General Concepts of Viral Pathogenesis PDF

General concepts of viral pathogenesis venerdì 30 settembre 2022 10:38 Molecular virology Unit: Molecular pathogenesis of viral diseases General concepts of viral pathogenesis What you will learn in this lecture 1. What is viral pathogenesis: host perspective and viral perspective and differe...

General concepts of viral pathogenesis venerdì 30 settembre 2022 10:38 Molecular virology Unit: Molecular pathogenesis of viral diseases General concepts of viral pathogenesis What you will learn in this lecture 1. What is viral pathogenesis: host perspective and viral perspective and differences between acute, chronic and latent virus infection 2. The concepts of disease and virulence 3. How viruses infect the host, and how they spread between tissues 4. The outcome of virus spreading: the fate of infected cell, tissue and host. How virus-infected cells die What is viral pathogenesis: host perspective and viral perspective and differences between acute, chronic and latent virus infection Definitions and general concepts in viral pathogenesis Viruses are obligate parasites of living cells that cannot live independent of an intricate relationship with an infected cell Viruses in the host are not homogeneous, since both viral and host RNA and DNA polymerases make errors (one every 10 3 to 105 nucleotides) that generate mutant viruses during infection They are defined as quasi-species Mutations contribute to pathogenesis of viral infections due to emerging of immune-escape mutants via a Darwinian process The viral perspective The outcomes of viral invasions: productive, abortive and latent infections Infection is the process by which a virus introduces its genome into a cell It may be: Productive if new infectious virus is made Abortive if no new infectious virus is produced Latent if the production of infectious virus does not occur immediately but the virus retains the potential to initiate productive infection at a later time It is an unique transcriptional and translational state where infectious virus is not present Reactivation is when the productive replication cycle can be reinitiated when the need arises A cell is permissive if it can support productive infection and non-permissive if infection cannot occur at all or is abortive Establishment of latency During latent viral infection, cell features restricted viral gene expression, which allows the viral genome to survive even when lytic replication is not occurring Latently infected cells express no viral proteins, making latency immunologically silent Latency is the ultimate form of immune evasion: During latency, the host has no known mechanisms for sensing the presence of the virus To survive and spread from the latently infected cell, the virus must be able to reactivate and reinitiate the lytic cycle of gene expression, potentially generating antigens that the immune system can respond to The host perspective The outcome of viral infection: acute and chronic infection Infection may be: 1. Acute infection (3-5 days) Occurs when a virus first infects a susceptible host Both the host response and virus infection change continuously until infection is resolved or progresses to death of the host or establishment of chronic infection 2. Chronic infection Turin Pagina 14  2. Chronic infection Is the continuation of infection beyond the time when the immune system might reasonably be expected to clear acute infection It is an equilibrium process with viral and host processes balancing each other The immune system of the host brings the acute infection under control and delays or prevents a chronic infection from killing the host 3. Progression of chronic infection to disease There is a change in this equilibrium which is disrupted When the virus infection is dangerous: the concept of disease and virus virulence Disease is a harmful pathologic consequence of infection (not all infections have as result a disease) Not all the individual are affected: even highly virulent viruses often establish infection in a greater number of hosts than they cause disease (Exception are viruses such as rabies, Ebola, or HIV, which cause significant disease in nearly all infected persons) How a virus causes disease: Cell and tissue destruction Induction or secretion of inflammatory cytokines (family of 50-60 proteins, both pro- or anti-inflammatory), causing an excessive and unbalanced immune reaction Cellular dysfunction induced by viral infection Paracrine effects of viral gene products The induction of malignant tumours The effects of the immune system as it responds to infection activating wrong reactive pathways (immunopathology) The concept of virulence Virulence is the relative capacity of a virus to cause disease The forms of virulence: Induction of rapid death (as for Variola major, the causative agent in smallpox) Induction of tumours over prolonged periods (Papilloma or Herpesviruses) Induction of organ failure over many years (chronic HBV or HCV infection) Virulence is related to: Efficiency of replication Tropism toward specific organs or tissues Host response to infection Interaction between the virus and the host tissue How viruses infect the host, and how they spread between tissues The transmission of viruses between the hosts and within the host: 1. Entering in the host a. Horizontal transmission of the virus and entry into the organism There are five primary portals of entry for viruses, each of them STRONGLY defended Penetration through epithelial surfaces: ▪ Skin ▪ Conjunctiva ▪ Respiratory tract ▪ Gastrointestinal tract ▪ Genitourinary tract Video: ▪ Immunology of the lung ▪ Immunology in the gut mucosa ▪ Immunology in the skin Model of viral invasion Turin Pagina 15 Model of viral invasion Steps: 1. The virus enters the intestine via the fecal-oral route 2. It binds to M cells overlying the Peyer's patch and enters into lymphatic system where it replicates in lymphoid cell leading to a primary viremia and infection of secondary site 3. It replicates in secondary sites, giving rise to a secondary viremia that reaches a level capable of transmission to more protected organs (CNS for example), that the virus reaches by: □ It may cross the blood–brain barrier (poorly understood) □ Via the blood to peripheral nerves and then spreading up the nerves □ White Blood Cells containing viruses may facilitate the transmission The virus often enters the organism using the immune system, such as cells whose role is to integrate the immune reaction of the host at peripheral level (e.g. M cells or macrophages), and then, once inside the cells, avoids to be killed M cells role is to uptake and transport of particulate antigens from lumen to dendritic cell rich sub-epithelial dome where they are presented to immune cells thereby