Gasdermin E is required for induction of pyroptosis and severe disease during enterovirus 71 infection.

Pyroptosis is an inflammatory form of programmed cell death that is executed by the gasdermin (GSDM)-N domain of GSDM family proteins, which form pores in the plasma membrane. Although pyroptosis acts as a host defense against invasive pathogen infection, its role in the pathogenesis of enterovirus 71 (EV71) infection is unclear. In the current study, we found that EV71 infection induces cleavage of GSDM E (GSDME) by using western blotting analysis, an essential step in the switch from caspase-3-mediated apoptosis to pyroptosis. We show that this cleavage is independent of the 3C and 2A proteases of EV71. However, caspase-3 activation is essential for this cleavage, as GSDME could not be cleaved in caspase-3-KO cells upon EV71 infection. Further analyses showed that EV71 infection induced pyroptosis in WT cells but not in caspase-3/GSDME double-KO cells. Importantly, GSDME is required to induce severe disease during EV71 infection, as GSDME deficiency in mice was shown to alleviate pathological symptoms. In conclusion, our results reveal that GSDME is important for the pathogenesis of EV71 via mediating initiation of pyroptosis.

Respiratory Syncytial Virus Seasonality, Beijing, China, 2007-2015.

During July 2007-June 2015, we enrolled 4,225 hospitalized children with pneumonia in a study to determine the seasonality of respiratory syncytial virus (RSV) infection in Beijing, China. We defined season as the period during which >10% of total PCRs performed each week were RSV positive. We identified 8 distinctive RSV seasons. On average, the season onset occurred at week 41 (mid-October) and lasted 33 weeks, through week 20 of the next year (mid-May); 97% of all RSV-positive cases occurred during the season. RSV seasons occurred 3-5 weeks earlier and lasted ≈6 weeks longer in RSV subgroup A-dominant years than in RSV subgroup B-dominant years. Our analysis indicates that monitoring such RSV subgroup shifts might provide better estimates for the onset of RSV transmission. PCR-based tests could be a flexible or complementary way of determining RSV seasonality in locations where RSV surveillance is less well-established, such as local hospitals throughout China.

3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7.

UNLABELLED: Human enterovirus 68 (EV-D68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, clusters of EV-D68 infections have occurred worldwide. A recent outbreak in the United States is the largest one associated with severe respiratory illness and neurological complication. Although clinical symptoms are recognized, the virus remains poorly understood. Here we report that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7), an immune factor with a pivotal role in viral pathogenesis. This process depends on 3C(pro), an EV-D68-encoded protease, to mediate IRF7 cleavage. When expressed in host cells, 3C(pro) targets Q167 and Q189 within the constitutive activation domain, resulting in cleavage of IRF7. Accordingly, wild-type IRF7 is fully active. However, IRF7 cleavage abrogated its capacity to activate type I interferon expression and limit replication of EV-D68. Notably, IRF7 cleavage strictly requires the protease activity of 3C(pro). Together, these results suggest that a dynamic interplay between 3C(pro) and IRF7 may determine the outcome of EV-D68 infection. IMPORTANCE: EV-D68 is a globally emerging pathogen, but the molecular basis of EV-D68 pathogenesis is unclear. Here we report that EV-D68 inhibits innate immune responses by targeting an immune factor, IRF7. This involves the 3C protease encoded by EV-D68, which mediates the cleavage of IRF7. These observations suggest that the 3C(pro)-IRF7 interaction may represent an interface that dictates EV-D68 infection.

Enterovirus D68 and Human Respiratory Infections.

