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Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA – protein interaction network.

Abstract Background Migraine is a neurological disorder characterized by complex, widespread, and sudden attacks with an unclear pathogenesis, particularly in chronic migraine (CM). Specific brain regions, including the insula, amygdala, thalamus, and cingulate, medial prefrontal, and anterior cingulate cortex, are commonly activated by pain stimuli in patients with CM and animal models. This study employs fluorescence microscopy optical sectioning tomography (fMOST) technology and AAV-PHP.eB whole-brain expression to map activation patterns of brain regions in CM mice, thus enhancing the understanding of CM pathogenesis and suggesting potential treatment targets. Methods By repeatedly administering nitroglycerin (NTG) to induce migraine-like pain in mice, a chronic migraine model (CMM) was established. Olcegepant (OLC) was then used as treatment and its effects on mechanical pain hypersensitivity and brain region activation were observed. All mice underwent mechanical withdrawal threshold, light-aversive, and elevated plus maze tests. Viral injections were administered to the mice one month prior to modelling, and brain samples were collected 2 h after the final NTG/vehicle control injection for whole-brain imaging using fMOST. Results In the NTG-induced CMM, mechanical pain threshold decreased, photophobia, and anxiety-like behavior were observed, and OLC was found to improve these manifestations. fMOST whole-brain imaging results suggest that the isocortex-cerebral cortex plate region, including somatomotor areas (MO), somatosensory areas (SS), and main olfactory bulb (MOB), appears to be the most sensitive area of activation in CM (P

Abstract Background Migraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated. Methods We established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1β, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C. Results At the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2. Conclusions Our data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine. Graphical Abstract

Abstract Background Energy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM. Methods Mice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-κB pathway activation were detected after the intervention. Results Repeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1β, IL-6, and TNF-α in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-κB pathway. Conclusions AMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine.

Abstract Background Diarrheal diseases caused by viral agents have led to a great morbidity, mortality, and economic loss in global pig industry. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and group A porcine rotavirus (RVA) are main causative agents of swine viral diarrhea with similar clinical signs on Chinese farms and their co-infection is also common. However, it is still lack of a convenient method to detect these four agents. Methods A TaqMan multiplex qPCR method was developed to detect PEDV, TGEV, PDCoV, and RVA, simultaneously. This method was then applied to investigate 7,342 swine fecal samples or rectal swabs, as well as 1,246 swine intestinal samples collected from 2075 farms in China in 2022. Results Minimum detection limits of this method were 3 copies/µL for PEDV, 4 copies/µL for TGEV, 8 copies/µL for RVA, and 8 copies/µL for PDCoV, suggesting a good sensitivity. No signals were observed by using this method detecting other viral agents commonly prevalent in pigs, which is suggestive of a good specificity. Application of this method on investigating clinical samples demonstrated a relatively high positive rate for PEDV (22.21%, 1907/8588) and RVA (44.00%, 3779/8588). In addition, co-infection between PEDV and RVA was observed on 360 investigated farms, accounting for 17.35% (360/2075) of the farms where co-infection events were screened. Conclusions A TaqMan multiplex qPCR method targeting PEDV, TGEV, PDCoV, and RVA was developed in this study. This method demonstrated a good specificity and sensitivity on investigating these four common viruses responsible for viral diarrhea on Chinese pig farms, which represents a convenient method for the monitoring and differential diagnosis of swine viral diarrhea.

Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and “collateral” promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT – qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.

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

The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.

Since it was first reported in 2013, the NADC30-like PRRSV has been epidemic in China. Hubei Province is known as China’s key hog-exporting region. To understand the prevalence and genetic variation of PRRSV, herein, we detected and analyzed 317 lung tissue samples from pigs with respiratory disease in Hubei Province, and demonstrated that the NADC30-like strain was the second-most predominant strain during 2017–2018, following the highly pathogenic PRRSV (HP-PRRSV). Additionally, we isolated a new NADC30-like PRRSV strain, named CHN-HB-2018, which could be stably passaged in Marc-145 cells. Genetic characterization analysis showed that compared with the NADC30 strain, the CHN-HB-2018 strain had several amino acid variations in glycoprotein (GP) 3, GP5, and nonstructural protein 2 (NSP2). Moreover, the CHN-HB-2018 strain showed a unique 5-amino acid (aa) deletion in NSP2, which has not previously been reported. Gene recombination analysis identified the CHN-HB-2018 strain as a potentially recombinant PRRSV of the NADC30-like strain and HP-PRRSV. Animal experiments indicated that the CHN-HB-2018 strain has a mild pathogenicity, with no mortality and only mild fever observed in piglets. This study contributes to defining the evolutionary characteristics of PRRSV and its molecular epidemiology in Hubei Province, and provides a potential candidate strain for PRRSV vaccine development.

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)3-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)3-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)3-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.

[This corrects the article DOI: 10.1371/journal.pone.0055838.].

