Polyphenol rich sugarcane extract (PRSE) has potential antiviral activity against influenza A virus in vitro.

Influenza A virus (IAV) is one of the major global public health concerns but the emerging resistance of IAV to currently available antivirals requires the need to identify potential alternatives. Polyphenol rich sugarcane extract (PRSE) is an extract prepared from the sugarcane plant Saccharum Officinarum. Herein we aimed to determine if PRSE had antiviral activity against IAV. We showed that treatment of IAV-infected cells with PRSE results in a dose-dependent inhibition of virus infection at concentrations that were non-cytotoxic. PRSE treatment limited the early stages of infection, reducing viral genome replication, mRNA transcription and viral protein expression. PRSE did not affect the ability of IAV to bind sialic acid or change the morphology of viral particles. Additionally, PRSE treatment attenuated the replication of multiple IAV strains of the H3N2 and H1N1 subtype. In conclusion, we show that PRSE displays antiviral activity against a broad range of IAV strains, in vitro.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Macrophage ACE2 is necessary for SARS-CoV-2 replication and subsequent cytokine responses that restrict continued virion release.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity.

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.

Cutting Edge: High-Dose Live Attenuated Influenza Vaccines Elicit Pulmonary Tissue-Resident Memory CD8+ T Cells in the Face of Pre-Existing Humoral Immunity.

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.

UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription.

More than half the world's population is infected with human cytomegalovirus (HCMV), causing congenital birth defects and impacting the immuno-compromised. Many of the >170 HCMV genes remain uncharacterized, and this gap in knowledge limits the development of novel antivirals. In this study, we investigated the essential viral protein UL49 and found it displayed leaky late expression kinetics, and localized to nuclear replication compartments. Cells infected with mutant UL49 virus were unable to produce infectious virions and phenocopied other beta-gamma viral pre-initiation complex (vPIC) subunit (UL79, UL87, UL91, UL92, and UL95) mutant infections. RNA-seq analysis of vPIC mutant infections revealed a consistent diminution of genes encoding capsid subunits, including TRX2/UL85 and MCP/UL86, envelope glycoproteins gM, gL and gO, and egress-associated tegument proteins UL99 and UL103. Therefore, as a member of the vPIC, UL49 serves as a fundamental HCMV effector that governs viral gene transcription required to complete the replication cycle.

IFITM Proteins That Restrict the Early Stages of Respiratory Virus Infection Do Not Influence Late-Stage Replication.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Downregulation of MHC Class I Expression by Influenza A and B Viruses.

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.

Mouse Norovirus Infection Arrests Host Cell Translation Uncoupled from the Stress Granule-PKR-eIF2α Axis.

The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study, we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in the suppression of host translation, and yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signaling cytokines such as beta interferon, interleukin-6, and tumor necrosis factor alpha. Our subsequent analyses revealed that the phosphorylation of eIF2α was mediated via protein kinase R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2α, and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection and that MNV can restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears that MNV utilizes G3BP1 to enhance replication but equally to prevent SG formation, suggesting an anti-MNV property of SGs. Overall, this study highlights MNV manipulation of SGs, PKR, and translational control to regulate cytokine translation and to promote viral replication.IMPORTANCE Viruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate, and cause disease. In retaliation, cells possess various defense mechanisms to detect, destroy, and clear infecting viruses, as well as signal to neighboring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and proinflammatory cytokines. However, virus replication and protein translation still ensue. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SGs), by recruiting the key host protein G3BP1 to the MNV replication complex, a recruitment that is crucial to establishing and maintaining virus replication. Thus, MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.

The Microtubule-Associated Innate Immune Sensor GEF-H1 Does Not Influence Mouse Norovirus Replication in Murine Macrophages.

Norovirus is an acute infection of the gastrointestinal tract causing rapid induction of vomiting and diarrhoea. The infection is sensed and controlled by the innate immune system, particularly by the RNA helicase MDA-5 and type I and III interferons (IFNs). We have observed that intracellular replication of murine norovirus (MNV) occurs in membranous clusters proximal to the microtubule organising centre, a localisation dependent on intact microtubules. Recently, it was shown that the host protein guanine nucleotide exchange factor-H1 (GEF-H1) is a microtubule-associated innate immune sensor that activates interferon Regulatory Factor 3 to induce the production of type I IFNs. Thus, we interrogated the potential role of GEF-H1 in controlling MNV infections. We observed that GEF-H1 was recruited to the MNV replication complex; however RNAi-mediated suppression of GEF-H1 did not outwardly affect replication. We furthered our studies to investigate the impact of GEF-H1 on MNV innate detection and observed that GEF-H1 did not contribute to type I IFN induction during MNV infection or influenza virus infection but did result in a small reduction of interferonâ»ß (IFNß) during West Nile virus infection. Intriguingly, we discovered an interaction of GEF-H1 with the viral MNV non-structural protein 3 (NS3), an interaction that altered the location of GEF-H1 within the cell and prevented the formation of GEF-H1-induced microtubule fibres. Thus, our results indicate that GEF-H1 does not contribute significantly to the innate immune sensing of MNV, although its function may be modulated via interaction with the viral NS3 protein.

