Prebiotic inulin ameliorates SARS-CoV-2 infection in hamsters by modulating the gut microbiome.

Current treatment options for COVID-19 are limited, with many antivirals and immunomodulators restricted to the most severe cases and preventative care limited to vaccination. As the SARS-CoV-2 virus and its increasing variants threaten to become a permanent fixture of our lives, this new reality necessitates the development of cost-effective and accessible treatment options for COVID-19. Studies have shown that there are correlations between the gut microbiome and severity of COVID-19, especially with regards to production of physiologically beneficial short-chain fatty acids (SCFAs) by gut microbes. In this study, we used a Syrian hamster model to study how dietary consumption of the prebiotic inulin affected morbidity and mortality resulting from SARS-CoV-2 infection. After two weeks of observation, we discovered that inulin supplementation attenuated morbid weight loss and increased survival rate in hamster subjects. An analysis of microbiome community structure showed significant alterations in 15 genera. Notably, there were also small increases in fecal DCA and a significant increase in serum DCA, perhaps highlighting a role for this secondary bile acid in conferring protection against SARS-CoV-2. In light of these results, inulin and other prebiotics are promising targets for future investigation as preventative treatment options for COVID-19.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection.

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.

Inactivation and spike protein denaturation of novel coronavirus variants by CuxO/TiO2 nano-photocatalysts.

In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO2 (4-8 nm) and CuxO (1-2 nm or less). Their extraordinarily small size leads to high dispersity and good optical transparency, besides large active surface area. Those photocatalysts can be applied to white and translucent latex paints. Although Cu2O clusters involved in the paint coating undergo gradual aerobic oxidation in the dark, the oxidized clusters are re-reduced under > 380 nm light. The paint coating inactivated the original and alpha variant of novel coronavirus under irradiation with fluorescent light for 3 h. The photocatalysts greatly suppressed binding ability of the receptor binding domain (RBD) of coronavirus (the original, alpha and delta variants) spike protein to the receptor of human cells. The coating also exhibited antivirus effects on influenza A virus, feline calicivirus, bacteriophage Qß and bacteriophage M13. The photocatalysts would be applied to practical coatings and lower the risk of coronavirus infection via solid surfaces.

Oral Bacteria Combined with an Intranasal Vaccine Protect from Influenza A Virus and SARS-CoV-2 Infection.

The gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here, we found that disruption of nasal bacteria by intranasal application of antibiotics before influenza virus infection enhanced the virus-specific antibody response in a MyD88-dependent manner. Similarly, disruption of nasal bacteria by lysozyme enhanced antibody responses to intranasally administered influenza virus hemagglutinin (HA) vaccine in a MyD88-dependent manner, suggesting that intranasal application of antibiotics or lysozyme could release bacterial pathogen-associated molecular patterns (PAMPs) from disrupted nasal bacteria that act as mucosal adjuvants by activating the MyD88 signaling pathway. Since commensal bacteria in the nasal mucosal surface were significantly lower than those in the oral cavity, intranasal administration of HA vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to an intranasally administered HA vaccine. Finally, we demonstrated that oral bacteria combined with an intranasal vaccine protect from influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our results reveal the role of nasal bacteria in the induction of the virus-specific adaptive immunity and provide clues for developing better intranasal vaccines. IMPORTANCE Intranasal vaccination induces the nasal IgA antibody which is protective against respiratory viruses, such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, understanding how mucosal immune responses are elicited following viral infection is important for developing better vaccines. Here, we focused on the role of nasal commensal bacteria in the induction of immune responses following influenza virus infection. To deplete nasal bacteria, we intranasally administered antibiotics to mice before influenza virus infection and found that antibiotic-induced disruption of nasal bacteria could release bacterial components which stimulate the virus-specific antibody responses. Since commensal bacteria in nasal mucosa were significantly lower than those in the oral cavity, intranasal administration of split virus vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to the intranasally administered vaccine. Therefore, both integrity and amounts of nasal bacteria may be critical for an effective intranasal vaccine.