ABSTRACT Endoribonuclease non-structural protein 15 (nsp15) (EndoU) is conserved among coronaviruses (CoVs) and is crucial for viral replication, evasion of the innate immune system, and virulence. EndoU-deficient CoVs can activate the interferon (IFN) response and attenuate their virulence, and nsp15 is considered the target of attenuated vaccine development. Among alpha-CoVs, transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) cause lethal diseases in piglets and cats, but the role of EndoU in viral propagation and virulence remains unclear. Here, we verified the TGEV and FIPV EndoU active sites His226 and His241 and found that the antagonization of SeV-induced IFN-β production by nsp15 depends on its EndoU activity. Furthermore, we constructed infectious clones of wild-type (WT) and EndoU-deficient (EnUmt) TGEV and FIPV. Unexpectedly, we found that both the WT and EnUmt viruses propagated efficiently in multiple types of immunocompetent (PK-15, IPI-2I, ST, CRFK, F81, and Fcwf-4) cells. Moreover, the results of infection experiments showed that compared with piglets and cats infected with the WT, the EnUmt virus-infected piglets and cats did not exhibit significantly reduced mortality, tissue injury, or viral shedding. Specially, the death of cats infected with EnUmt-FIPV occurred earlier than that of cats infected with WT. Hence, our results suggest that the function of EndoU is conserved, but nsp15-mediated regulation of the propagation and pathogenesis of CoVs are diverse. Our findings provide a reference for an in-depth understanding of EndoU-mediated immune escape and pathogenicity in CoVs. IMPORTANCE Understanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-β (IFN-β) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-β response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell–cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell–cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

Abstract Background Headache during hemodialysis (HDH) is prevalent but not negligible. Despite the high prevalence of dialysis headaches, they have rarely been studied. Therefore, this study aimed to evaluate the prevalence, risk factors, and clinical characteristics of HDH and reappraise the HDH diagnostic criteria in the International Classification of Headache Disorders 3 (ICHD-3). Methods One hundred and fifty-four patients completed this randomized cross-sectional study. Consecutive patients who underwent haemodialysis were assessed using a semi-structured questionnaire. The patients were administered face-to-face questionnaires while undergoing dialysis. Results This study included 154 patients. Before commencing dialysis, 3.24% (5/154) of the patients had migraine without aura, 1.29% (2/154) had menstrual-related migraine, 0.6% (1/154) had tension-type headaches, and 0.6% (1/154) had an unclassifiable headache. One case (0.6%) of headache resolved after dialysis treatment. HDH was diagnosed in 9.09% (14/154) of the patients. Headache after haemodialysis (HAH) was reported in 6.49% (10/154) of patients. The most prevalent features of HDH were frontal or temporal location, bilateral headaches, dull and throbbing nature, and moderate severity. HDH started at a mean of 2.33 ± 0.79 h after dialysis commenced. The average headache duration was 6.56 ± 1.57 h (median = 3.0 h), with 66.67% of the patients reporting a duration of ≤4 h. HDH was more prevalent in females than males (P = 0.01, P

ObjectiveThis cross-sectional survey aimed to investigate the prevalence of depression among medical staff and its risk factors as well as the association between depression, anxiety, headache, and sleep disorders.MethodsStratified random cluster sampling was used to select medical staff from various departments of four hospitals in Sanya City. The Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), and Pittsburgh Sleep Quality Index (PSQI) were used to quantitatively assess depression, anxiety, and sleep disorders. Correlation and regression analyses were performed to determine factors affecting the depression occurrence and scores.ResultsAmong 645 medical staff members, 548 (85%) responded. The 1-year prevalence of depression was 42.7% and the prevalence of depression combined with anxiety, headache, and sleep disorders was 23, 27, and 34.5%, respectively. The prevalence of depression in women, nurses, the unmarried or single group, and the rotating-shift population was significantly higher than that in men (48.3% vs. 27.1%, odds ratio OR = 2.512), doctors (55.2% vs. 26.7%, OR = 3.388), the married group (50.5% vs. 35.8%, OR = 1.900), and the day-shift population (35.2% vs. 7.5%, OR = 1.719). The occurrence of depression was correlated with anxiety, sleep disorders, headache, and migraines, with anxiety having the highest correlation (Spearman’s Rho = 0.531). The SDS was significantly correlated with the SAS and PSQI (Spearman’s Rho = 0.801, 0.503) and was also related to the presence of headache and migraine (Spearman Rho = 0.228, 0.159). Multiple logistic regression indicated that nurse occupation and anxiety were risk factors for depression, while grades of anxiety, sleep disorders and nurse occupation were risk factors for the degree of depression in multiple linear regression.ConclusionThe prevalence of depression among medical staff was higher than that in the general population, especially among women, nurses, unmarried people, and rotating-shift workers. Depression is associated with anxiety, sleep disorders, headache, and migraines. Anxiety and nursing occupation are risk factors for depression. This study provides a reference for the promotion of occupational health among medical professionals.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2′ site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2′ site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2′ site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2′JS2008, in which the S2 (amino acids 894–1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2′JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2′JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.

