral signal axis of NLRP1-IL-11-ERK. This study expands our understanding of the regulatory network of NLRP1 in the host defense against virus infection and provides a new insight into the treatment of coronaviruses and the development of corresponding drugs.IMPORTANCECoronavirus, which mainly infects gastrointestinal and respiratory epithelial cells in vivo, poses a huge threat to both humans and animals. Although porcine deltacoronavirus (PDCoV) is known to primarily cause fatal diarrhea in piglets, reports detected in plasma samples from Haitian children emphasize the potential risk of animal-to-human spillover. Finding effective therapeutics against coronaviruses is crucial for controlling viral infection. Nucleotide-binding oligomerization-like receptor (NLR) family pyrin domain-containing 1 (NLRP1), a key regulatory factor in the innate immune system, is highly expressed in epithelial cells and associated with the pathogenesis of viruses. We demonstrate here that NLRP1 inhibits the infection of the intestinal coronavirus PDCoV through IL-11-mediated phosphorylation inhibition of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor can control the infection of PDCoV in pigs. Our study emphasizes the importance of NLRP1 as an immune regulatory factor and may open up new avenues for the treatment of coronavirus infection.

Broad antagonism of coronaviruses nsp5 to evade the host antiviral responses by cleaving POLDIP3.

Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Combined transcriptome and metabolome analysis reveals breed-specific regulatory mechanisms in Dorper and Tan sheep.

BACKGROUND: Dorper and Tan sheep are renowned for their rapid growth and exceptional meat quality, respectively. Previous research has provided evidence of the impact of gut microbiota on breed characteristics. The precise correlation between the gastrointestinal tract and peripheral organs in each breed is still unclear. Investigating the metabolic network of the intestinal organ has the potential to improve animal growth performance and enhance economic benefits through the regulation of intestinal metabolites. RESULTS: In this study, we identified the growth advantage of Dorper sheep and the high fat content of Tan sheep. A transcriptome study of the brain, liver, skeletal muscle, and intestinal tissues of both breeds revealed 3,750 differentially expressed genes (DEGs). The genes PPARGC1A, LPL, and PHGDH were found to be highly expressed in Doper, resulting in the up-regulation of pathways related to lipid oxidation, glycerophospholipid metabolism, and amino acid anabolism. Tan sheep highly express the BSEP, LDLR, and ACHE genes, which up-regulate the pathways involved in bile transport and cholesterol homeostasis. Hindgut content analysis identified 200 differentially accumulated metabolites (DAMs). Purines, pyrimidines, bile acids, and fatty acid substances were more abundant in Dorper sheep. Based on combined gene and metabolite analyses, we have identified glycine, serine, and threonine metabolism, tryptophan metabolism, bile secretion, cholesterol metabolism, and neuroactive ligand-receptor interaction as key factors contributing to the differences among the breeds. CONCLUSIONS: This study indicates that different breeds of sheep exhibit unique breed characteristics through various physiological regulatory methods. Dorper sheep upregulate metabolic signals related to glycine, serine, and threonine, resulting in an increase in purine and pyrimidine substances. This, in turn, promotes the synthesis of amino acids and facilitates body development, resulting in a faster rate of weight gain. Tan sheep accelerate bile transport, reduce bile accumulation in the intestine, and upregulate cholesterol homeostasis signals in skeletal muscles. This promotes the accumulation of peripheral and intramuscular fat, resulting in improved meat quality. This work adopts a joint analysis method of multi-tissue transcriptome and gut metabolome, providing a successful case for analyzing the mechanisms underlying the formation of various traits.

Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1ß (IL-1ß) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1ß and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

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.

E2F5 Targeted by Let-7d-5p Facilitates Cell Proliferation, Metastasis and Immune Escape in Gallbladder Cancer.