initiating an immune response or tolerance Penetration Through Epithelial Barriers To invade an organism, a virus must pass: ▪ Superficial epithelial barriers, a set of intracellular barriers to infection collectively referred to as intrinsic cellular resistance to infection ▪ Confront the innate and adaptive immune response Viruses cross epithelial barriers: ▪ Through mechanical breaches (e.g., vector-borne delivery or bite wound delivery) ▪ Accessing specialized cells and then being shuttled inside the organism How epithelia defences inhibit viral entry: ▪ Epithelial cells are constantly turning over and being replenished (cells that are contacted by a virus are shed continuously) ▪ The surface of skin is made by several layers of cells that are metabolically inactive and cannot support viral replication ▪ Barriers may be protected by low pH or secretions, including mucus Immunity related to epithelia Immunity related to epithelia Epithelial tissues are highly active immune organs containing several immune-related cells: Turin Pagina 16  Epithelial tissues are highly active immune organs containing several immune-related cells: 1. Dendritic cells (DC) (e.g. Langerhans cells in the skin) In all epithelia, DC serve as sentinels for invasion After activation by virus, they relocate to lymph nodes to induce immune responses DCs lie beneath the intestinal columnar epithelium DC play a dual role in infection: □ They induce immunity □ They are targeted by viruses as an initial site of Infection, providing the virus access to the lymphatic system 2. Intraepithelial lymphocytes Are present in sub-epithelial and epithelial tissues, providing cells capable of protective immune responses in the most superficial layers of the body 3. Epithelial cells themselves May be activated to express interferons or other antiviral molecules In many sites, an invading virus is subject to inactivation by antibodies and complement b. Vertical transmission of the virus: mother-foetus/newborn There are two mechanisms for entry into the developing fetus: ▪ Placental penetration, as when a virus enters the foetus after invasion of the foetal circulation or amniotic fluid ▪ Via the germ line (sperm) 2. Systemic spread of virus infection The viruses spread via three host systems that can provide access to a large number of tissues and cells: ○ Blood, the major highway for spread of viruses through the host ○ Lymphatics ○ Nerves (Rabies for example: from periphery to CNS, at a rate of 50-100 mm per day) The level of viremia is important, being related to: ○ Severity of the disease during acute viral disease ○ Prognosis during chronic viral disease (as in HIV) Ways of systemic spreading via blood: ○ Indirect: after entering into and replicating in tissues (draining lymph nodes or in lymphoid structures such as Peyer’s patches or tonsils) Entry into lymphoid tissue is a two-edged sword for the virus, as a facile route to access the viscera of the host but one that passes through the very tissues that generate adaptive antiviral immune responses ○ Direct: via introduction into the circulation, as by a needle or an insect bite 3. The distribution of virus between and within host tissues Distribution of virus in tissues is a dynamic process determined by competing processes including: ○ The speed of viral replication ○ The presence of specific viral receptors or other pro-viral factors the permit viral entry or replication ○ Viral mutation rate ○ Viral virulence genes ○ Host susceptibility and resistance genes ○ Innate and adaptive immunity The outcome of virus spreading: the fate of infected cell, tissue and host. How virus-infected cells die Turin Pagina 17 The outcome of virus spreading: the fate of infected cell, tissue and host. How virus-infected cells die The outcome of virus spreading The outcome of virus spreading is the result of an ongoing battle between the virus and the host being played out in different tissues and at different ranges: Local battle, such as the contact between a virus and a specific cell or the contact between an NK cell or cytolytic T cell and a virus- infected cell Middle range battle due to the effects of host cytokines or virus-encoded soluble proteins that evade or subvert host responses Long-range battle, including: ○ Production of antibody ○ Synthesis of stress steroids ○ Activation of the bone marrow to produce inflammatory cells, stimulation of the liver to synthesize and release acute-phase reactants ○ Stimulation of autonomic centers in the brain to produce fever The presence of a virus in a tissue may or may not result in damage depending on: The regenerative capacities of different primary cell types Whether the immune system damages the tissue as part of the protective response; Whether the virus induces autoimmune responses; Whether the virus is ever cleared from the tissue and, if not, the nature of residual virus infection. Tissue damages due to virus may be: Local effects, such as death of infected cells caused by: ○ Viral cytopathic effects ○ Killing by immune cells Local effects: direct killing of cells by virus and virus cytopathic effects Cytopathic effect is defined as the destructive consequences of virus infection on cells Direct killing of cells is caused by: ○ Viral subversion of cell metabolism for replication ○ Cell-intrinsic programmed cell death pathways such as necrosis, apoptosis, non-necrotic and non-apoptotic cell death Local effects: killing of cells due to immune system The immune system has many mechanisms for killing infected cells, arguing that the death of infected cells can benefit the host: ○ Direct: killing of cells by viruses death of infected cells is harmful if those cells are essential to the host; BUT death of an infected cell may inhibit viral replication with the sacrifice of one cell contributing to the protection of other cells ○ Indirect: killing of cells by immune system (cytokines, apoptosis induction, bystander killing by leukocytes) The host immune system has many mechanisms related to the killing of infected cells: ○ Complement: the complement cascade is a two-edged sword important for antiviral immunity but can also contribute to virus induced tissue damage Complement proteins are involved in: ▪ T-cell responses ▪ trapping of viral antigen on antigen-presenting cells ▪ induction of chemotactic and vascular permeability changes in infected tissues ▪ Activation of leukocytes ▪ complement can directly lyse both virus-infected cells and lyse or neutralize virions ○ Cell-extrinsic mechanisms: for initiating apoptosis, such as the granzymes that are injected via perforin into infected cells by leukocytes Systemic effects over longer distances, caused by: ○ Vascular damage leading to ischemia ○ Induction of fever or cachexia ○ Cytokine induction of the death of