Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo.

RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.

Enhanced antigen-specific CD8 T cells contribute to early protection against FMDV through swine DC vaccination.

Foot-and-mouth disease virus (FMDV) remains a challenge for cloven-hooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines.IMPORTANCEAlthough the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines.

Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6).

Hand, foot, and mouth disease (HFMD) is caused by more than 20 pathogenic enteroviruses belonging to the Picornaviridae family and Enterovirus genus. Since the introduction of the enterovirus-71 (EV71) vaccine in 2016, the number of HFMD cases caused by EV71 has decreased. However, cases of infections caused by other enteroviruses, such as coxsackievirus A6 (CA6) and coxsackievirus A10, have been increasing accordingly. In this study, we used a clinical isolate of CA6 to establish an intragastric infection mouse model using 7-day-old mice to mimic the natural transmission route, by which we investigated the differential gene expression profiles associated with virus infection and pathogenicity. After intragastric infection, mice exhibited hind limb paralysis symptoms and weight loss, similar to those reported for EV71 infection in mice. The skeletal muscle was identified as the main site of virus replication, with a peak viral load reaching 2.31 × 107 copies/mg at 5 dpi and increased infiltration of inflammatory cells. RNA sequencing analysis identified differentially expressed genes (DEGs) after CA6 infection. DEGs in the blood, muscle, brain, spleen, and thymus were predominantly enriched in immune system responses, including pathways such as Toll-like receptor signaling and PI3K-Akt signaling. Our study has unveiled the genes involved in the host immune response during CA6 infection, thereby enhancing our comprehension of the pathological mechanism of HFMD.IMPORTANCEThis study holds great significance for the field of hand, foot, and mouth disease (HFMD). It not only delves into the disease's etiology, transmission pathways, and severe complications but also establishes a novel mouse model that mimics the natural coxsackievirus A6 infection process, providing a pivotal platform to delve deeper into virus replication and pathogenic mechanisms. Additionally, utilizing RNA-seq technology, it unveils the dynamic gene expression changes during infection, offering valuable leads for identifying novel therapeutic drug targets. This research has the potential to enhance our understanding of HFMD, offering fresh perspectives for disease prevention and treatment and positively impacting children's health worldwide.

Foot-and-mouth disease virus (FMDV) negatively regulates ZFP36 protein expression to alleviate its antiviral activity.

Zinc finger protein 36 (ZFP36) is a key regulator of inflammatory and cytokine production. However, the interplay between swine zinc-finger protein 36 (sZFP36) and foot-and-mouth disease virus (FMDV) has not yet been reported. Here, we demonstrate that overexpression of sZFP36 restricted FMDV replication, while the knockdown of sZFP36 facilitated FMDV replication. To subvert the antagonism of sZFP36, FMDV decreased sZFP36 protein expression through its non-structural protein 3C protease (3Cpro). Our results also suggested that 3Cpro-mediated sZFP36 degradation was dependent on its protease activity. Further investigation revealed that both N-terminal and C-terminal-sZFP36 could be degraded by FMDV and FMDV 3Cpro. In addition, both N-terminal and C-terminal-sZFP36 decreased FMDV replication. Moreover, sZFP36 promotes the degradation of FMDV structural proteins VP3 and VP4 via the CCCH-type zinc finger and NES domains of sZFP36. Together, our results confirm that sZFP36 is a host restriction factor that negatively regulates FMDV replication.IMPORTANCEFoot-and-mouth disease (FMD) is an infectious disease of animals caused by the pathogen foot-and-mouth disease virus (FMDV). FMD is difficult to prevent and control because there is no cross-protection between its serotypes. Thus, we designed this study to investigate virus-host interactions. We first demonstrate that swine zinc-finger protein 36 (sZFP36) impaired FMDV structural proteins VP3 and VP4 to suppress viral replication. To subvert the antagonism of sZFP36, FMDV and FMDV 3Cpro downregulate sZFP36 expression to facilitate FMDV replication. Taken together, the present study reveals a previously unrecognized antiviral mechanism for ZFP36 and elucidates the role of FMDV in counteracting host antiviral activity.

The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases.

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322-330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection.

Bayesian nonparametric inference for heterogeneously mixing infectious disease models.

SignificanceMathematical models of infectious disease transmission continue to play a vital role in understanding, mitigating, and preventing outbreaks. The vast majority of epidemic models in the literature are parametric, meaning that they contain inherent assumptions about how transmission occurs in a population. However, such assumptions can be lacking in appropriate biological or epidemiological justification and in consequence lead to erroneous scientific conclusions and misleading predictions. We propose a flexible Bayesian nonparametric framework that avoids the need to make strict model assumptions about the infection process and enables a far more data-driven modeling approach for inferring the mechanisms governing transmission. We use our methods to enhance our understanding of the transmission mechanisms of the 2001 UK foot and mouth disease outbreak.