BACKGROUND: Gallbladder cancer (GBC) remains a serious cause of cancer-related mortality across the globe. E2F5 has been identified to as a known oncogene in various cancers. However, the special functions of E2F5 have not been investigated in GBC. AIMS: To explore the regulatory functions of E2F5 and its related molecular regulatory mechanism in GBC progression. METHODS: The expression of genes were examined through qRT-PCR, western blot and IHC assay. The cell proliferation was assessed through CCK-8 and EDU assays. The cytotoxicity was tested through LDH assay. The percentage of CD8+ T cells and cell apoptosis were evaluated through flow cytometry. The binding ability was detected through luciferase reporter assay. The tumor growth was assessed through in vivo assays. RESULTS: In this study, it was demonstrated that E2F5 expression was evaluated in GBC, and resulted into poor prognosis. Bioinformatics analysis revealed E2F5 as a target for let-7d-5p, which when overexpressed, suppressed the metastasis and proliferation of GBC through the downregulation of E2F5. It was discovered that E2F5 activates JAK2/STAT3 signaling which is suppressed by let-7d-5p, implicating this pathway as one of the effectors of the oncogenic effects of ESF5 in GBC. E2F5 had been confirmed to aggravate tumor growth in vivo. CONCLUSION: E2F5 targeted by let-7d-5p facilitated cell proliferation, metastasis and immune escape in GBC through the JAK2/STAT3 pathway.

Current status of electronic health literacy among pregnant women with gestational diabetes mellitus and their perceptions of online health information: a mixed-methods study.

BACKGROUND: Women diagnosed with gestational diabetes mellitus often rely on internet-based health information for managing their condition. This study aims to investigate the present state of electronic health literacy among women with gestational diabetes mellitus, analyze the influencing factors, and explore their experiences regarding accessing, comprehending, evaluating, and applying online health information pertinent to gestational diabetes mellitus. METHODS: A sequential explanatory mixed methods research design was adopted in this study. Initially, 235 women with gestational diabetes mellitus participated in a cross-sectional survey. The research tools included general information and the Chinese version of the electronic Health Literacy Scale (eHEALS). Descriptive analyses were conducted to describe the characteristics of the sample, and multiple linear regression analyses were used to explore the factors influencing electronic health literacy among women with gestational diabetes mellitus. Secondly, 11 women with gestational diabetes mellitus joined semi-structured in-depth interviews to obtain their perceptions about online health information. The data were analyzed using inductive content analysis to develop themes. RESULTS: The median score of eHEALS in the Chinese version among 235 women diagnosed with gestational diabetes mellitus was 29 (interquartile range [IQR], 26 to 32). Factors influencing electronic health literacy among these women included accessing health information from medical professionals (ß = 0.137, p = 0.029) and utilizing health information from applications (ß = 0.159, p = 0.013). From the qualitative phase of the study, four thematic categories emerged: reasons and basis for accessing health information from the Internet; address barriers to accessing and applying online health information; desires for a higher level of online health information services; outcomes of accessing and applying online health information. CONCLUSION: The electronic health literacy of women diagnosed with gestational diabetes mellitus remains suboptimal and warrants improvement. The sources of access to health information affect electronic health literacy in women with gestational diabetes mellitus. Moreover, women facing gestational diabetes encounter numerous impediments when attempting to access health-related information online, underscoring the necessity for enhanced online health information services to meet their needs.

Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens.

The genetic foundation of chicken body plumage color has been extensively studied. However, little attention has been paid to the inheritance patterns and molecular mechanisms underlying the formation of distal feather colors (tail and wingtip). Differences in these colors are common; for example, the Chinese Huiyang Beard chicken has black tail feathers, but yellow body plumage. Here, the hybrid offspring of Huiyang Beard and White Leghorn chickens were used to study the inheritance patterns of tail-feather color. The expression levels of pigment genes in differently colored feather follicles were analyzed using quantitative real-time PCR. The results showed that genetic regulation of tail-feather color was independent of body-plumage color. The Dominant White locus inhibited eumelanin synthesis in tail feathers without affecting the formation of yellow body plumage, whereas the Silver locus had the opposite effect. The expression of agouti signaling protein (ASIP) gene class 1 transcripts was significantly lower in black tail-feather follicles than in yellow body follicles, whereas tyrosinase-related protein 1 (TYRP1) gene expression was significantly higher in black tail feathers. These differentially expressed genes were confirmed to exert an effect on eumelanin and pheomelanin formation in feathers, thus influencing the regulation of chicken tail-feather color. In conclusion, this study lays the foundation for further research on the genetic mechanisms of regional differences in feather color, contributing to a better understanding of plumage pigmentation in chickens.

Development, validation and reliability testing of the hospice care environment scale.