Abstract Background The infection and spread of porcine reproductive and respiratory syndrome virus (PRRSV) pose a serious threat to the global pig industry, and inhibiting the viral infection process is a promising treatment strategy. Nanomaterials can interact with viruses and have attracted much attention due to their large specific surface area and unique physicochemical properties. Ferrous sulfide nanoparticles (FeS NPs) with the characteristics of high reactivity, large specific surface area, and low cost are widely applied to environmental remediation, catalysis, energy storage and medicine. However, there is no report on the application of FeS NPs in the antiviral field. In this study, gelatin stabilized FeS nanoparticles (Gel-FeS NPs) were large-scale synthesized rapidly by the one-pot method of co-precipitation of Fe2+ and S2‒. Results The prepared Gel-FeS NPs exhibited good stability and dispersibility with an average diameter of 47.3 nm. Additionally, they were characterized with good biocompatibility and high antiviral activity against PRRSV proliferation in the stages of adsorption, invasion, and replication. Conclusions We reported for the first time the virucidal and antiviral activity of Gel-FeS NPs. The synthesized Gel-FeS NPs exhibited good dispersibility and biocompatibility as well as effective inhibition on PRRSV proliferation. Moreover, the Fe2+ released from degraded Gel-FeS NPs still displayed an antiviral effect, demonstrating the advantage of Gel-FeS NPs as an antiviral nanomaterial compared to other nanomaterials. This work highlighted the antiviral effect of Gel-FeS NPs and provided a new strategy for ferrous-based nanoparticles against PRRSV. Graphical Abstract

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CLpro) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CLpro exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CLpro remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CLpro using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CLpro numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CLpro and EAV 3CLpro formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CLpro and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CLpro toward P1-Gln. In turn, the T139K exchange of EAV 3CLpro showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CLpro to trigger a substrate specificity switch.

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Severe interstitial pneumonia is the typical pathological characteristic of PRRSV-infected swine. Accumulating evidence has suggested that hypoxia-inducible factor 1α (HIF-1α) plays vital roles in the development of inflammation and the viral life cycle. However, the role and the underlying mechanism of HIF-1α in PRRSV infection remain elusive. Here, we found that PRRSV infection elevated HIF-1α expression. Furthermore, overexpression of HIF-1α increased PRRSV replication, whereas knockdown of HIF-1α inhibited PRRSV infection. Our further mechanistic analysis revealed that PRRSV-encoded nonstructural protein 1β (nsp1β) promoted HIF-1α transcription via its N-terminal nuclease activity and degraded the polyubiquitin chain of HIF-1α via its C-terminal deubiquitylation (DUB) enzyme activity, collectively stabilizing HIF-1α. Meanwhile, nsp1β interacted with both HIF-1α and von Hippel-Lindau tumor suppressor (pVHL) to form a ternary complex, which may have hindered pVHL-mediated ubiquitination degradation of HIF-1α by impairing the interaction between HIF-1α and pVHL. Interestingly, pVHL also stabilized nsp1β via K63-linked ubiquitination, forming a positive feedback loop to stabilize HIF-1α. Taken together, these results indicate that PRRSV infection stabilizes HIF-1α to facilitate viral proliferation and that viral nsp1β plays a vital role in enhancing the expression and stabilization of HIF-1α. The regulation of HIF-1α may have great therapeutic potential for the development of novel drugs against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry worldwide for over 30 years and shows no signs of slowing down. In this study, we found that PRRSV infection elevated hypoxia-inducible factor 1α (HIF-1α) expression. In addition, overexpressed HIF-1α contributed to PRRSV replication, whereas knockdown of HIF-1α reduced PRRSV growth. The PRRSV-encoded nonstructural protein 1β (nsp1β) exerted a stabilizing effect on HIF-1α through its nuclease protease and papain-like cysteine protease enzymatic domains. PRRSV nsp1β also interacted with von Hippel-Lindau tumor suppressor (pVHL) and HIF-1α, whereby nsp1β impaired the interaction between HIF-1α and pVHL. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection and provides new insights for the development of HIF-1α-based anti-PRRSV therapies.