Experiential grounding of abstract concepts: Processing of abstract mental state concepts engages brain regions involved in mentalizing, automatic speech, and lip movements.

concepts like mental state concepts lack a physical referent, which can be directly perceived. Classical theories therefore claim that abstract concepts require amodal representations detached from experiential brain systems. However, grounded cognition approaches suggest an involvement of modal experiential brain regions in the processing of abstract concepts. In the present functional magnetic resonance imaging study, we investigated the relation of the processing of abstract mental state concepts to modal experiential brain systems in a fine-grained fashion. Participants performed lexical decisions on abstract mental state as well as on verbal association concepts as control category. Experiential brain systems related to the processing of mental states, generating verbal associations, automatic speech as well as hand and lip movements were determined by corresponding localizer tasks. Processing of abstract mental state concepts neuroanatomically overlapped with activity patterns associated with processing of mental states, generating verbal associations, automatic speech and lip movements. Hence, mental state concepts activate the mentalizing brain network, complemented by perceptual-motor brain regions involved in simulation of visual or action features associated with social interactions, linguistic brain regions as well as face-motor brain regions recruited for articulation. The present results provide compelling evidence for the rich grounding of abstract mental state concepts in experiential brain systems related to mentalizing, verbal communication and mouth action.

Emerging Enterovirus A71 Subgenogroup B5 Causing Severe Hand, Foot, and Mouth Disease, Vietnam, 2023.

We report on a 2023 outbreak of severe hand, foot, and mouth disease in southern Vietnam caused by an emerging lineage of enterovirus A71 subgenogroup B5. Affected children were significantly older than those reported during previous outbreaks. The virus should be closely monitored to assess its potential for global dispersal.

Foot-and-Mouth Disease Virus Induces Porcine Gasdermin E-Mediated Pyroptosis through the Protease Activity of 3Cpro.

Foot-and-mouth disease (FMD) is an acute, highly contagious disease of cloven-hoofed animals caused by FMD virus (FMDV). Currently, the molecular pathogenesis of FMDV infection remains poorly understood. Here, we demonstrated that FMDV infection induced gasdermin E (GSDME)-mediated pyroptosis independent of caspase-3 activity. Further studies showed that FMDV 3Cpro cleaved porcine GSDME (pGSDME) at the Q271-G272 junction adjacent to the cleavage site (D268-A269) of porcine caspase-3 (pCASP3). The inhibition of enzyme activity of 3Cpro failed to cleave pGSDME and induce pyroptosis. Furthermore, overexpression of pCASP3 or 3Cpro-mediated cleavage fragment pGSDME-NT was sufficient to induce pyroptosis. Moreover, the knockdown of GSDME attenuated the pyroptosis caused by FMDV infection. Our study reveals a novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the pathogenesis of FMDV and the design of antiviral drugs. IMPORTANCE Although FMDV is an important virulent infectious disease virus, few reports have addressed its relationship with pyroptosis or pyroptosis factors, and most studies focus on the immune escape mechanism of FMDV. GSDME (DFNA5) was initially identified as being associated with deafness disorders. Accumulating evidence indicates that GSDME is a key executioner for pyroptosis. Here, we first demonstrate that pGSDME is a novel cleavage substrate of FMDV 3Cpro and can induce pyroptosis. Thus, this study reveals a previously unrecognized novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the design of anti-FMDV therapies and the mechanisms of pyroptosis induced by other picornavirus infections.

Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.

Study on the epidemiological characteristics of enterovirus among pediatric patients in Hangzhou, China: A comparison between the pre-COVID-19, COVID-19 pandemic, and post-COVID-19 periods.