uninfected cells ○ Virus induced autoimmunity Systemic effects: tissue damages due to viral infection Tissue damage: ○ Is directly caused by the virus ○ Is indirectly caused by the immune system The perfect reaction balances he protective and destructive capacities of the immune system Immunopathology occurs when the immune system goes too far Turin Pagina 18  For example: ○ Circulating viral antigens are deposited in the kidney (alone of after binding with specific antibodies) ○ Solubility is lost and they precipitate in the kidney, activating complement and cellular inflammation Host cells can be killed by: ○ Activated immune cells ○ Cytokines ○ Adaptive immunity (antibodies) Antiviral immune response can be: ○ Beneficial, curing the infection ○ Harmful, inducing tissue pathology and systemic toxicity In some cases, the balance between protective effects of immunity and harmful effects shifts to pathology and the death of the host Systemic effects: tissue damages due to excessive immune reaction ○ CD8 T cells They can kill cells also when they are not that cytopathic --> CD8 T cell kill cells that are infected BUT would not otherwise die ○ Autoimmunity Molecular mimicry between virus and host-antigens --> Knock-down of self-tolerance ○ Virus-induced immunosuppression Some viruses kill immune system cells directly (CD4+ during HIV) Alteration in cytokines’ secretion by infected cells --> Upregulation of immunosuppressive cytokines How virus kill the cells (and the host)? Viruses kill: Directly, by killing infected cells as they replicate Indirectly, by Altering fluid and electrolyte balance, as in the case of dehydration induced by gastroenteritis Indirectly, by damaging the host by making the host susceptible to secondary infections Indirectly, by triggering an unbalanced inflammation (e.g. viral pneumonia) Outcome of virus infection Three options: Clearing of virus by immune system Latency: virus persists in a small portion of the host cells Establishing of chronic infection What is the rationale at the basis of these three options? It depends on how the host innate and adaptive immunity reacts and engages viruses What you should remember from this lecture… Turin Pagina 19 Immune response to viral infection mercoledì 5 ottobre 2022 12:42 Immune response to viral infection What will you learn in this lecture 1. How the host recognizes the presence of a virus 2. How the host reacts consistently against a virus infection. The antiviral defense strategies: cell intrinsic and extrinsic me chanisms and the cells involved in defense against virus, and their main functions 3. How virus evades and/or manipulates innate immunity. Interaction between innate and adaptive immunity The immunity against a virus at a glance Virus enters into the cell and it is recognized as “not self” Virus peptides are presented as Major Histocompatibility Complex: ALL the cells activate MHCI --> Infected cells apoptosis The professional Antigen Presenting Cells activate MHCII --> Production of specific antibodies Once infected, cells develop an anti-viral defense strategy Host defense strategy: Physical approach: the blockade of viral entry into the host cells Molecular approach, using innate immunity and neutralizing antibodies (acquired immunity) Aim of the strategy is to eliminate the infected cells (and the virus within, possibly) This can be achieved by: Cell-intrinsic mechanisms that are induced by type-I IFNs and operate in the infected cells Cell-extrinsic mechanisms, with the help of cytotoxic lymphocytes: natural killer (NK) cells and CD8 T cells The reaction of immunity against virus The immune system is organized in: 1. SENSING VIRAL INFECTIONS (sensing arm) The microbial sensing mechanism is based on pattern recognition receptors (PRRs). that specifically recognize Pathogen Associ ated Molecular Pattern (PAMP) - viral RNA and DNA - including: ○ Long double-stranded RNA ○ RNAs containing 5'-triphosphate ○ Unmethylated CpG motifs in viral DNA genomes 2. FIGHTING VIRAL INFECTIONS (effector arm): a. Step 1: It leads to the induction of the innate antiviral mechanisms, most of which are mediated primarily by innate anti-viral cytokines, such as type-I interferons (IFNs) b. Step 2: It leads to the activation of the adaptive immune response that can provide a more directed, antigen-specific, and long- lasting antiviral immunity Turin Pagina 20  lasting antiviral immunity 1. Sensing of viral infection Mammalian immune system detects viruses though several mechanism The best understood is Viral Sensing Through Pattern Recognition Receptors (PRR) Most common molecular patterns associated with virus infection are the features associated with viral nucleic acids Innate immune recognition can be: In infected cells: cell INTRINSIC recognition In non-infected cells: cell EXTRINSIC recognition Intrinsic and extrinsic innate immune recognition Cell-intrinsic innate immune recognition is mediated by cytosolic sensors: RIG-I-like receptors (RLRs) NOD-like receptors (NLRs) Activation of these receptors generally occurs in infected cells PRRs involved in cell-intrinsic recognition are broadly expressed because viral pathogens target a variety of cell types for replication Cell-extrinsic innate immune recognition is mediated by transmembrane receptors: Toll-like receptors (TLRs) C-type lectins (CLRs) Their activation does not require the cells expressing these receptors to be infected Cell-extrinsic recognition is mainly mediated by specialized cells of the immune system, such as the plasmacytoid dendritic cell (pDC), macrophage, and dendritic cell (DC) Intrinsic innate recognition RIG-I-Like Receptors (RLRs): RIG-I and MDA5 They have an helicase domains that recognize RNA viruses, both ssRNA and dsRNA In a virally infected cell, RNA structures unique to viruses are recognized by RIG-I (ssRNA) and MDA-5 (dsRNA) RNA binding to RLRs induces conformational changes, enabling their binding to MAVS adaptor protein expressed on the surface of mitochondria and peroxisomes MAVS activates signaling to activate NF-kB, MAP kinases, and IRF3, leading to target gene expression including IFN-b and cytokines Type I IFNs increase the levels of RLRs in bystander cells, enabling robust antiviral signalling upon infection Intrinsic Innate recognition NOD-Like Receptors (NLRs) They are activated by endogenous signals from: Dying cells (uric acid) Turin Pagina 21  Dying cells (uric acid) Crystals (asbestos, silica, alum) Microbial signals (whole bacteria, bacterial RNA, extracellular ATP, pore-forming toxins, viral infections, cytosolic dsDNA) NLRs play a key role in the activation of caspase-1 by forming a multiprotein complex known as the INFLAMMASOME Caspase-1 is an essential mediator of inflammatory response through its capacity to cleave and generate active forms