Due to the worldwide impact of viruses such as SARS‐CoV‐2, researchers have paid extensive attention to antiviral reagents against viruses. Despite extensive research on two‐dimensional (2D) transition metal carbides (MXenes) in the field of biomaterials, their antiviral effects have received little attention. In this work, heparan sulfate analogue (sodium 3‐mercapto‐1‐propanesulfonate, MPS) modified 2D MXene nanocomposites (Ti3C2‐Au‐MPS) for prevention of viral infection are prepared and investigated using severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pseudovirus and porcine reproductive and respiratory syndrome virus (PRRSV) as two model viruses. Ti3C2‐Au‐MPS nanocomposites are shown to possess antiviral properties in the different stages of PRRSV proliferation, such as direct interaction with PRRS virions and inhibiting their adsorption and penetration in the host cell. Additionally, Ti3C2‐Au‐MPS nanocomposites can strongly inhibit the infection of SARS‐CoV‐2 pseudovirus as shown by the contents of its reporter gene GFP and luciferase. These results demonstrate the potential broad‐spectrum antiviral property of Ti3C2‐Au‐MPS nanocomposites against viruses with the receptor of heparin sulfate. This work sheds light on the specific antiviral effects of MXene‐based nanocomposites against viruses and may facilitate further exploration of their antiviral applications.

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

DEAD-box RNA helicase 21 (DDX21), also known as RHII/Gu, is an ATP-dependent RNA helicase. In addition to playing a vital role in regulating cellular RNA splicing, transcription, and translation, accumulated evidence has suggested that DDX21 is also involved in the regulation of innate immunity. However, whether DDX21 induces or antagonizes type I interferon (IFN-I) production has not been clear and most studies have been performed through ectopic overexpression or RNA interference-mediated knockdown. In this study, we generated DDX21 knockout cell lines and found that knockout of DDX21 enhanced Sendai virus (SeV)-induced IFN-β production and IFN-stimulated gene (ISG) expression, suggesting that DDX21 is a negative regulator of IFN-β. Mechanistically, DDX21 competes with retinoic acid-inducible gene I (RIG-I) for binding to double-stranded RNA (dsRNA), thereby attenuating RIG-I-mediated IFN-β production. We also identified that the 217–784 amino acid region of DDX21 is essential for binding dsRNA and associated with its ability to antagonize IFN production. Taken together, our results clearly demonstrated that DDX21 negatively regulates IFN-β production and functions to maintain immune homeostasis.

Porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent of PRRS, is prevalent worldwide, causing substantial and immense economic losses to the global swine industry. While current commercial vaccines fail to efficiently control PRRS, the development of safe and effective antiviral drugs against PRRSV is urgently required. Alkaloids are natural products with wide pharmacological and biological activities. Herein, sanguinarine, a benzophenanthridine alkaloid that occurs in many plants such as Macleaya cordata, was demonstrated as a potent antagonist of PRRSV. Sanguinarine attenuated PRRSV proliferation by targeting the internalization, replication, and release stages of the viral life cycle. Furthermore, ALB, AR, MAPK8, MAPK14, IGF1, GSK3B, PTGS2, and NOS2 were found as potential key targets related to the anti-PRRSV effect of sanguinarine as revealed by network pharmacology and molecular docking. Significantly, we demonstrated that the combination of sanguinarine with chelerythrine, another key bioactive alkaloid derived from Macleaya cordata, improved the antiviral activity. In summary, our findings reveal the promising potential of sanguinarine as a novel candidate for the development of anti-PRRSV agents.

The spike protein of coronaviruses (S-protein) is an envelope-anchored trimeric type I transmembrane glycoprotein that mediates receptor binding and the fusion of the viral and host cell membranes. Here the authors report the conformational states of HCoV-229E S trimer and observe the conformational changes in S1 subunit from closed state to activated state for receptor binding.

Coronaviruses (CoVs) are potential pandemic pathogens that can infect a variety of hosts and cause respiratory, enteric, hepatic and neurological diseases. Nonstructural protein 3 (nsp3), an essential component of the replication/transcription complex, is one of the most important antiviral targets. Here, we report the first crystal structure of multiple functional domains from porcine delta-coronavirus (PDCoV) nsp3, including the macro domain (Macro), ubiquitin-like domain 2 (Ubl2) and papain-like protease (PLpro) catalytic domain. In the asymmetric unit, two of the subunits form the head-to-tail homodimer with an interaction interface between Macro and PLpro. However, PDCoV Macro-Ubl2-PLpro mainly exists as a monomer in solution. Then, we conducted fluorescent resonance energy transfer-based protease assays and found that PDCoV PLpro can cleave a peptide by mimicking the cognate nsp2/nsp3 cleavage site in peptide substrates and exhibits deubiquitinating and de-interferon stimulated gene(deISGylating) activities by hydrolysing ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and ISG15-AMC substrates. Moreover, the deletion of Macro or Macro-Ubl2 decreased the enzyme activity of PLpro, indicating that Macro and Ubl2 play important roles in maintaining the stability of the PLpro domain. Two active sites of PLpro, Cys260 and His398, were determined; unexpectedly, the conserved site Asp412 was not the third active site. Furthermore, the motif “NGYDT” (amino acids 409–413) was important for stabilizing the enzyme activity of PLpro, and the N409A mutant significantly decreased the enzyme activity of PLpro. These results provide novel insights into the replication mechanism of CoV and new clues for future drug design.