BACKGROUND: WHO stated the environment is an important factor affecting the development of hospice care. The environment is the sum of factors affecting behavior besides the individual factors. Currently, a scale to comprehensively assess the hospice environment of nurse is still lacking. This study aimed to develop an instrument to investigate the environmental factors affecting hospice care of nurses. METHODS: Literature review and a semi-structured interview were conducted to form the items pool of the Hospice Care Environment Scale. Two rounds of Delphi expert consultation were conducted by 16 experts to revise the scale dimensions and entries to form the Hospice Care Environment Scale. A psychometric evaluation was then performed among 530 oncology nurses in a large tertiary oncology hospital in Hubei Province. The 500 valid questionnaires were randomly divided into two groups in a 1:1 ratio, sample 1 (n1 = 250) for item screening and sample 2 (n2 = 250) for quality evaluation of the resulting scale. Item analysis, reliability analysis, validity analysis and acceptability analysis were performed. RESULT: The Hospice Care Environment Scale consists of two dimensions and 13 entries. The Cronbach's α coefficient of the Hospice Care Environment Scale was 0.970, and the Cronbach's α coefficient of the two dimensions were 0.952 and 0.969, respectively, with the Item-content validity index and average Scale- content validity index of the scale was both 1.000. The validation factor analysis showed the standardized path coefficients of each item were basically above 0.5, and the factor structure model was stable and suitable. The average completion time of the scale was about 3 min, which had good feasibility. CONCLUSION: The Hospice Care Environment Scale to assess the environment of hospice care services, has good content and construct validity and reliability. This scale can provide guidance to evaluate the hospice care environment.

Behaviors and influencing factors of Chinese oncology nurses towards hospice care: a cross-sectional study based on social cognitive theory in 2022.

BACKGROUND: Although there is growing demand for hospice care in China due to its aging population and increasing cancer rates, the sector remains slow to expand. Oncology nurses are the primary providers of hospice care, but little is known about their behaviors towards hospice care and related factors. METHODS: This cross-sectional study conveniently sampled 933 oncology nurses from six grade A tertiary hospitals in Hubei Province between January to March 2022. The questionnaire was composed of seven parts: general information (including sociodemographic and work-related information), hospice care behaviors, hospice care knowledge, hospice care attitudes, hospice care self-efficacy, hospice care outcome expectancy, and hospice care environment. Data were analyzed using descriptive analysis, independent sample t-tests, one-way ANOVA, Pearson's correlation, multiple linear regression, random forest regression, and BP neural network model analysis. RESULTS: A total of 852 questionnaires were valid. The mean score of hospice care behaviors was 50.47 ± 10.56, with a mean item score of 3.61 ± 0.75. The three highest scoring behaviors were "pain assessment of patients (4.21 ± 0.91)", "satisfying the physical and mental needs of dying patients (4.04 ± 0.92)", and "creating good relationships between the medical staff and family members (4.02 ± 0.87)". The two lowest-scoring behaviors were "proactively recommending medical institutions for hospice care to terminally ill patients and their families (2.55 ± 1.10)" and "proactively talking to patients and families about death-related topics for patients who are critically ill and cannot be reversed (2.87 ± 1.03)." Multiple linear regression, random forest regression, and BP neural network models all showed that the frequency of sharing hospice care experiences with colleagues, hospice care attitudes, hospice care self-efficacy, and hospice care environments were positively associated with hospice care behaviors. CONCLUSIONS: The frequency of hospice care behaviors among Chinese oncology nurses is generally at a moderate to high level. The results provide a basis for promoting hospice care behaviors among oncology nurses in order to improve the quality of life for terminally ill cancer patients.

Effect of simethicone for the management of early abdominal distension after laparoscopic cholecystectomy: a multicenter retrospective propensity score matching study.

OBJECTIVE: To investigate whether simethicone expediates the remission of abdominal distension after laparoscopic cholecystectomy (LC). METHODS: This retrospective study involved LC patients who either received perioperative simethicone treatment or not. Propensity score matching (PSM) was employed to minimize bias. The primary endpoint was the remission rate of abdominal distension within 24 h after LC. Univariable and multivariable logistic regression analyses were conducted to identify independent risk factors affecting the early remission of abdominal distension after LC. Subsequently, a prediction model was established and validated. RESULTS: A total of 1,286 patients were divided into simethicone (n = 811) and non-simethicone groups (n = 475) as 2:1 PSM. The patients receiving simethicone had better remission rates of abdominal distension at both 24 h and 48 h after LC (49.2% vs. 34.7%, 83.9% vs. 74.8%, respectively), along with shorter time to the first flatus (14.6 ± 11.1 h vs. 17.2 ± 9.1 h, P < 0.001) compared to those without. Multiple logistic regression identified gallstone (OR = 0.33, P = 0.001), cholecystic polyp (OR = 0.53, P = 0.050), preoperative abdominal distention (OR = 0.63, P = 0.002) and simethicone use (OR = 1.89, P < 0.001) as independent factors contributing to the early remission of abdominal distension following LC. The prognosis model developed for predicting remission rates of abdominal distension within 24 h after LC yielded an area under the curve of 0.643 and internal validation a value of 0.644. CONCLUSIONS: Simethicone administration significantly enhanced the early remission of post-LC abdominal distension, particularly for patients who had gallstones, cholecystic polyp, prolonged anesthesia or preoperative abdominal distention. TRIAL REGISTRATION: ChiCTR2200064964 (24/10/2022).