of IL-1b and IL-18 Extrinsic innate recognition: Toll Like receptors External domains containing leucine rich repeat (LRR) that mediate the recognition of PAMPs The transmembrane domains of TLRs dictate the localization of the receptor The internal domains of TLRs activate downstream signals Some TLRs recognize bacterial, fungal, and protozoan pathogens, while others are specific for viral recognition Extrinsic innate recognition: Toll Like receptors' mechanism of action The cytosolic domain contains the toll/interleukin-1 receptor (TIR) domain, which binds to adaptor proteins and leads to expression of a variety of gene TLR activation, following virus detection, catalyses a cascade that splits into two main synthetic pathways: Synthesis of proinflammatory cytokines (NF-kB-dependent) Synthesis of antiviral cytokines, type I interferons (IFNs) (IRF-dependent) IRF=(Interferon regulatory factor) In addition, TLR signalling results in the activation of the mitogen-activated protein kinases (MAPKs) Type I IFNs in turn induce a battery of genes that directly suppress viral replication The Toll Like Receptors TLRs can be categorized into two major types: Those that are expressed on the cell surface (TLR1, 2, 4, 5, 6, 11, and 12) Those that are expressed in the endolysosomes (TLR3, 7, 8, and 9) Viruses are recognized mostly by this group, since they can gain access to viral nucleic acids upon endocytosis of virions TLR3 is expressed by DCs, B cells, fibroblasts, and epithelial cells Activates of both NF-kB and IRF3, leading to proinflammatory cytokines and type I IFN gene expression, respectively Recognize dsDNA, possibly in the context of phagocytized apoptotic cells TLR7/8, in humans TLR7 is expressed in plasmacytoid DCs (pDC) and B cells TLR8 is expressed by myeloid DCs and monocytes Turin Pagina 22 TLR8 is expressed by myeloid DCs and monocytes Recognize ssRNA and induce innate immune responses to ssRNA viruses TLR9 is expressed in pDCs and B cells in humans Recognise CpG (previously thought), but more recently it is supposed to recognize the sugar-base-backbone, 2-deoxyribose, of phosphodiester DNA irrespective of the CpG content C-Type lectins C-type lectin receptors (CLRs) expressed on the plasma membrane can bind to certain viruses Example: Langerin, which is expressed on Langerhans cells, is capable of clearing HIV by receptor-mediated endocytosis and degradation 2. Fighting viral infection The Effector arm Cell-intrinsic anti-viral mechanisms a. Innate anti-viral cytokines (Type I IFN) b. Inflammatory cytokines c. Retroviral Restriction Factors d. RNA Interference e. Autophagy Cell-extrinsic anti-viral mechanisms: Immune cells a. NK Cells b. Plasmacytoid DC c. Monocytes d. Macrophages e. Dendritic cells Cell-intrinsic anti-viral defense mechanisms 1. Type I IFN-Dependent innate - Antiviral cytokines Antiviral mechanisms in vertebrates are highly dependent on type I IFN Type I IFNs are a family of cytokines that act early in the innate immune response, and capable of: Turin Pagina 23 Type I IFNs are a family of cytokines that act early in the innate immune response, and capable of: a. inducing an antiviral state in infected and uninfected neighbouring cells b. regulating the ensuing adaptive immune response Stimulation of various PRRs including TLRs (TLR3, 4, 7, 8, and 9) RLRs (RIG-I, MDA-5) upregulate the production of type I IFNs --> Upregulation of Interferon-stimulated genes (ISG) The type I IFN family ○ IFN-α: produced by leukocytes 13 subtypes (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21) ○ IFN-β: produced by leukocytes and fibroblasts IFN-β1 (IFNB1) and IFN-β3 (IFNB3) Stimulate both macrophages and NK cells to elicit an anti-viral response ○ Other IFN Type I IFN-ε, -κ, -τ, -δ, and -ζ IFN-ω released by leukocytes at the site of viral infection The biological role of IFN Type I and their targets ISG and their targets: ○ Cholesterol-25-hydroxylase (CH25H) affects viruses early, presumably at ISG and their targets the host-membrane fusion event; at protein maturation of viral structural proteins by prenylation; and at protein maturation of viral replication enzymes ○ IFN-induced transmembrane (IFITM) protein members inhibit endocytic fusion events of a broad spectrum of viruses ○ Tripartite motif protein 5 α (TRIM5 α) inhibits human immunodeficiency virus 1 (HIV-1) uncoating of the viral RNA ○ The myxoma resistance protein 1 (Mx1) inhibits a wide range of viruses by blocking endocytic traffic of incoming virus particles and uncoating of ribonucleocapsids ○ 2′,5′-oligoadenylate synthetase (OAS) and latent ribonuclease L (RNase L), protein kinase R (PKR), Moloney leukemia virus 10 homolog (MOV10), and zinc-finger antiviral protein (ZAP) inhibit viruses by degrading viral RNA and/or blocking translation of viral mRNAs ○ IFN-induced proteins with tetratricopeptide repeats (IFIT) inhibit protein translation and have been implicated in viral RNA degradation as well ○ TRIM22 inhibits viral transcription, replication, or trafficking of viral proteins to the plasma membrane ○ ISG15 can inhibit viral translation, replication, or egress ○ Viperin has been shown to inhibit viral replication or virus budding at the plasma membrane ○ Tetherin traps mature virus particles on the plasma membrane and thus inhibits viral release IFN-stimulated gene (ISG) products (stella arancione) interfere with different stages of different viral life cycles (they are >300) Turin Pagina 24  IFN-stimulated gene (ISG) products (stella arancione) interfere with different stages of different viral life cycles (they are >300) Type I IFN Dependent Antiviral defense (ISG) ○ Type I IFN engagement of its receptors leads to the expression of over 300 ISG ○ Most of their function is unknown ○ Signalling is different in INFECTED or NON INFECTED cells In INFECTED CELLS, IFN acts in an autocrine way. IFN induces ISG that encode proteins that interfere with multi -steps of viral infection cycles such as: ○ Interfere with processing and presentation of Antigens ○ Cytotoxic activity of NK and CD8 T cells In NON INFECTED CELLS, IFN acts in a paracrine way and induce an antiviral state in neighbouring cells Some ISG genes and their functions: ○ 2′,5′-oligoadenylate synthetase (OAS) activates RNAse L which to degrades viral ssRNA in the cytosol ○ Protein Kinase R (PKR) Is a Ser/Thr kinase. It is activated by dsRNA resulting in a decreasing in TOTAL (cellular and viral) proteins ○ ISG15 It is an ubiquitin-like molecule. ISGylation inhibits newly translated viral proteins ○ Tetherin is associated with lipid rafts and inhibits retrovirus particle release ○ Viperin (Virus Inhibitory Protein) endoplasmic reticulum-associated, interferon inducible Viperin disrupts lipid rafts 2. Inflammatory Cytokines In addition to type I IFNs, many inflammatory