Search

Coronaviruses (CoVs) can infect a variety of hosts, including humans, livestock and companion animals, and pose a serious threat to human health and the economy. The current COVID-19 pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed millions of people. Unfortunately, effective treatments for CoVs infection are still lacking, suggesting the importance of coronavirus vaccines. Our previous work showed that CoV nonstuctural protein 14 (nsp14) functions as (guanine-N7)-methyltransferase (N7-MTase), which is involved in RNA cap formation. Moreover, we found that N7-MTase is well conserved among different CoVs and is a universal target for developing antivirals against CoVs. Here, we show that N7-MTase of CoVs can be an ideal target for designing live attenuated vaccines. Using murine hepatitis virus strain A59 (MHV-A59), a representative and well-studied model of coronaviruses, we constructed N7-MTase-deficient recombinant MHV D330A and Y414A. These two mutants are highly attenuated in mice and exhibit similar replication efficiency to the wild-type (WT) virus in the cell culture. Furthermore, a single dose immunization of D330A or Y414A can induce long-term humoral immune responses and robust CD4+ and CD8+ T cell responses, which can provide full protection against the challenge of a lethal-dose of MHV-A59. Collectively, this study provides an ideal strategy to design live attenuated vaccines for coronavirus by abolishing viral RNA N7-MTase activity. This approach may apply to other RNA viruses that encode their own conservative viral N7-methyltransferase.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions., This work is supported in part by grants from the National Institutes of Health USA (NIH; P01 AI060699 (S.P. and P.B.M.) and R01 AI129269 (S.P.)) and BIOAGE Labs (S.P.). The Pathology Core is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH grant P30 DK-54759) and the Cystic Fibrosis Foundation. P.B.M. is supported by the Roy J. Carver Charitable Trust. L.-Y.R.W. is supported by Mechanism of Parasitism Training Grant (T32 AI007511). We thank M. Gelb (University of Washington) for Pla2g2d−/− mice.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice. We now demonstrate that mRNA expression of PLA2G2D and PTGDR, a PGD2 receptor, and production of PGD2 increase in human PBMC-derived dendritic cells with aging and after SARS-CoV-2 infection. Using our virulent mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR, or PLA2G2D are protected from severe disease. Further, treatment with a PTGDR antagonist, Asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions. This work is supported in part by grants from the National Institutes of Health USA (NIH) (P01 AI060699 (SP, PBM) and RO1 AI129269 (SP) and BIOAGE Labs (SP). The Pathology Core, is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH Grant P30 DK-54759), and the Cystic Fibrosis Foundation. PBM is supported by the Roy J. Carver Charitable Trust.

Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Highlights • The research of this paper showed that some suspected SFTS cases are confused with HV infection due to similar symptoms. • No case of simultaneous infection with two or more pathogens was found. • The distribution of SFTSV has a marked regional aggregation in Hubei Province. • Combining qPCR and ELISA can increase sensitivity markedly for diagnosis of SFTSV infection., To evaluate the aetiological agents and epidemiologic features of severe fever with thrombocytopenia syndrome (SFTS) in Hubei province, China, sera from patients were collected from January to December 2011. All cases occurred from April to December, and the epidemic peaked from May to August. The ages of patients ranged from 10 to 86 years (median = 55 years), and the incidence of SFTS increased with age. The female:male ratio of cases was 1.008:1, and 54.6% (77/141) and 1.4% (2/141) of the cases were confirmed by qPCR to be SFTSV and Hantavirus (HV) infection, respectively. No case of simultaneous infection with two or more pathogens was found. The research in this paper showed that some suspected SFTS cases are confused with HV infection due to similar symptoms. The analysis showed that the distribution of SFTSV has a marked regional aggregation in Hubei province.

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-β production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

The 5′ cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′- O -methyltransferases (2′- O -MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5′ end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2′- O -MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2′- O -MTase activity (E. Decroly et al., J Virol 82:8071–8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08 ; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863 ; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059 ; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294 ) . In this study, we demonstrate that stimulation of nsp16 2′- O -MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2′- O -MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2′- O -MTase activity can be targeted in vitro and in vivo . IMPORTANCE Coronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2′- O -MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2′- O -MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2′- O -MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2′- O -MTase of coronaviruses.

To gain insights into the pathogenesis of influenza A virus (IAV) infections, this study focused on characterizing the inflammatory network and identifying key proteins by combining high-throughput data and computational techniques. We constructed the cell-specific normal and inflammatory networks for H5N1 and H1N1 infections through integrating high-throughput data. We demonstrated that better discrimination between normal and inflammatory networks by network entropy than by other topological metrics. Moreover, we identified different dynamical interactions among TLR2, IL-1β, IL10 and NFκB between normal and inflammatory networks using optimization algorithm. In particular, good robustness and multistability of inflammatory sub-networks were discovered. Furthermore, we identified a complex, TNFSF10/HDAC4/HDAC5, which may play important roles in controlling inflammation, and demonstrated that changes in network entropy of this complex negatively correlated to those of three proteins: TNFα, NFκB and COX-2. These findings provide significant hypotheses for further exploring the molecular mechanisms of infectious diseases and developing control strategies.