Mouse Norovirus Infection Reduces the Surface Expression of Major Histocompatibility Complex Class I Proteins and Inhibits CD8+ T Cell Recognition and Activation.

Human noroviruses are highly infectious single-stranded RNA (ssRNA) viruses and the major cause of nonbacterial gastroenteritis worldwide. With the discovery of murine norovirus (MNV) and the introduction of an effective model for norovirus infection and replication, knowledge about infection mechanisms and their impact on the host immune response has progressed. A major player in the immune response against viral infections is the group of major histocompatibility complex (MHC) class I proteins, which present viral antigen to immune cells. We have observed that MNV interferes with the antigen presentation pathway in infected cells by reducing the surface expression of MHC class I proteins. We have shown that MNV-infected dendritic cells or macrophages have lower levels of surface expression of MHC class I proteins than uninfected and bystander cells. Transcriptional analysis revealed that this defect is not due to a decreased amount of mRNA but is reflected at the protein level. We have determined that this defect is mediated via the MNV NS3 protein. Significantly, treatment of MNV-infected cells with the endocytic recycling inhibitor dynasore completely restored the surface expression of MHC class I proteins, whereas treatment with the proteasome inhibitor MG132 partly restored such expression. These observations indicate a role for endocytic recycling and proteasome-mediated degradation of these proteins. Importantly, we show that due to the reduced surface expression of MHC class I proteins, antigen presentation is inhibited, resulting in the inability of CD8+ T cells to become activated in the presence of MNV-infected cells.IMPORTANCE Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis worldwide and impose a great burden on patients and health systems every year. So far, no antiviral treatment or vaccine is available. We show that MNV evades the host immune response by reducing the amount of MHC class I proteins displayed on the cell surface. This reduction leads to a decrease in viral antigen presentation and interferes with the CD8+ T cell response. CD8+ T cells respond to foreign antigen by activating cytotoxic pathways and inducing immune memory to the infection. By evading this immune response, MNV is able to replicate efficiently in the host, and the ability of cells to respond to consecutive infections is impaired. These findings have a major impact on our understanding of the ways in which noroviruses interact with the host immune response and manipulate immune memory.

Norovirus Infection: Replication, Manipulation of Host, and Interaction with the Host Immune Response.

Noroviruses (NoVs) belong to the Caliciviridae family of viruses and are responsible for causing the majority of gastroenteritis outbreaks worldwide. In the past decade, research on NoV biology has intensified because of the discovery of murine NoV and subsequently the first cell culture system and small animal model for NoV replication and pathogenesis. In this review, we discuss the current literature on NoV biology, focusing particularly on NoV replication and the interaction between NoV and the host immune response. Understanding the NoV replication cycle and its interaction with cellular processes and innate immune immunity will help develop molecular targets to control human NoV infection and prevent outbreaks. In addition to the innate immune response, we have documented the current efforts to develop NoV vaccines to control outbreaks.

Increased survival in the red flour beetle after oral priming with bacteria-conditioned media.

Immune priming is defined as enhanced protection upon secondary exposure to a pathogen. Such enhanced resistance after prior exposure has been demonstrated for a number of insect species including the red flour beetle, Tribolium castaneum. In testing this phenomenon, the majority of studies have focused on introducing the pathogen into the insect's hemocoel via septic wounding through the cuticle. Although such septic injury can occur in nature, many pathogens enter their hosts via the oral route, i.e. by ingestion. Bacillus thuringiensis bacteria are well-known insect pathogens that infect their host orally. We found that T. castaneum larvae showed increased survival after oral exposure to B. thuringiensis, when they had been orally primed with filter-sterilized media in which spores of B. thuringiensis had been raised. Such priming was achieved only with a naturally pathogenic strain of B. thuringiensis and a strain that was made pathogenic by transfer of plasmids. Moreover, primed larvae were smaller in size 24 h after priming and had a longer developmental time, indicating that investment in such a response comes at a cost. However, the increased survival in primed larvae was not caused by larval size differences upon challenge.