cytokines play an important role in antiviral defense: ○ Directly by inducing antiviral effector molecules ○ Indirectly by stimulating: ▪ Cellular recruitment ▪ Phagocytosis of infected cells ▪ Activating adaptive immune responses such as CTL and neutralizing antibodies Cytokines can act: ○ Locally Through autocrine and paracrine mechanisms ○ At systemic level if produced at high enough levels, they can gain access to the circulation and induce, such as acute-phase response The systemic response to inflammation: The acute phase reaction Turin Pagina 25 Cytokines involved in anti-viral activity: ○ IL-6 ▪ Triggers APP production ▪ Activates T cell and antibody producing proteins ○ IL-1b/IL-18 Their secretion requires the activation of inflammasome ▪ IL-1b is mainly produced by the sentinel cells of the innate immune system, including macrophages and DCs, although fibroblasts and keratinocytes can also synthesize IL-1b in response to tissue injury or stress signals Induces the expression of hundreds of genes, including cytokines (IL-6 and TNF-a), chemokines (e.g., IL-8), and adhesion molecules that are important for leukocyte trafficking In adaptive antiviral immunity, IL-1b plays an important role in the antigen-driven expansion and differentiation of CD4 T cells ▪ IL-18 is expressed mainly by macrophages and DCs Prime NK cells for the production of IFN-g and enhance their cytolytic in combination with IL-12 or IL-2 alone can also induce differentiation of either Th1 or Th2 cell types depending on the cytokine milieu IL-18 is also required for optimal cytokine production by CTLs, including IFN-g, TNF-a, and IL-2 Activation of NF-kB: ▪ > expression of adhesion factors on endothelial cells increasing diapedesis of immunocompetent cells to sites of infection. ▪ > body temperature (fever) ○ TNF-a Upon engagement of the ligand TNF, TNFR forms a trimer TNFRI contains a cytoplasmic death domain, which recruits the adaptor TRADD through its death domain TRADD can assemble two different signaling complexes, involving: ▪ FADD pro-caspase 8 or RIP1 ▪ TRAF2 Turin Pagina 26  TNFR signaling TNFR signaling leads to three distinct cell fates after TNF binding that trimerizes the TNFR Through the cytoplasmic death domain, TNFR recruits TRADD, which also contains a Death Domain (DD) TRADD can assemble different signaling complexes: i. TRADD can recruit FADD through DD-DD interaction, which results in caspase8 activation, leading to apoptosis ii. TRADD can also recruit RIP-1 and TRAF2, resulting in signal transduction and activation of NF-kB and MAP kinase pathways iii. In cells in which caspase-8 is rendered non-functional, RIP-3 is recruited to the complex to induce necrosis ○ IL-15 It is produced by DC and Macrophages after viral infection The main target are CTL (CD8 T cells and NK) It is a growth factor and a survival factor ○ IFN-g It is produced by three lymphocyte types after induction of adaptive immunity: ▪ NK cells (during early phase of viral infection) ▪ CD8 T cells ▪ Th1 cells Induce the transcription of genes encoding anti-viral effectors such as PKR and viperin Induce the activation of genes involved in antigen processing and presentation 3. Retroviral Restriction Factors TRIM5 a It is thought to perturb the controlled uncoating of the sub-viral particles by recognizing and degrading the capsid proteins APOBEC family It is a cytidine deaminase It becomes encapsidated in retroviral virions in infected cells After viral fusion and entry in infected cells of new host, APOBEC deaminates cytosine residues in nascent retroviral cDNA 4. RNA Interference Two steps a. Viral dsRNA is recognized by members of the Dicer endonuclease, which processes it into siRNA b. These siRNA are incorporated in RNA-induced silencing complex (RISC), which guides the RNAse enzyme AGO to degrade the viral RNA 5. Xenophagy/Autophagy Turin Pagina 27  5. Xenophagy/Autophagy Authophagy is a pathway for lysosomal degradation of cytosolic constituents and organelles that is critical for cellular home ostasis Pathogens can be cleared via autophagy (called in this case Xenophagy) Upon xenophagocytosis, virions or viral replication products in cytosol are engulfed into autophagosomes and degraded in the lysosome of clearance Cell-extrinsic anti-viral mechanisms Cell types involved in innate antiviral responses: 1. NK Cells Are cytotoxic lymphocytes that detect and eliminate virally infected cells and produce IFNg NK produce two classes of receptors that induce or inhibit the target cell lysis and IFNg production, reacting with: ○ Activating ligands: are induced in virally infected cells ○ Inhibitory ligands: are MHCI (and other molecules) constitutively expressed on most cells, and downregulated after viral infection Through their capability to detect missing self MHCI, NK cells fill the gap in immuno -surveillance when CD8 T cells are not working NK cell activity ○ When a NK cell recognizes a healthy cell, NK cell lysis of the target cell is prevented by engagement of the inhibitory receptor by abnormal level of MHC class I on the target cell ○ When a NK cell interacts with virally infected cells that have reduced MHC class I on the cell surface (“missing self”), NK cells induce apoptosis in the target cell ○ When a NK cell recognizes stressed virally infected cell expressing high levels of stimulating ligands (“induced self”), negative signals from inhibitory receptor is overridden by the activating signal, leading to lysis of target cells 2. Plasmacytoid DC (pDC) Are regarded as professional viral sensors: a) pDC are equipped with special cellular machinery to endocytose virus and transport them to endolysosomes, where PAMP are directed toward TLR b) pDC constitutively express molecules (such as IRF7) that induce type I IFN genes pDC are the main cells to secrete Type I IFN 3. Monocytes Are circulating monocytes divided (roughly) in two groups: ○ Inflammatory monocytes: are recruited to inflamed tissues and lymph nodes, differentiate in macrophages and produce TNFa and IL-1 during infection ○ Patrolling monocytes: can differentiate into macrophages as well and are probably associated with tissue repair 4. Macrophages Versatile cells playing important roles in inflammation, wound healing tissue homeostasis and remodelling Macrophages are professional phagocytes, clearing dying cells and degrading incoming pathogens Turin Pagina 28  Macrophages are professional phagocytes, clearing dying cells and degrading incoming pathogens Infected macrophages produce the highest levels of type I IFNs Infected macrophages also serve as “viral sink” to prevent more vulnerable cell types from becoming infected 5. Dendritic cells Dendritic cells are professional antigen-presenting cells capable of stimulating naïve antigen-specific lymphocytes in secondary lymphoid