Nonpharmaceutical interventions (NPIs) for coronavirus disease 2019 (COVID-19) not only reduce the prevalence of this disease among children but also influence the transmission of other viruses. This retrospective study investigated the impact of NPIs on human enterovirus (HEV) infection in children diagnosed with hand, foot, and mouth disease (HFMD) or herpangina (HA) in Hangzhou, China. We collected and analyzed the laboratory results and clinical data of children diagnosed with HFMD or HA during the following periods: pre-COVID-19 (January 2019 to December 2019), the COVID-19 pandemic (January 2020 to December 2022), and post-COVID-19 (January to December 2023). A total of 41 742 specimens that met the inclusion criteria were obtained, of which 1998 (4.79%) tested positive for enterovirus. In comparison to those in the pre-COVID-19 period, which had 695 (5.63%) HEV-positive specimens, the numbers dramatically decreased to 69 (1.19%), 398 (5.12%), and 112 (1.58%) in 2020, 2021, and 2022, respectively, but significantly increased to 724 (8.27%) in 2023. Seasonal peaks of infections occurred in May, June, July, and August each year, with the total detection rate ranging from 2019 to 2023 being 9.41% in May, 22.47% in June, 28.23% in July, and 12.16% in August, respectively. The difference in the detection rates of HEV infection between males and females was statistically significant (p < 0.005), with 5.11% (1221/23 898) of males and 4.35% (777/17 844) of females testing positive, resulting in a male-to-female positive ratio of 1.57:1. Among the age groups, 11.25% (378/3360) of the children aged 3-5 years had the highest detection rate, which steadily decreased with increasing or decreasing age. The detection of HEV indicated that >95% of the viruses were other types than the previously commonly reported enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16). In conclusion, NPIs for COVID-19 may be effective at reducing the transmission of HEV. However, with the relaxation of NPIs, the detection rate of HEVs increased slowly to a certain extent. Active awareness and surveillance of the epidemiological characteristics of HEV are essential for preventing, controlling, and managing the development of HFMD and HA, as well as contributing to the development of a multivalent HFMD vaccine.

A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022.

Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.

Single case of lymphogranuloma venereum on the tongue.

Traditionally, lymphogranuloma venereum (LGV) has been associated with disease of the genital area. However, atypical presentations and proctitis are increasingly observed. We report a case of LGV affecting the dorsum of the tongue, which presented as a very painful ulcer. The response to doxycycline (100 mg two times per day for 21 days) was satisfactory. This case may represent a paradigm shift in the differential diagnosis of lingual ulcers.

Can interventional radiotherapy (brachytherapy) be an alternative to surgery in early-stage oral cavity cancer? A systematic review.

PURPOSE: Brachytherapy (BT), also known as interventional radiotherapy (IRT), has proven its utility in the treatment of localized tumors. The aim of this review was to examine the efficacy of modern BT in early-stage oral cavity cancer (OCC) in terms of local control (LC), overall survival (OS), disease-free survival (DFS), cancer-specific survival (CSS), and safety. METHODS: The SPIDER framework was used, with sample (S), phenomena of interest (PI), design (D), evaluation (E), and research type (R) corresponding to early-stage oral cavity cancer (S); BT (PI); named types of qualitative data collection and analysis (D); LC, OS, DFS, CSS, and toxicity (E); qualitative method (R). Systematic research using PubMed and Scopus was performed to identify full articles evaluating the efficacy of BT in patients with early-stage OCC. The studies were identified using medical subject headings (MeSH). We also performed a PubMed search with the keywords "brachytherapy oral cavity cancer, surgery." The search was restricted to the English language. The timeframe 2002-2022 as year of publication was considered. We analyzed clinical studies of patients with OCC treated with BT alone only as full text; conference papers, surveys, letters, editorials, book chapters, and reviews were excluded. RESULTS: The literature search resulted in 517 articles. After the selection process, 7 studies fulfilled the inclusion criteria and were included in this review, totaling 456 patients with early-stage node-negative OCC who were treated with BT alone (304 patients). Five-year LC, DFS, and OS for the BT group were 60-100%, 82-91%, and 50-84%, respectively. CONCLUSION: In conclusion, our review suggests that BT is effective in the treatment of early-stage OCC, particularly for T1N0 of the lip, mobile tongue, and buccal mucosa cancers, with good functional and toxicity profiles.

Effects of educational interventions based on the theory of planned behavior on oral cancer-related knowledge and tobacco smoking in adults: a cluster randomized controlled trial.