ABSTRACT Type I interferons (IFN-Is) are critical antiviral cytokine in innate immunity but with limited direct defense ability against bacterial infections in mammals. In bony fish, despite all the IFN-Is (IFN1-4) act in antiviral immunity, studies demonstrate that IFN1 can remarkably contribute to host defense against bacterial infections. In this study, we found that IFN1 from grass carp (Ctenopharyngodon idella) contains an unusual cationic and amphipathic α-helical region (named as gcIFN-20, sequence: SYEKKINRHFKILKKNLKKK). The synthesized peptide gcIFN-20 could form α-helical structure in a membrane environment and exerts potent antimicrobial activity against multiple species of Gram-negative (G−) and Gram-positive (G+) bacteria with negligible toxicity. Mechanism studies showed gcIFN-20 kills G+ bacteria through membrane disruption and cytoplasm outflow while G− bacteria through membrane permeation and protein synthesis inhibition. In two mouse bacterial infection models, gcIFN-20 therapy could significantly reduce tissue bacterial loads and mortalities. In addition to the direct antibacterial activity, we also found that gcIFN-20 could significantly suppress the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro and in vivo, obviously alleviated lung lesions in a mouse endotoxemia model. The mechanism is that gcIFN-20 interacts with LPS, causes LPS aggregation and neutralization. The antimicrobial and anti-inflammatory activities in vivo of gcIFN-20 in mammalian models suggested a promising agent for developing peptide-based antibacterial therapy. IMPORTANCE Type I interferons play crucial role in antiviral immunity in both vertebrates and invertebrates. The powerful antimicrobial activity is recently reported in nonmammalian vertebrates. The present study identified a novel antimicrobial peptide (gcIFN-20) derived from grass carp interferon 1, found gcIFN-20 exhibits forceful bactericidal and anti-inflammatory activity in mammals, and efficient therapeutic effect against two clinical severe extraintestinal pathogenic Escherichia coli and a mouse endotoxemia models. The antimicrobial mechanisms are membrane disruption and cytoplasm overflow for Gram-positive bacteria, while membrane permeation and protein synthesis inhibition for Gram-negative bacteria. The anti-inflammatory mechanisms can be aggregating and neutralizing lipopolysaccharide to attenuate the binding with receptors and facilitate phagocytosis. The results indicate that gcIFN-20 can be a promising novel therapeutic agent for bacterial diseases and inflammatory disorders, especially as a potential weapon for multidrug resistant strain infections.

Abstract Deltacoronavirus is the last identified Coronaviridae subfamily genus. Differing from other coronavirus (CoV) genera, which mainly infect birds or mammals, deltacoronaviruses (δ‐CoVs) reportedly infect both animal types. Recent studies show that a novel δ‐CoV, porcine deltacoronavirus (PDCoV), can also infect calves and chickens with the potential to infect humans, raising the possibility of cross‐species transmission of δ‐CoVs. Here, we explored the deep phylogenetic history and cross‐species transmission of δ‐CoVs. Virus–host cophylogenetic analyses showed that δ‐CoVs have undergone frequent host switches in birds, and sparrows may serve as the unappreciated hubs for avian to mammal transmission. Our molecular clock analyses show that PDCoV possibly originated in Southeast Asia in the 1990s and that the PDCoV cluster shares a common ancestor with Sparrow‐CoV of around 1,810. Our findings contribute valuable insights into the diversification, evolution, and interspecies transmission of δ‐CoVs and the origin of PDCoV, providing a model for exploring the relationships of δ‐CoVs in birds and mammals.

Cellular cholesterol plays an important role in the life cycles of enveloped viruses. Previous studies by our group and other groups have demonstrated that the depletion of cellular cholesterol by methyl-β-cyclodextrin (MβCD) reduces the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine Arterivirus that has been devastating the swine industry worldwide for over two decades. However, how PRRSV infection regulates cholesterol synthesis is not fully understood. In this study, we showed that PRRSV infection upregulated the activity of protein phosphatase 2 (PP2A), which subsequently activated 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the cholesterol synthesis pathway, to increase the levels of cellular cholesterol. By screening the PRRSV-encoded proteins, we showed that nsp4 dominated the upregulation of cellular cholesterol, independently of the 3C-like protease activity of nsp4. A mutation analysis showed that domain I (amino acids 1–80) of PRRSV nsp4 interacted with PR65 alpha (PR65α), the structural subunit, and PP2Ac, the catalytic subunit, of PP2A. Importantly, domain I of nsp4 inhibited Sendai virus-induced interferon β production, and this inhibitory effect was eliminated by Lovastatin, an HMGCR inhibitor, indicating that the upregulation of cellular cholesterol by nsp4 is a strategy used by PRRSV to suppress the antiviral innate immunity of its host. Collectively, we here demonstrated the mechanism by which PRRSV regulates cellular cholesterol synthesis and reported a novel strategy by which PRRSV evades its host's antiviral innate immune response.

Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

The intestinal tract is a crucial part of the body for growth and development, and its dysregulation can cause several diseases. The lack of appropriate in vitro models hampers the development of effective preventions and treatments against these intestinal tract diseases. Intestinal organoids are three-dimensional (3D) polarized structures composed of different types of cells capable of self-organization and self-renewal, resembling their organ of origin in architecture and function. Porcine intestinal organoids (PIOs) have been cultured and are used widely in agricultural, veterinary, and biomedical research. Based on the similarity of the genomic sequence, anatomic morphology, and drug metabolism with humans and the difficulty in obtaining healthy human tissue, PIOs are also considered ideal models relative to rodents. In this review, we summarize the current knowledge on PIOs, emphasizing their culturing, establishment and development, and applications in the study of host–microbe interactions, nutritional development, drug discovery, and gene editing potential.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-β (IFN-β) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-β promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-β production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-β production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.

The porcine reproductive and respiratory syndrome virus (PRRSV) remains a persistent hazard in the global pig industry. DEAD (Glu-Asp-Ala-Glu) box helicase 21 (DDX21) is a member of the DDX family. In addition to its function of regulating cellular RNA metabolism, DDX21 also regulates innate immunity and is involved in the replication cycle of some viruses. However, the relationship between DDX21 and PRRSV has not yet been explored. Here, we found that a DDX21 overexpression promoted PRRSV replication, whereas knockdown of DDX21 reduced PRRSV proliferation. Mechanistically, DDX21 promoted PRRSV replication independently of its ATPase, RNA helicase, and foldase activities. Furthermore, overexpression of DDX21 stabilized the expressions of PRRSV nsp1α, nsp1β, and nucleocapsid proteins, three known antagonists of interferon β (IFN-β). Knockdown of DDX21 activated the IFN-β signaling pathway in PRRSV-infected cells, suggesting that the effect of DDX21 on PRRSV-encoded IFN-β antagonists may be a driving factor for its contribution to viral proliferation. We also found that PRRSV infection enhanced DDX21 expression and promoted its nucleus-to-cytoplasm translocation. Screening PRRSV-encoded proteins showed that nsp1β interacted with the C-terminus of DDX21 and enhanced the expression of DDX21. Taken together, these findings reveal that DDX21 plays an important role in regulating PRRSV proliferation through multiple mechanisms.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen in the swine industry, is a genetically highly diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus are not yet fully understood. In this study, we performed an integrated analysis of all available whole-genome sequences of type 2 PRRSV (n = 901) to reveal its evolutionary dynamics. The results showed that there were three distinct phylogenetic lineages of PRRSV in their distribution patterns. We identified that sublineage 2.7 (L2.7), associated with a NADC30 cluster, had the highest substitution rate and higher viral genetic diversity, and inter-lineage recombination is observed more frequently in L2.7 PRRSV compared to other sublineages. Most inter-lineage recombination events detected are observed between L2.7 PRRSVs (as major parents) and L3.4 (a JXA1-R-related cluster)/L3.7 (a WUH3-related cluster) PRRSVs (as minor parents). Moreover, the recombination hotspots are located in the structural protein gene ORF2 and ORF4, or in the non-structural protein gene nsp7. In addition, a GM2-related cluster, L3.2, shows inconsistent recombination modes compared to those of L2.7, suggesting that it may have undergone extensive and unique recombination in their evolutionary history. We also identified several amino acids under positive selection in GP2, GP4 and GP5, the major glycoproteins of PRRSV, showing the driving force behind adaptive evolution. Taken together, our results provide new insights into the evolutionary dynamics of PPRSV that contribute to our understanding of the critical factors involved in its evolution and guide future efforts to develop effective preventive measures against PRRSV.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Coronavirus accessory proteins are a unique set of proteins whose genes are interspersed among or within the genes encoding structural proteins. Different coronavirus genera, or even different species within the same coronavirus genus, encode varying amounts of accessory proteins, leading to genus- or species-specificity. Though accessory proteins are dispensable for the replication of coronavirus in vitro, they play important roles in regulating innate immunity, viral proliferation, and pathogenicity. The function of accessory proteins on virus infection and pathogenesis is an area of particular interest. In this review, we summarize the current knowledge on accessory proteins of several representative coronaviruses that infect humans or animals, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with an emphasis on their roles in interaction between virus and host, mainly involving stress response, innate immunity, autophagy, and apoptosis. The cross-talking among these pathways is also discussed.