organs DC are situated at different sites of pathogen entry and are among the firsts to recognize incoming pathogens via pathogens recognising receptors (PRR) DC initiate adaptive immune response by activating naïve T cells. They overexpress chemokine receptor CCR7, enabling them to migrate to lymph-nodes where T cell recirculate. During transit, DC upregulate MHCII and co-stimulatory molecules Once on the lymph node, DC cells present the antigens to T cell, with the aim to develop a coherent immune reaction Innate instruction to adaptive immunity 1. A T cell must recognize viral peptide presented within MHC (signal 1). CD4 T cells bind to MHCII, and CD8 T cells bind to MHC I 2. Engaged cells must receive co-stimulatory signals from DC: PRR stimulation overexpress CD 80 and CD86, which bind to CD28 on naïve T cells (second signal) and provide clonal expansion 3. The third signal dictates the differentiation into different effector types (Th1, Th2, Th17), depending on the cytokines prov ided by DC (or other accessory cells) 3 video: The MCH I activation and CTL-induced apoptosis of infected cells (innate immunity) CTL Killing of virus infected cells MHCII activation and processing: antibodies are produced (adaptive immunity) The development of adaptive immunity Turin Pagina 29 Antibody activities against free virus particles and virally infected cells 3. Viral evasion of the innate immunity Viral evasion of the innate immunity can be categorized into four major strategies: 1. Global inhibition of cellular gene expression 2. Evasion from innate recognition 3. Inhibition of molecules involved in the IFN induction and signalling 4. Inactivation of the IFN induced effector molecules Differences in innate immunities are balanced between: Protective immune responses --> Aggressive response (Clearing infection) Pathogenic immune responses --> Exuberant innate immune activity (Induces host tissue damage) The common themes in innate immune responses In viral infections in humans INNATE IMMUNE RESPONSES underlie severe disease manifestations Examples: 1. Influenza A and SARS PMN and monocyte dysregulation The viruses cause and excessive chemokine and cytokine response --> Excessive chemotaxis of PMN and monocytes into the site of infection --> Wide tissue damages 2. SARS CoV: INF type I production Excessive IFN type I and chemokine production --> Type I IFN, when induced at high levels, can inhibit the formation of optimal antibody response 3. Ebola VV: APC (DC and macrophage virus infection) Turin Pagina 30  3. Ebola VV: APC (DC and macrophage virus infection) Antibody presenting cells (DC and MF) are infected by Ebola virus --> High production of Cytokine, Chemokine, Tissue factor --> Septic shock and DIC (disseminate intravascular coagulation) Septic shock DIC Disseminated intravascular Coagulation Disseminated Intravascular Coagulation is a the generation of thrombi in Micro -circulation, and consumption of coagulation factors and platelets Condizioni particolari come: ○ Cancer ○ Serious haemolysis ○ Infections (Ag-Ab complexes) ○ Endotoxemia (LPS) ○ Vascular stasis ○ Burns … possono causare: ○ Wide release of tissue factor From leukocytes, platelets and endothelial cells Gram- sepsis ○ Endothelium damages Immunocomplexes 4. HIV: erosion of immunocompetence HIV infection induces a persistent yet ineffective host response --> Active virus replication in CD4+ T cell population induces --> Chronic pleiotropic immune activation (increased activation, proliferation apoptosis, of immune effector cell populations) --> Unrelenting virus application --> Hyperactivation of innate immunity (IFN type I and Proinflammatory cytokines) --> Damaging effects (intestine) --> Translocation of intestinal bacteria and virus What you should remember from this lecture… Turin Pagina 31 What you should remember from this lecture… Once virus has infected a cell, the host reacts in at least two ways: Protecting the infected cell (that reacts in an autocrine way) Protecting neighbouring cells (infected cells act in a paracrine way) Apoptosis of infected cell is a way to protect the host Call for support from other immune related ells The immune response against viral infection is usually effective in destroying the virus (innate immunity and adaptive immunity) and providing a protection (adaptive immunity) but… Exuberant immune reaction may do more harm than good Turin Pagina 32 Host immune responses to emerging zoonotic diseases martedì 18 ottobre 2022 10:29 Host immune responses to emerging zoonotic diseases The transmission risk of viruses between wild animals and humans What will you learn in this lecture 1. Emerging disease concept 2. General pathways of transmission between animals and humans 3. The concept of reservoir host and virus tolerance: why some hosts are susceptible and other are not? Most of the emerging viruses have a zoonotic origin, such as Ebola virus, HIV, SARS Coronavirus, Influenza A virus, Hantaviru s, Hendra and Nipah virus How transmission occurs: 1. Direct transmission: directly to people from living reservoir hosts Example: fruit bats forage at night in various trees that are producing ripe fruit and often drink from palm sap collection vessels 2. Butchering of reservoir hosts The contact with bush meat was responsible of SIV ( Simian Immunodepression Virus) transmission (via blood Bush meat is one of the major source of meat in several non developed countries in the world Huntind wild animals may expose humans to the risk of viruses these animals are reservoirs of (e.g. they are adapted to), triggering the spilling over process (examples: Ebola virus outbreak in Democratic Republic of Congo and maybe also SARS Covid-19) 3. Intermediate hosts Examples: Nipah virus, swine influenza, SARS Covid-19, West Nile disease Factors that increase dissemination of viruses: 1. Growth in size of human population 2. Environmental and climate change 3. Spread of agricultural practice promoting human and animal contact , in a context of lack of hygiene and animal welfare respect 4. Poaching protected species and eating animals that have been for a long segregated in the wild The outcome of infection in humans is related to the immune system of the host Different effects on different hosts: the example of SIV in chimpanzees SIV is the viral precursor of HIV in humans: Chimpanzees Detectable depletion of CD4+ Animals have shorter lifespan Reduced reproduction success Sooty mangabeys (Cercocebus atys) It is the natural reservoir host of SIV No detectable effects on host Patterns of reservoir hosts and viral association Bats are reservoirs for many viral diseases, including: SARS Corona virus MERS Corona virus Turin Pagina 33  MERS Corona virus Nipah and Hendra virus Rabies Marburg and Ebola virus Transmission from bat to humans Directly: from live (or death…) reservoir hosts to people Example: fruit bats forage at night in various trees that are producing ripe fruit and often drink from palm sap collection vessels Indirectly: via a spillover event SPILLOVER EVENT: when a reservoir population with a high pathogen prevalence comes into contact with a novel host population The pathogen is transmitted from the reservoir population and may or may not be transmitted within the host population Why bats are regarded as involved as virus reservoirs? Bats can very efficiently regulate host responses to infection Bats' immune system can prevent excessive immunopathology in response to most vital pathogens Bats are many and flexible The relationship between reservoir host physiology metabolism and infection in bats Bats feature: Sustained flight Long lifespan Small body size High metabolic rate The consequence is a positive selection for mitochondrial and nuclear genes Involved in the detoxification of ROS Is it conceivable that these features confer to infected cells a specific resistance to damages (including those caused by virus) What you may want to know about bats (and could explain why bats harbors so many viruses) The environment The mechanism of survival to viral infection of Bats Bats can survive to viral infection (and spread the virus) because they can balance the host defence tolerance system by enhancing: ○ Host defences ○ Immune tolerance The metabolism Turin Pagina 34 Bats have different inflammasome sensors Recent genomic analysis of two bat species (Pteropus alecto and Myotis davidii) revealed the absence of the PYHIN gene family (AIM2, IFI16), an important immune sensor of intracellular self and foreign DNA and activators of the inflammasome and/or interferon pathway s This removal of inflammasome DNA sensors may indicate an important adaptation that is flight induced and related, at least in part, to pathogen host co existence The concept of reservoirs hosts and virus tolerance: why viruses cause disease in humans but not in their animal hosts? Because: 1. The virus is cytopathic in the non-natural host but not on its reservoir 2. Differences in virus tropism between the natural and the non-natural host Example: ○ Sooty mangabey is a natural host of SIV: CCR5 is downregulated on the surface of memory T cells of sooty mangabeys (CCR5 is a co-receptor of SIV) --> reduced cell infections ○ CD4 expression is downregulated in CD4+ T cells in African green monkey making them resistant to SIV infection --> reduced cell infections 3. Differences in the interactions between the viral proteins and host resistance mechanisms Tetherin is a lipid raft-associated protein which reduces the release of newly produced HIV It is antagonized by the HIV Accessory protein Vpu Turin Pagina 35 Of the four groups of HIV (M, N, O, P) ONLY M group became pandemic because it has the most potent anti-tetherin activity Induction of responses that actively suppress damaging responses is caused by: ○ Specific viral proteins may interact with immune pathways to limit proinflammatory responses, and preventing the clearance ○ Changes in innate recognition could directly lead to reduction in inflammatory mediators The African primate species sensitivity to SIV is the best-studied reservoir host response Unlike humans, the African Primates do not develop the: ○ Severe immune dysfunctions of AIDS ○ Susceptibility to opportunistic Infections of AIDS Why? Simian immunodeficiency viruses (SIVs) are retroviruses infecting non-human primates causing disease only in some of them (e.g. Rhesus macaque) Through contact with the blood of chimps that are often hunted for bushmeat in Africa SIVsmm is non-pathogenic, while SIVcpz develop later a syndrome that resembles AIDS The ISG gene expression Current knowledge on cell-intrinsic restriction of HIV replication by ISGs HIV nucleic acids react with PAMP receptors which induce expression of IFN stimulatory genes (ISGs) TETHERIN reduces the release of newly produced HIV SLFN11 targets tRNAs and reducing their cellular abundance, thus interfering with mRNA translation SAMHD1 blocks the replication of HIV in DC and macrophages Turin Pagina 36  SAMHD1 blocks the replication of HIV in DC and macrophages APOBEC3G blocks HIV replication by deaminating viral cDNA and causes direct destruction of viral cDNA Pathogenic HIV and SIV have a different ISG (interferon-stimulated genes) signature 4. Interactions between virus and other parasites or microbiome Helminth infection can reactivate Herpes viruses from latency through generation of cytokines that activate viral promoter, or inhibit T cell responses In general, microbiome shapes the immune reactivity of mucosae (intestinal, respiratory, vaginal, …) 5. The reservoir host responses control viral replication more effectively 6. Viral loads are better tolerated by natural host The factors that influence the transmission to other species include: ○ Chronic/repeated virus shedding produces greater number of virus particles --> Increases the likehood of transmission ○ Immunological effector mechanisms affect viral genetic diversity and evolution --> Reduces spillover ○ Viral diversity --> More choices to establish in non-natural hosts ○ Clinical manifestation of infection (sneezing, coughing, diarrhea) --> Facilitates transmission Turin Pagina 37 Coronavirus-related diseases mercoledì 19 ottobre 2022 15:50 Coronavirus-related diseases What will you learn in this lecture An overview of Coronaviruses as pathogens in animals A description of the structure of Corona viruses' structure, the agents of SARS and other related diseases Corona virus replication: how virus bind to the cells and enter in the cytoplasm Pathogenesis of the Corona virus diseases and evasion of the immune response The post-COVID Syndrome Emerging Corona virus Diseases: MERS Diagnosis Animal Models to study COVID An overview of Coronaviruses as pathogens in animals How to call it: SARS-CoV-2: is the causative virus named as Severe Acute Respiratory Syndrome Coronavirus 2 COVID-19: COronaVIrus Disease 2019 is the name of the disease Pathogenesis and Pathology: the Coronavirus diseases General features of the infection Most Coronaviruses spread to susceptible hosts by respiratory tracts, or fecal-oral route and replicate in epithelial cells Coronavirus-related diseases in animals Studied as early as 1930s as causative agents of: Infectious bronchitis in chickens (IBV) Turkey COV (TCoV) Pheasant COV (PhCoV) Other Avian Corona Viruses: pigeons, peafowl, waterfowl, penguins, quail, teal, duck, swan Coronavirus in other species: Transmissible gastroenteritis in pigs Hepatitis in mice Feline infectious peritonitis in cats Upper airways infection in humans (inconsequential pathogens before SARS and COVID 2019) Turin Pagina 38 Coronavirus