Enterovirus D68 (EV-D68) is a member of the species Enterovirus D in the genus Enterovirus of the Picornaviridae family. EV-D68 was first isolated in the United States in 1962 and is primarily an agent of respiratory disease. Infections with EV-D68 have been rarely reported until recently, when reports of EV-D68 associated with respiratory disease increased notably worldwide. An outbreak in 2014 in the United States, for example, involved more than 1,000 cases of severe respiratory disease that occurred across almost all states. Phylogenetic analysis of all EV-D68 sequences indicates that the circulating strains of EV-D68 can be classified into two lineages, lineage 1 and lineage 2. In contrast to the prototype Fermon strain, all circulating strains have deletions in their genomes. Respiratory illness associated with EV-D68 infection ranges from mild illness that just needs outpatient service to severe illness requiring intensive care and mechanical ventilation. To date, there are no specific medicines and vaccines to treat or prevent EV-D68 infection. This review provides a detailed overview about our current understanding of EV-D68-related virology, epidemiology and clinical syndromes, pathogenesis, and laboratory diagnostics.

Enterovirus 68 3C protease cleaves TRIF to attenuate antiviral responses mediated by Toll-like receptor 3.

UNLABELLED: Human enterovirus 68 (EV68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, there have been increasingly documented outbreaks of respiratory disease associated with EV68. As a globally emerging pathogen, EV68 infects both adults and children. However, the molecular basis of EV68 pathogenesis is unknown. Here we report that EV68 inhibits Toll-like receptor 3 (TLR3)-mediated innate immune responses by targeting the TIR domain-containing adaptor inducing beta interferon (TRIF). In infected HeLa cells, EV68 inhibits poly(I·C)-induced interferon regulatory factor 3 (IRF3) activation and beta interferon (IFN-ß) expression. Further investigations revealed that TRIF, a critical adaptor downstream of TLR3, is targeted by EV68. When expressed alone, 3C(pro), an EV68-encoded protease, cleaves TRIF. 3C(pro) mediates TRIF cleavage at Q312 and Q653, which are sites in the amino- and carboxyl-terminal domains, respectively. This cleavage relies on 3C(pro)'s cysteine protease activity. Cleavage of TRIF abolishes the capacity of TRIF to activate NF-κB and IFN-ß signaling. These results suggest that control of TRIF by 3C(pro) may be a mechanism by which EV68 subverts host innate immune responses. IMPORTANCE: EV68 is a globally emerging pathogen, but the molecular basis of EV68 pathogenesis is unclear. Here we report that EV68 inhibits TLR3-mediated innate immune responses by targeting TRIF. Further investigations revealed that TRIF is cleaved by 3C(pro). These results suggest that control of TRIF by 3C(pro) may be a mechanism by which EV68 impairs type I IFN production in response to TLR3 activation.

Prevalence and clinical characteristics of human respiratory syncytial virus in Chinese adults with acute respiratory tract infection.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract illnesses worldwide. Although the prevalence and clinical manifestations of the two subtypes, RSV-A and RSV-B, have been studied in some detail in infants and young children, they have not been determined in adults. To evaluate the prevalence of the RSV subtypes and disease severity between RSV-A and RSV-B infections in adults, nasal and throat swabs that were collected from patients ≥15 years old who sought medical care for acute respiratory infections at the Fever Clinic of the Peking Union Medical College Hospital in Beijing, China between May 2005 and April 2010. The samples were tested for RSV infection using PCR and sequencing analysis. RSV was detected in 95 (1%) of the adult patients, of whom 53 (55.8%) were positive for RSV-A and 42 (44.2%) for RSV-B. The incidence of RSV infections increased with age (χ(2) = 37.17, P = 1.66E-07). Demographic data and clinical manifestations of RSV-A were similar to those of RSV-B. Although RSV-A and RSV-B co-circulated during the 2005-2006 and 2008-2009 seasons, RSV-A was predominant in the 2006-2008 seasons, whereas RSV-B was predominant in the 2009-2010 season. Upper respiratory tract infections were diagnosed in most RSV-infected patients (n = 80, 84.2%), and three patients suffered from pulmonary infection. This is the first study to provide data on the prevalence and clinical manifestations of RSV subgroups among Chinese adults with fever and acute illness, over five successive epidemic seasons.

Proteomic and phosphoproteomic analysis of responses to enterovirus A71 infection reveals novel targets for antiviral and viral replication.