BACKGROUND: The theory of planned behavior (TPB) is an effective model for facilitating behavioral change. The aim of the present study was to evaluate the impact of TPB-based educational interventions on oral cancer-related knowledge and tobacco smoking behavior in an Iranian adult population in 2022. METHODS: In this randomized controlled trial, a total of 400 healthy individuals were enrolled. The study was implemented in 20 urban health centers in the south of Tehran, Iran. The health centers were randomly allocated into two intervention groups. In group PowerPoint (PP), the participants received education through a 20-minute PowerPoint presentation complemented by a pamphlet. Group WhatsApp (WA) was educated via WhatsApp messages and images. Data was collected using a structured questionnaire at baseline, and at one- and three-month follow-ups. The outcomes were evaluated in terms of knowledge, tobacco smoking behavior, and the related model constructs i.e. intention, attitude, subjective norm, and perceived behavioral control. Generalized estimating equations (GEE) regression models were applied to assess the effect of interventions on repeated measurements of the outcomes. All analyses were conducted using STATA Software Version 17. RESULTS: Out of all the participants, 249 (62%) were women. The mean and standard deviation (SD) of age were 39.67 and 13.80 years. Overall, group PP had a significantly higher score of knowledge compared to group WA (ß = 0.43, p = 0.005). No significant differences were found between the groups with regard to tobacco smoking and the related TPB constructs, except for attitude with a higher score in group PP compared to group WA (ß = 0.50, p = 0.004). At the three-month follow-up, both interventions had significant effects on increasing knowledge (ß = 4.41), decreasing tobacco smoking (OR = 0.54), and increasing intention (ß = 1.11), attitude (ß = 1.22), subjective norm (ß = 1.37), and perceived behavioral control (ß = 1.08) (P < 0.001). CONCLUSIONS: Both interventions were effective in improving knowledge, tobacco smoking, and the TPB constructs after three months. Therefore, the application of both methods could be considered in the design and implementation of oral cancer prevention programs. TRIAL REGISTRATION: The trial protocol was registered in the Iranian Registry of Clinical Trials (IRCT) on 04/03/2022 (registration number: IRCT20220221054086N1).

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis.

BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

The Novel OMERACT Ultrasound Scoring System for Salivary Gland Changes in Patients With Sjögren Syndrome Is Associated With MRI and Salivary Flow Rates.

OBJECTIVE: To assess the construct validity of the novel Outcome Measures in Rheumatology (OMERACT) ultrasound (US) semiquantitative scoring system for morphological lesions in major salivary glands by comparing it with magnetic resonance imaging (MRI) and unstimulated whole salivary flow rates (U-WSFRs) in patients with primary Sjögren syndrome (pSS). METHODS: Nine sonographers applied the OMERACT 0-3 grayscale scoring system for parotid (PGs) and submandibular glands (SMGs) in 11 patients with pSS who also had MRIs performed. These were evaluated by 2 radiologists using a semiquantitative 0-3 scoring system for morphological lesions. The agreement between US and MRI and the association between U-WSFRs and imaging structural lesions was determined. A score ≥ 2 for both US and MRI was defined as gland pathology. RESULTS: The prevalence of US morphological lesions in 11 patients with a score ≥ 2 was 58% for PGs and 76% for SMGs, and 46% and 41% for PGs and SMGs, respectively, for MRI. The agreement between OMERACT US scores and MRI scores was 73-91% (median 82%) in the right PG and 73-91% (median 91%) in the left PG, 55-91% (median 55%) in the right SMG and 55-82% (median 55%) in the left SMG. When relations between the presence of hyposalivation and an US score ≥ 2 were examined, agreement was 91-100% (median 83%) in both PGs and 55-91% (median 67%) in both SMGs. CONCLUSION: There is moderate to strong agreement between the OMERACT US and MRI scores for major salivary glands in patients with pSS. Similar agreement ratios were observed between the higher OMERACT US scores and presence of hyposalivation.

Genomic epidemiology of CVA10 in Guangdong, China, 2013-2021.

Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness primarily affecting children globally. A significant epidemiological transition has been noted in mainland China, characterized by a substantial increase in HFMD cases caused by non-Enterovirus A71 (EV-A71) and non-Coxsackievirus A16 (CVA16) enteroviruses (EVs). Our study conducts a retrospective examination of 36,461 EV-positive specimens collected from Guangdong, China, from 2013 to 2021. Epidemiological trends suggest that, following 2013, Coxsackievirus A6 (CVA6) and Coxsackievirus A10 (CVA10) have emerged as the primary etiological agents for HFMD. In stark contrast, the incidence of EV-A71 has sharply declined, nearing extinction after 2018. Notably, cases of CVA10 infection were considerably younger, with a median age of 1.8 years, compared to 2.3 years for those with EV-A71 infections, possibly indicating accumulated EV-A71-specific herd immunity among young children. Through extensive genomic sequencing and analysis, we identified the N136D mutation in the 2 A protein, contributing to a predominant subcluster within genogroup C of CVA10 circulating in Guangdong since 2017. Additionally, a high frequency of recombination events was observed in genogroup F of CVA10, suggesting that the prevalence of this lineage might be underrecognized. The dynamic landscape of EV genotypes, along with their potential to cause outbreaks, underscores the need to broaden surveillance efforts to include a more diverse spectrum of EV genotypes. Moreover, given the shifting dominance of EV genotypes, it may be prudent to re-evaluate and optimize existing vaccination strategies, which are currently focused primarily target EV-A71.

Correlations of PSGL-1 VNTR polymorphism with the susceptibility to severe HFMD associated with EV-71 and the immune status after infection.

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71-induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71-associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71-induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.