Whole-Genome Resequencing of Worldwide Wild and Domestic Sheep Elucidates Genetic Diversity, Introgression, and Agronomically Important Loci.

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (∼16.10×) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, ∼121.2 million single nucleotide polymorphisms, ∼61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3'-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.

Coronavirus Porcine Deltacoronavirus Upregulates MHC Class I Expression through RIG-I/IRF1-Mediated NLRC5 Induction.

Major histocompatibility complex class I (MHC-I) and MHC-II molecules, mainly being responsible for the processing and presentation of intracellular or extracellular antigen, respectively, are critical for antiviral immunity. Here, we reported that porcine deltacoronavirus (PDCoV) with the zoonotic potential and potential spillover from pigs to humans, upregulated the expressions of porcine MHC-I (swine leukocyte antigen class I, SLA-I) molecules and SLA-I antigen presentation associated genes instead of porcine MHC-II (SLA-II) molecules both in primary porcine enteroids and swine testicular (ST) cells at the late stage of infection, and this finding was verified in vivo. Moreover, the induction of SLA-I molecules by PDCoV infection was mediated through enhancing the expression of NOD-like receptor (NLR) family caspase recruitment domain-containing 5 (NLRC5). Mechanistic studies demonstrated that PDCoV infection robustly elevated retinoic acid-inducible gene I (RIG-I) expression, and further initiated the downstream type I interferon beta (IFN-ß) production, which led to the upregulation of NLRC5 and SLA-I genes. Likewise, interferon regulatory factor 1 (IRF1) elicited by PDCoV infection directly activated the promoter activity of NLRC5, resulting in an increased expression of NLRC5 and SLA-I upregulation. Taken together, our findings advance our understanding of how PDCoV manipulates MHC molecules, and knowledge that could help inform the development of therapies and vaccines against PDCoV. IMPORTANCE MHC-I molecules play a crucial role in antiviral immunity by presenting intracellular antigens to CD8+T lymphocytes and eliminating virus-infected cells by natural killer cells' "missing-self recognition." However, the manipulation of MHC molecules by coronaviruses remains poorly understood. Here, we demonstrated that PDCoV, a zoonotic potential coronavirus efficiently infecting cells from broad species, greatly increased the expressions of porcine MHC-I (SLA-I) molecules and MHC-I antigen presentation associated genes but not porcine MHC-II (SLA-II) molecules both in vitro and in vivo. Mechanistically, the upregulation of MHC-I molecules by PDCoV infection required the master transactivator of MHC-I, NLRC5, which was mediated not only by RIG-I-initiated type I IFN signaling pathway but also by IRF1 induced by PDCoV as it could activate NLRC5 promoter activity. These results provide significant insights into the modification of the MHC class I pathway and may provide a potential therapeutic intervention for PDCoV.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/ß Nuclear Import Pathway.

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of the FBXW7 Protein via the Ubiquitin-Proteasome Pathway.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Longitudinal study of multidimensional factors influencing maternal and offspring health outcomes: a study protocol.