As an adaptor molecule in the retinoic acid-inducible gene-I (RIG-I) signaling pathway, the virus-induced signaling adaptor (VISA) molecule activates NF-κB and IRF3 and thereby leads to the production of type I interferons (IFNs). To explore the potential of VISA as a genetic adjuvant for DNA vaccines, a eukaryotic expression plasmid, pVISA, was generated by cloning the VISA gene into the pVAX1vector. For comparison, the pTRIF plasmid was similarly constructed, encoding the known genetic adjuvant TRIF (TIR-domain-containing adapter-inducing interferon-β), an adapter in the Toll-like receptor (TLR) signaling pathway. Mice were immunized with the chimeric DNA vaccine pHA/NP147–155, which encodes the HA (hemagglutinin) fused with NP (nucleoprotein) CTL epitope (NP147–155) of H5N1 influenza virus, either alone or in combination with pVISA or pTRIF. Antigen-specific immune responses were examined in immunized mice. Our results demonstrate that co-immunization of the pHA/NP147–155 plasmid with the VISA adjuvant augmented DNA-raised cellular immune responses and provided protection against H5N1 influenza virus challenge in mice. In addition, our data suggest that VISA acts as a stronger adjuvant for DNA immunization than TRIF. We conclude that co-inoculation with a vector expressing the adaptor molecule VISA enhanced the protective immunity against H5N1 infection induced by pHA/NP147–155 and that VISA could be developed as a novel genetic adjuvant for DNA vaccines.

Host cells sense double-stranded RNA (dsRNA) produced during viral replication and initiate type I interferon (IFN-alpha/beta) production, leading to subsequent antiviral responses. Many viruses, including classical swine fever virus (CSFV), have developed strategies for counteracting the IFN-alpha/beta response. In this study, we explored the role of the CSFV E(rns) glycoprotein in the inhibition of IFN-beta production induced by dsRNA [poly(IC)]. Our results demonstrated that CSFV E(rns) could bind to exogenous dsRNA and inhibit dsRNA-induced IFN-beta production but failed to inhibit TRIF-triggered IFN-beta production. Our data suggest that the inhibition of IFN-beta induction occurred at the initial step of the TLR3 signaling pathway. We also showed that deglycosylation of E(rns) rendered it unable to bind to dsRNA, and thus unable to inhibit dsRNA-induced IFN-beta production. Taken together, these results indicated that N-glycan of CSFV E(rns) is essential for E(rns) blocking of IFN-beta induction.

Virus-like particles (VLPs), which produce from the baculovirus insect cell expression system, constitute potential vaccines for prevention of the epidemic diseases. The expression efficiency of the baculovirus insect cell system used for VLPs production is closely related with the dynamics of infection. In a large scale production process for VLPs with a low multiplicity of infection (MOI), how do we quantitatively determine the relation of the highest virus concentration and culture conditions? When is the optimal time of harvest (TOH)? To address these issues, several new mathematical models that describe the dynamic process of cell infection with baculovirus at low MOI have been developed and numerical results are given. The results indicate a quantitative relationship among the uninfected cells concentration, the infected cells concentration and virus concentration, and also show that the harvest time is dependent on the MOI and the attachment coefficient. These results provide useful instruction for the industrial scale production of VLPs vaccines.

DNA vaccination is an effective means of eliciting both humoral and cellular immune responses. The hemagglutinin (HA) surface protein of influenza A virus is a major target of protective antibody responses induced by virus infection or by vaccination and is widely considered to be the antigen of choice for an influenza vaccine. Cytotoxic T lymphocyte (CTL) responses directed against the conserved nucleoprotein (NP) are thought to play an important role in clearing virus and promoting survival and recovery from influenza. In this study, we developed a novel DNA vaccine approach using a chimeric plasmid consisting of the HA of H5N1 influenza virus in which an MHC class I-restricted NP-specific CTL epitope (NP147-155) was inserted. Immunogenicity and antiviral efficacy of this vaccine was assessed in mouse models. A similar level of HA expression was achieved in 293T cells transfected with pHA/NP(147-155) compared to that with pHA. Besides eliciting the specific anti-HA antibody responses, vaccination using pHA/NP(147-155) in mice induced NP epitope-specific CD8(+) T cell responses, which are generally not inducible by vaccination with pHA alone. After H5N1 influenza virus challenge, BALB/c mice vaccinated with pHA/NP(147-155) exhibited reduced inflammation severity and lung viral titers compared to those vaccinated with pHA. Our work may contribute to improvement of HA-based influenza DNA vaccines.

The investigation of the dynamics and regulation of virus-triggered innate immune signaling pathways at a system level will enable comprehensive analysis of the complex interactions that maintain the delicate balance between resistance to infection and viral disease. In this study, we developed a delayed mathematical model to describe the virus-induced interferon (IFN) signaling process by considering several key players in the innate immune response. Using dynamic analysis and numerical simulation, we evaluated the following predictions regarding the antiviral responses: (1) When the replication ratio of virus is less than 1, the infectious virus will be eliminated by the immune system’s defenses regardless of how the time delays are changed. (2) The IFN positive feedback regulation enhances the stability of the innate immune response and causes the immune system to present the bistability phenomenon. (3) The appropriate duration of viral replication and IFN feedback processes stabilizes the innate immune response. The predictions from the model were confirmed by monitoring the virus titer and IFN expression in infected cells. The results suggest that the balance between viral replication and IFN-induced feedback regulation coordinates the dynamical behavior of virus-triggered signaling and antiviral responses. This work will help clarify the mechanisms of the virus-induced innate immune response at a system level and provide instruction for further biological experiments.