Non-structural protein 1 (nsp1) is only characterized in alphacoronaviruses (α-CoVs) and betacoronaviruses (β-CoVs). There have been extensive researches on how the β-CoVs nsp1 regulates viral virulence by inhibiting host protein synthesis, but the regulatory mechanism of the α-CoVs nsp1 is still unclear. Here, we report the 2.1-Å full-length crystal structure of nsp1 in emerging porcine SADS-CoV and the 1.8-Å full-length crystal structure of nsp1 in the highly lethal cat FIPV. Although they belong to different subtypes of α-CoVs, these viruses all have a bucket-shaped fold composed of six β-sheets, similar to the crystal structure of PEDV and TGEV nsp1. Comparing the above four structures, we found that the structure of α-CoVs nsp1 in the same subtype was more conserved. We then selected mammalian cells that were treated with SADS-CoV and FIPV nsp1 for RNA sequencing analysis and found that nsp1 had a specific inhibitory effect on interferon (IFN) and cell cycle genes. Using the Renilla luciferase (Rluc) assay and Western blotting, we confirmed that seven representative α-CoVs nsp1s could significantly inhibit the phosphorylation of STAT1-S727 and interfere with the effect of IFN-I. Moreover, the cell cycle experiment confirmed that α-CoVs nsp1 could encourage host cells to stay in the G0/G1 phase. Based on these findings, we not only greatly improved the crystal structure data on α-CoVs nsp1, but we also speculated that α-CoVs nsp1 regulated host proliferation and immune evasion-related biological functions by inhibiting the synthesis of host proteins, thus creating an environment conducive to the virus.

Abstract In a previous work, gold nanoclusters (Au NCs) are found to inactivate RNA virus, but the effect of surface modification of Au NCs on its proliferation is still largely unknown. Here, the effect of surface modification of Au NCs on the proliferation of pseudorabies virus (PRV) by synthesizing two types of gold clusters with different surface modification, histidine stabilized Au NCs (His‐Au NCs) and mercaptoethane sulfonate and histidine stabilized Au NCs (MES‐Au NCs), is investigated. His‐Au NCs rather than MES‐Au NCs could strongly inhibit the proliferation of PRV, as indicated by the results of plaque assay, confocal microscopic analysis, Western blot assay, and quantitative real‐time polymerase chain reaction (PCR) assay. Further study reveals that His‐Au NCs perform the function via blockage of the viral replication process rather than the processes of attachment, penetration, or release. Additionally, His‐Au NCs are found to be mainly localized to nucleus, while MES‐Au NCs are strictly distributed in cytoplasm, which may explain why His‐Au NCs can suppress the proliferation of PRV, but not MES‐Au NCs. These results demonstrate that surface modification plays a key role in the antiviral effects of Au NCs and a potential antiviral agent can be developed by changing the Au NC surface modification.

Foot-and-mouth disease (FMD) is a highly contagious, severe viral illness notifiable to the World Organization for Animal Health. The causative agent, FMD virus (FMDV), replicates rapidly and efficiently inhibits host translation and the innate immune response for it has developed multiple tactics to evade host defenses and takes over gene expression machinery in the host cell. Here, we report a systemic analysis of the proteome and phosphoproteome of FMDV-infected cells. Bioinformatics analysis suggested that FMDV infection shuts off host cap-dependent translation, but leaves intact internal ribosome entry site (IRES)-mediated translation for viral proteins. Interestingly, several FMDV IRES-transacting factors, including G3BP stress granule assembly factor 1 (G3BP1), were dephosphorylated during FMDV infection. Ectopic expression of G3BP1 inhibited FMDV IRES activity, promoted assembly of stress granules, and activated innate immune responses, collectively suppressing FMDV replication. To counteract these host protective responses, FMDV-induced dephosphorylation of G3BP1, compromising its inhibitory effect on viral IRES. In addition, FMDV also proteolytically cleaved G3BP1 by its 3C protease (3Cpro). G3BP1 was cleaved at glutamic acid-284 (E284) by FMDV 3Cpro, and this cleavage completely lost the abilities of G3BP1 to activate innate immunity and to inhibit FMDV replication. Together, these data provide new insights into the post-translational mechanisms by which FMDV limits host stress and antiviral responses and indicate that G3BP1 dephosphorylation and its proteolysis by viral protease are important factors in the failure of host defense against FMDV infection.