Skewed endosomal RNA responses from TLR7 to TLR3 in RNase T2-deficient macrophages.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.

The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6-2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

Influenza Virus-Induced Oxidized DNA Activates Inflammasomes.

Influenza virus M2 and PB1-F2 proteins have been proposed to activate the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome in macrophages by altering intracellular ionic balance or mitochondrial reactive oxygen species (ROS) production. However, the precise mechanism by which these viral proteins trigger the NLRP3 inflammasome activation remains unclear. Here we show that influenza virus stimulates oxidized DNA release from macrophages. Ion channel activity of the M2 protein or mitochondrial localization of the PB1-F2 protein was required for oxidized DNA release. The oxidized DNA enhanced influenza virus-induced IL-1ß secretion, whereas inhibition of mitochondrial ROS production by antioxidant Mito-TEMPO decreased the virus-induced IL-1ß secretion. In addition, we show that influenza virus stimulates IL-1ß secretion from macrophages in an AIM2-dependent manner. These results provide a missing link between influenza viral proteins and the NLRP3 inflammasome activation and reveal the importance of influenza virus-induced oxidized DNA in inflammasomes activation.

Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.

Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses.

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome.

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1ß) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1ß secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1ß secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

High ambient temperature dampens adaptive immune responses to influenza A virus infection.

Although climate change may expand the geographical distribution of several vector-borne diseases, the effects of environmental temperature in host defense to viral infection in vivo are unknown. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C impaired adaptive immune responses against infection with viral pathogens, influenza, Zika, and severe fever with thrombocytopenia syndrome phlebovirus. Following influenza virus infection, the high heat-exposed mice failed to stimulate inflammasome-dependent cytokine secretion and respiratory dendritic cell migration to lymph nodes. Although commensal microbiota composition remained intact, the high heat-exposed mice decreased their food intake and increased autophagy in lung tissue. Induction of autophagy in room temperature-exposed mice severely impaired virus-specific CD8 T cells and antibody responses following respiratory influenza virus infection. In addition, we found that administration of glucose or dietary short-chain fatty acids restored influenza virus-specific adaptive immune responses in high heat-exposed mice. These findings uncover an unexpected mechanism by which ambient temperature and nutritional status control virus-specific adaptive immune responses.

Cytidine deaminase enables Toll-like receptor 8 activation by cytidine or its analogs.

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.

Two Conserved Amino Acids within the NSs of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Are Essential for Anti-interferon Activity.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-ß) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-ß responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-ß mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-ß promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1ß (IL-1ß) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-ß) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-ß production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

Herpes Simplex Virus 1 VP22 Inhibits AIM2-Dependent Inflammasome Activation to Enable Efficient Viral Replication.

The AIM2 inflammasome is activated by DNA, leading to caspase-1 activation and release of pro-inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18, which are critical mediators in host innate immune responses against various pathogens. Some viruses employ strategies to counteract inflammasome-mediated induction of pro-inflammatory cytokines, but their in vivo relevance is less well understood. Here we show that the herpes simplex virus 1 (HSV-1) tegument protein VP22 inhibits AIM2-dependent inflammasome activation. VP22 interacts with AIM2 and prevents its oligomerization, an initial step in AIM2 inflammasome activation. A mutant virus lacking VP22 (HSV-1ΔVP22) activates AIM2 and induces IL-1ß and IL-18 secretion, but these responses are lost in the absence of AIM2. Additionally, HSV-1ΔVP22 infection results in diminished viral yields in vivo, but HSV-1ΔVP22 replication is largely restored in AIM2-deficient mice. Collectively, these findings reveal a mechanism of HSV-1 evasion of the host immune response that enables efficient viral replication in vivo.

Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes.

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via ß-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.

TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells.