BACKGROUND: Reducing preventable adverse maternal and offspring outcomes is a global priority. The causes of adverse maternal and fetal outcomes are complex with multidimensional influencing factors. In addition, the Covid-19 epidemic has had a significant psychological and physical impact on people. China is now stepping into the post-epidemic era. We are curious about the psychological and physical situation of maternity in China at this stage. Therefore, we plan to initiate a prospective longitudinal study to investigate the multidimensional influences and mechanisms that affect maternal and offspring health. METHOD: We will recruit eligible pregnant women at Renmin Hospital of Hubei Province, China. The expected sample size is 1490. We will assess socio-demographics, Covid-19 related information, social capital, sleep, mental health and medical records, including clinical examination and biochemical tests. Eligible pregnant women will be enrolled in the study with less than 14 weeks of gestation. Participants will receive a total of nine follow-up visits between mid-pregnancy and one year postpartum. The offspring will be followed up at birth, 6 weeks, 3 months, 6 months and one year. In addition, a qualitative study will be conducted to understand the underlying causes that affect maternal and offspring health outcomes. DISCUSSION: This is the first longitudinal study of maternity in Wuhan, Hubei Province which integrates physical, psychological and social capital dimensions. Wuhan is the first city to be affected by Covid-19 in China. As China moves into the post-epidemic era, this study will provide us with a better understanding of the long-term impact of the epidemic on maternal and offspring health outcomes. We will implement a range of rigorous measures to enhance participants' retention rate and ensure the quality of data. The study will provide empirical results for maternal health in the post-epidemic era.

Identification and integrated analysis of lncRNAs and miRNAs in IPEC-J2 cells provide novel insight into the regulation of the innate immune response by PDCoV infection.

BACKGROUND: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are pivotal regulators involved in the pathogenic mechanism of multiple coronaviruses. Porcine deltacoronavirus (PDCoV) has evolved multiple strategies to escape the innate immune response of host cells, but whether ncRNAs are involved in this process during PDCoV infection is still unknown. RESULTS: In this study, the expression profiles of miRNAs, lncRNAs and mRNAs in IPEC-J2 cells infected with PDCoV at 0, 12 and 24 hours postinfection (hpi) were identified through small RNA and RNA sequencing. The differentially expressed miRNAs (DEmiRNAs), lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were screened from the comparison group of IPEC-J2 cells at 0 and 12 hpi as well as the comparison group of IPEC-J2 cells at 12 and 24 hpi. The target genes of these DEncRNAs were predicted. The bioinformatics analysis of the target genes revealed multiple significantly enriched functions and pathways. Among them, the genes that were associated with innate immunity were specifically screened. The expression of innate immunity-related ncRNAs and mRNAs was validated by RT-qPCR. Competing endogenous RNA (ceRNA) regulatory networks among innate immunity-related ncRNAs and their target mRNAs were established. Moreover, we found that the replication of PDCoV was significantly inhibited by two innate immunity-related miRNAs, ssc-miR-30c-3p and ssc-miR-374b-3p, in IPEC-J2 cells. CONCLUSIONS: This study provides a data platform to conduct studies of the pathogenic mechanism of PDCoV from a new perspective and will be helpful for further elucidation of the functional role of ncRNAs involved in PDCoV escaping the innate immune response.

Anaerobic sulfamethoxazole-degrading bacterial consortia in antibiotic-contaminated wetland sediments identified by DNA-stable isotope probing and metagenomics analysis.

Anaerobic degradation has been demonstrated as an important pathway for the removal of sulfonamide (SA) in contaminated environments, and identifying the microorganisms responsible for the degradation of SA is a key step in developing bioaugmentation approaches. In this study, we investigated the anaerobic degradation activity of three SA [sulfadiazine (SDZ), sulfamethazine (SMZ) and sulfamethoxazole (SMX)] and the associated bacterial community in wetland sediments contaminated by aquaculture (in Fujian Province, coded with FJ), livestock farming (in Sichuan Province, coded with SC), or rural wastewaters (in Guangdong Province, coded with GD). Additionally, the combination of DNA-stable isotope probing (SIP) with metagenomics was further applied to assess the active SA-degrading microbes using SMX as a model SA. Among SDZ, SMZ and SMX, only SMX could be effectively dissipated, and the degradation of SMX was relatively fast in the microcosms of sediments with higher levels of SA contamination (FJ and SC). The anaerobic biotransformation pathway of SMX was initiated by hydrogenation with the cleavage of the N-O bond on the isoxazole ring. DNA-SIP revealed that the in situ active anaerobic SMX-degraders (5, 18 and 3 genera in sediments FJ, SC and GD respectively) were dominated by Proteobacteria in sediments FJ and SC, but by Firmicutes (two Family XVIII members) in sediment GD. Mycobacterium, unclassified Burkholderiaceae and Rhodocyclaceae were identified as the dominant active SMX-degrading bacteria in both sediments FJ and SC. Higher proportions of antibiotic resistance gene and genes involved in various functional categories were observed in sediments FJ and SC.