Hand, foot, and mouth disease (HFMD) is a common infectious disease in infants and children, especially those under five years of age. EV-A71 is a common pathogen that causes HFMD and the primary pathogen leading to severe or fatal HFMD, which is characterized by neurological complications. However, the underlying mechanisms of EV-A71 pathogenesis remain largely unknown. In this report, we used proteomic and phosphorylated proteomic methods to characterize the proteome and phosphoproteome profiles of EV-A71-infected human neuroblastoma SK-N-SH cells. More than 7744 host proteins and 10069 phosphorylation modification sites were successfully quantified. Among them, 974 proteins and 3648 phosphorylation modification sites were regulated significantly during EV-A71 infection. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that EV-A71 altered cell biological processes, including protein synthesis, RNA splicing and metabolism in SK-N-SH cells. Notably, based on the prediction of upregulated kinases during EV-A71 infection, we identified specific kinase inhibitors approved by the FDA, with ceralasertib, bosutinib, flavin mononucleotide, minocycline, pimasertib and acetylcysteine inhibiting EV-A71 infection. Finally, EV-A71 proteins were found to be phosphorylated during infection, with one site (S184 on 3D polymerase) observed to be crucial for viral replication because a S184A mutation knocked out viral replication. The results improve our understanding of the host response to EV-A71 infection of neuroblastoma cells and provide potential targets for developing anti-EV-A71 strategies.

CD74 Interacts with Proteins of Enterovirus D68 To Inhibit Virus Replication.

Enterovirus D68 (EV-D68) is a member of the species Enterovirus D in the genus Enterovirus of the family Picornaviridae. As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Although the intrinsic restriction factors in the cell provide a frontline defense, the molecular nature of virus-host interactions remains elusive. Here, we provide evidence that the major histocompatibility complex class II chaperone, CD74, inhibits EV-D68 replication in infected cells by interacting with the second hydrophobic region of 2B protein, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. 3Cpro cleaves CD74 at Gln-125. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection. IMPORTANCE As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Here, we report that CD74 inhibits viral replication in infected cells by targeting 2B protein of EV-D68, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection.

Statistical Analysis of Common Respiratory Viruses Reveals the Binary of Virus-Virus Interaction.

Respiratory viruses may interfere with each other and affect the epidemic trend of the virus. However, the understanding of the interactions between respiratory viruses at the population level is still very limited. We here conducted a prospective laboratory-based etiological study by enrolling 14,426 patients suffered from acute respiratory infection (ARI) in Beijing, China during 2005 to 2015. All 18 respiratory viruses were simultaneously tested for each nasal and throat swabs collected from enrolled patients using molecular tests. The virus correlations were quantitatively evaluated, and the respiratory viruses could be divided into two panels according to the positive and negative correlations. One included influenza viruses (IFVs) A, B, and respiratory syncytial virus (RSV), while the other included human parainfluenza viruses (HPIVs) 1/3, 2/4, adenovirus (Adv), human metapneumovirus (hMPV), and enterovirus (including rhinovirus, named picoRNA), α and ß human coronaviruses (HCoVs). The viruses were positive-correlated in each panel, while negative-correlated between panels. After adjusting the confounding factors by vector autoregressive model, positive interaction between IFV-A and RSV and negative interaction between IFV-A and picoRNA are still be observed. The asynchronous interference of IFV-A significantly delayed the peak of ß human coronaviruses epidemic. The binary property of the respiratory virus interactions provides new insights into the viral epidemic dynamics in human population, facilitating the development of infectious disease control and prevention strategies. IMPORTANCE Systematic quantitative assessment of the interactions between different respiratory viruses is pivotal for the prevention of infectious diseases and the development of vaccine strategies. Our data showed stable interactions among respiratory viruses at human population level, which are season irrelevant. Respiratory viruses could be divided into two panels according to their positive and negative correlations. One included influenza virus and respiratory syncytial virus, while the other included other common respiratory viruses. It showed negative correlations between the two panels. The asynchronous interference between influenza virus and ß human coronaviruses significantly delayed the peak of ß human coronaviruses epidemic. The binary property of the viruses indicated transient immunity induced by one kind of virus would play role on subsequent infection, which provides important data for the development of epidemic surveillance strategies.