Plasmacytoid dendritic cells (pDC) sense viral RNA through toll-like receptor 7 (TLR7), form self-adhesive pDC-pDC clusters, and produce type I interferons. This cell adhesion enhances type I interferon production, but little is known about the underlying mechanisms. Here we show that MyD88-dependent TLR7 signaling activates CD11a/CD18 integrin to induce microtubule elongation. TLR7+ lysosomes then become linked with these microtubules through the GTPase Arl8b and its effector SKIP/Plekhm2, resulting in perinuclear to peripheral relocalization of TLR7. The type I interferon signaling molecules TRAF3, IKKα, and mTORC1 are constitutively associated in pDCs. TLR7 localizes to mTORC1 and induces association of TRAF3 with the upstream molecule TRAF6. Finally, type I interferons are secreted in the vicinity of cell-cell contacts between clustered pDCs. These results suggest that TLR7 needs to move to the cell periphery to induce robust type I interferon responses in pDCs.

Induction of lung CD8+ T cell responses by consecutive inoculations of a poly(I:C) influenza vaccine.

The cytotoxic T lymphocyte (CTL) response plays a key role in host recovery from influenza virus infection and in subsequent immunity. Compared to natural infection with influenza virus, however, intranasal vaccination with adjuvant-combined inactivated vaccine elicits only moderate CTL responses. Here we demonstrate that 5 days of consecutive, intranasal vaccination with a combination of inactivated influenza vaccine and poly(I:C) elicits a strong CTL response in the lung. Antigen-captured respiratory DCs did efficiently migrate from the lung to the mediastinal lymph node (mLN) after the 5 day series of inoculations with vaccine and poly(I:C). Importantly, formalin-inactivated whole virus vaccine and poly(I:C) adjuvant have synergic effects on consecutive vaccinations to elicit a strong CTL response in the lung. Although the CTL response was less effective against heterologous influenza virus, we show for the first time that intranasal administration of inactivated influenza virus vaccine and poly(I:C) for 5 consecutive days can elicit high levels of influenza virus-specific CD8+ T cells in the lung.

Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis.

Herpes simplex virus-1 (HSV-1) is the most common cause of sporadic viral encephalitis, which can be lethal or result in severe neurological defects even with antiviral therapy. While HSV-1 causes encephalitis in spite of HSV-1-specific humoral and cellular immunity, the mechanism by which HSV-1 evades the immune system in the central nervous system (CNS) remains unknown. Here we describe a strategy by which HSV-1 avoids immune targeting in the CNS. The HSV-1 UL13 kinase promotes evasion of HSV-1-specific CD8+ T cell accumulation in infection sites by downregulating expression of the CD8+ T cell attractant chemokine CXCL9 in the CNS of infected mice, leading to increased HSV-1 mortality due to encephalitis. Direct injection of CXCL9 into the CNS infection site enhanced HSV-1-specific CD8+ T cell accumulation, leading to marked improvements in the survival of infected mice. This previously uncharacterized strategy for HSV-1 evasion of CD8+ T cell accumulation in the CNS has important implications for understanding the pathogenesis and clinical treatment of HSV-1 encephalitis.

Consecutive inoculations of influenza virus vaccine and poly(I:C) protects mice against homologous and heterologous virus challenge.

Mucosal immunity induced through natural infection by influenza virus has potent cross-protective activity, compared to subcutaneous vaccination-induced systemic immunity. Compared to natural infection with influenza virus, however, a single intranasal vaccination with an inactivated influenza virus vaccine and poly(I:C) is not sufficient to induce primary immune response in naïve animals. The reasons for this moderate effect are not fully understood. Here, we demonstrated that intranasal vaccination with formalin-inactivated influenza virus vaccine and poly(I:C) for five consecutive days elicits high levels of virus-specific nasal IgA and serum IgG responses, while vaccination without poly(I:C) induced little response. Mice immunized with influenza virus vaccine and poly(I:C) for five consecutive days sustained high levels of virus-specific IgA in nasal wash and IgG in serum until at least 6months after vaccination. Furthermore, intranasal vaccination with influenza virus vaccine and poly(I:C) protected mice against homologous and heterologous influenza virus challenge. These results suggest that consecutive inoculations of influenza virus vaccine and poly(I:C) is an alternative method to induce primary immune responses in naïve subjects.