DExD/H-box helicase 36 (DHX36) is known to be an ATP-dependent RNA helicase that unwinds the guanine-quadruplexes DNA or RNA, but emerging data suggest that it also functions as pattern recognition receptor in innate immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide. Interstitial pneumonia is considered to be one of the most obvious clinical signs of PRRSV infection, suggesting that the inflammatory response plays an important role in PRRSV pathogenesis. However, whether DHX36 is involved in PRRSV-induced inflammatory cytokine expression remains unclear. In this study, we found that PRRSV infection increased the expression of DHX36. Knockdown of DHX36 and its adaptor myeloid differentiation primary response gene 88 (MyD88) by small-interfering RNA in MARC-145 cells significantly reduced NF-κB activation and pro-inflammatory cytokine expression after PRRSV infection. Further investigation revealed that PRRSV nucleocapsid protein interacted with the N-terminal quadruplex binding domain of DHX36, which in turn augmented nucleocapsid protein-induced NF-κB activation. Taken together, our results suggest that DHX36–MyD88 has a relevant role in the recognition of PRRSV nucleocapsid protein and in the subsequent activation of pro-inflammatory NF-κB pathway.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus (CoV) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. Unlike most CoVs that antagonize type I interferon (IFN) production, previous studies showed that TGEV infection induces IFN-I production both in vivo and in vitro. However, the underlying mechanism(s) remain largely unknown. In this study, we found that TGEV infection significantly facilitated IFN-β production as well as activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor-kappaB (NF-κB) in porcine kidney (PK-15) cells. Screening of TGEV-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent IFN-β inducer and induced IFN-β production mainly by activating NF-κB but not IRF3. Further analysis showed that nsp14 interacted with DDX1, a member of the DExD/H helicase family. Knockdown of DDX1 by specific small interfering RNA (siRNA) significantly decreased nsp14-induced IFN-β production and NF-κB activation. Furthermore, TGEV-induced IFN-β production and IFN-stimulated gene (ISG) expression were decreased in cells transfected with DDX1-specific siRNA, indicating the vital role of DDX1 to TGEV-induced IFN-β responses. In summary, our data revealed a potential coactivator role of host RNA helicase DDX1 to the induction of IFN-β response initiated by TGEV and demonstrated that nsp14 is an important IFN inducer among the TGEV-encoded proteins.

Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.

Em2007, a porcine reproductive and respiratory syndrome virus (PRRSV) variant with a unique 68 aa deletion in Nsp2, was recently isolated in China. Phylogenetic and molecular evolutionary analyses indicated that Em2007 is a natural recombinant between a vaccine strain of PRRSV and circulating virus. We also tested its pathogenicity in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Previous studies have reported that PRRSV infection induced the production of IL-1β. However, the cellular sensors and signaling pathways involved in this process have not been elucidated yet. Here, we studied the mechanisms responsible for the production of IL-1β in response to highly pathogenic PRRSV. Upon PRRSV infection of primary porcine alveolar macrophages, both mRNA expression and secretion of IL-1β were significantly increased in a time- and dose-dependent manner. We also investigated the role of several pattern-recognition receptors and adaptor molecules in this response and showed that the TLR4/MyD88 pathway and its downstream signaling molecules, NF-κB, ERK1/2, and p38 MAPKs, were involved in IL-1β production during PRRSV infection. Treatment with specific inhibitors or siRNA knockdown assays demonstrated that components of the NLRP3 inflammasome were crucial for IL-1β secretion but not for IL-1β mRNA expression. Furthermore, TLR4/MyD88/NF-κB signaling pathway was involved in PRRSV-induced expression of NLRP3 inflammasome components. Together, our results deciphered the pathways leading from recognition of PRRSV to the production and release of IL-1β, providing a deeper knowledge of the mechanisms of PRRSV-induced inflammation responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. To investigate the impact of cellular microRNAs (miRNAs) on the replication of PRRSV, we screened 10 highly conserved miRNAs implicated in innate immunity or antiviral function and identified miR-125b as an inhibitor of PRRSV replication. Virus titer and western blot assays demonstrated that miR-125b reduced PRRSV replication and viral gene expression in a dose-dependent manner in both MARC-145 cell line and primary porcine alveolar macrophages. Mechanistically, miR-125b did not target the PRRSV genome. Rather, it inhibited activation of NF-κB, which we found to be required for PRRSV replication. PRRSV, in turn, down-regulated miR-125b expression post-infection to promote viral replication. Collectively, miR-125b is an antiviral host factor against PRRSV, but it is subject to manipulation by PRRSV. Our study reveals an example of manipulation of a cellular miRNA by an arterivirus to re-orchestrate host gene expression for viral propagation and sheds new light on targeting host factors to develop effective control measures for PRRS.

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.

Porcine epidemic diarrhea virus (PEDV), a member of Alphacoronavirus, has caused huge economic losses for the global pork industry recently. The spike (S) protein mediates PEDV entry into host cells. Herein, we investigated the interactions between the S protein and its receptor porcine aminopeptidase N (pAPN) or co-receptor sugars. The C-terminal domain (CTD) of the S1 domain is bound to pAPN. The prototype strain demonstrated similar receptor-binding activity compared with the variant field isolate. Three loops at the tips of the β-barrel domains did not play crucial roles in the PEDV S-pAPN association, indicating that PEDV conforms to a different receptor recognition model compared with transmissible gastroenteritis virus (TGEV), porcine respiratory CoV (PRCV), and human coronavirus NL63 (HCoV-NL63). The N-terminal domain (NTD) of the PEDV S1 domain could bind sugar, a possible co-receptor for PEDV. The prototype strain exhibited weaker sugar-binding activity compared with the variant field isolate. Strategies targeting the receptor binding domain (RBD) may be helpful for developing vaccines or antiviral drugs for PEDV. Understanding the differences in receptor binding between the prototype and the variant strains may provide insight into PEDV pathogenesis.