Coxsackievirus A21, enterovirus 68, and acute respiratory tract infection, China.

During August 2006-April 2010, in Beijing, China, 2 rare human enterovirus serotypes, coxsackievirus A21 and enterovirus 68, were detected most frequently in human enterovirus-positive adults with acute respiratory tract infections. Thus, during some years, these 2 viruses cause a substantial proportion of enterovirus-associated adult acute respiratory tract infections.

Viral infections of the lower respiratory tract.

Lower respiratory tract infections (LRTIs) are a global burden to public health and are frequently caused by respiratory viruses. Advances in molecular diagnostic techniques have allowed the identification of previously undetected viral pathogens and have improved our understanding of respiratory virus infections. Here we review the epidemiological and clinical characteristics of recently identified viruses including human metapneumovirus, human coronaviruses NL63 and HKU1, human rhinovirus C, bocavirus, WU and KI polyomaviruses, and parechovirus. The roles of these viruses in LRTIs in children and adults are discussed.

ID2 inhibits innate antiviral immunity by blocking TBK1- and IKKε-induced activation of IRF3.

The transcription regulator ID2 plays an essential role in the development and differentiation of immune cells. Here, we report that ID2 also negatively regulates antiviral innate immune responses. During viral infection of human epithelial cells, ID2 bound to TANK-binding kinase 1 (TBK1) and to inhibitor of nuclear factor κB kinase ε (IKKε). These interactions inhibited the recruitment and activation of interferon (IFN) regulatory factor 3 (IRF3) by TBK1 or IKKε, leading to a reduction in the expression of IFN-ß1 (IFNB1). IFN-ß induced the nuclear export of ID2 to form a negative feedback loop. Knocking out ID2 in human cells enhanced innate immune responses and suppressed infection by different viruses, including SARS-CoV-2. Mice with a myeloid-specific deficiency of ID2 produced more IFN-α in response to viral infection and were more resistant to viral infection than wild-type mice. Our findings not only establish ID2 as a modulator of IRF3 activation induced by TBK1 and/or IKKε but also introduce a mechanism for cross-talk between innate immunity and cell development and differentiation.

SARS-CoV-2 nsp12 attenuates type I interferon production by inhibiting IRF3 nuclear translocation.

SARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-ß promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

Activation and evasion of type I interferon responses by SARS-CoV-2.

The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-ß promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-ß treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study.

BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown ß-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

Upsurge of Enterovirus D68 and Circulation of the New Subclade D3 and Subclade B3 in Beijing, China, 2016.

We conducted a surveillance among acute respiratory tract infection (ARTI) cases to define the epidemiology, clinical characteristics and genetic variations of enterovirus D68 (EV-D68) in Beijing, China from 2015 to 2017. Nasopharyngeal swabs and sputum were collected from 30 sentinel hospitals in Beijing and subjected to EV and EV-D68 detection by real-time PCR. The VP1 gene region and complete genome sequences of EV-D68 positive cases were analyzed. Of 21816 ARTI cases, 619 (2.84%) were EV positive and 42 cases were EV-D68 positive. The detection rates of EV-D68 were 0 (0/6644) in 2015, 0.53% (40/7522) in 2016 and 0.03% (2/7650) in 2017, respectively. Two peaks of EV-D68 infections occurred in late summer and early-winter. Ten cases (23.81%) with upper respiratory tract infection and 32 cases (76.19%) presented with pneumonia, including 3 cases with severe pneumonia. The phylogenetic analysis suggested 15 subclade D3 strains and 27 subclade B3 strains of EV-D68 were circulated in China from 2016 to 2017. A total of 52 amino acid polymorphisms were identified between subclades D1 and D3. These data suggest an upsurge of EV-D68 occurred in Beijing in 2016, the new subclade D3 emerged in 2016 and co-circulated with subclade B3 between 2016 and 2017.