in ruminants: Almost all livestock (cows, sheep, goats water buffaloes, alpaca, llama): gastroenteritis Camelidae: mild respiratory disease Coronavirus in bats: they're likely the natural reservoir Transmission of Coronavirus-related diseases The transmission between species probably needs an intermediate host between natural reservoir and the final host Transmission from bat to humans: Directly from live (or dead) reservoir hosts to people Example: fruit bats forage at night in various trees that are producing ripe fruit and often drink from palm sap collection vessels Indirectly through an intermediate host due to a spillover event Spillover event: when a reservoir population with a high pathogen prevalence comes into contact with a novel host population The pathogen is transmitted from the reservoir population and may or may not be transmitted within the host population Bats as reservoir animals? Bats are reservoirs for: Coronavirus Nipah and Hendra virus Rabies Marburg and Ebola virus Bats feature: Sustained flight Long lifespan Small body size High metabolic rate The consequence is a positive selection for mitochondrial and nuclear genes Involved in the detoxification of ROS Is it conceivable that these features confer to infected cells a specific resistance to damages (including those caused by virus) Bats have different inflammasome sensors Turin Pagina 39 Bats have different inflammasome sensors Recent genomic analysis of two bat species (Pteropus alecto and Myotis davidii) revealed the absence of the PYHIN gene family (AIM2, IFI16), an important immune sensor of intracellular self and foreign DNA and activators of the inflammasome and/or interferon pathways This removal of inflammasome DNA sensors may indicate an important adaptation that is flight induced and related, at least in part, to pathogen host co-existence Features of SARS-CoV-2 that makes so permissive to spillover Coronaviruses feature exceptional plasticity They evolve rapidly, changing: Their antigenic profile Tissue tropism The host range Antigenic drift increases viral fitness: the viral replicase (an RNA dependent-RNA polymerase) does not possess a good proofreading activity, incorporating wrong nucleotides at each replication cycle and accumulating mutations in the viral genome, leading to a progressive differentiation of the viral progeny from the parental strain Antigenic shift (like Influenza) increases viral fitness: the particular replicating machinery of CoVs facilitates recombination events due to the presence of consensus sequences upstream of each gene Therefore, in other CoV strains that are present in the same cell, the RNA polymerase can jump from the RNA of one strain to that of the other one, synthesizing a hybrid RNA containing sequences from both viruses (homologous recombination) It works also with RNAs of different viruses and other organisms (heterologous recombination) CoVs acquire novel biological properties: Virulence Host range Tissue tropism CoV strains, which are non-pathogenic or low pathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species rapidly spreading in the new host Coronavirus occurs as quasispecies Having high error rates in their replication, Coronavirus occur as quasispecies (i.e. groups of related genotypes) With every RNA replication of CoV, several point mutations or shifts occur, whose consequences are: Several viral quasispecies coexists in the same individual The virus is flexible and can more easily evade the immune system Difficult to find a target for vaccines Virus variant issues SARS-CoV-2 is continuing to evolve, emerging novel variants with spike protein mutations Although most mutations emerged in the SARS-CoV-2 genome are neutral or mildly deleterious A small number of mutations can affect virus phenotype that confers the virus a fitness advantage The history of SARS-CoV-2 variants Since December 2020, the SARS-CoV-2 has emerged five quickly spreading strains, designated variants of concern (VOCs): Alpha (B.1.1.7), Turin Pagina 40 Since December 2020, the SARS-CoV-2 has emerged five quickly spreading strains, designated variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529) The variants have a high number of the mutations in the spike protein that promotes viral cell entry through the angiotensin-converting enzyme-2 (ACE2) Mutations that have arisen in the receptor binding domain (RBD) of the spike protein are of great concern due to their potential to evade neutralizing antibodies triggered by previous infection and vaccines SARS-CoV-2 variant mutations The molecular evolution of the virus trend is toward the infectivity , involving mostly the spike protein Taxonomy Corona viruses are large, enveloped, single-strand RNA viruses The structure of Coronaviruses The structure of SARS-CoV-2 viruses The SARS-CoV-2 genome consists of a single-stranded positive-sense RNA molecule of approximately 29.900 nucleotides arranged into 14 open reading frames (ORFs) encoding 31 proteins ORF1a and ORF1b: that proteolytically cleaves by a virus-encoded protease into individual replicase complex non-structural proteins to form 1-6 non-structural proteins (nsp1-16), involved in genome replication and early transcription regulation 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), which are common to all Coronaviruses Turin Pagina 41 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), which are common to all Coronaviruses 11 accessory proteins: ORF3a, ORF3b, ORF3c, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10, the function of which is not completely characterised Virion structural proteins Roughly spherical and moderately pleomorphic, Ø 80-120 nm, spiked Viral structural proteins: The spikes Made of trimers of S protein monomer of 128-160 kDa Class I viral proteins that bind to the cell receptors and mediate the earliest steps of infection The surface spike (S) glycoprotein is critical for binding of host cell receptors and is believed to represent a key determinant of host range restriction The nucleocapsid The corona viruses contain the largest single strand molecule RNA virus (approximately 30,000 nucleotides) It is a ribonucleoprotein containing viral genome Helically symmetric nucleocapsid M protein M protein is the most abundant protein in Coronavirus It gives the shape to the virion envelope M monomer MW ranges from 25 to 30 kDa Is a membrane protein Conserved within Coronavirus genera E protein E protein is found in limited amount in the virion envelope Critical for virion infectivity E protein MW ranges from 8 to 12 kDa Is an integral membrane protein Conserved within Coronavirus genera The non structural (NS) proteins: Other accessory proteins: Turin Pagina 42 Coronavirus replication: how the virus binds to the cells and enter in the cytoplasm Steps: 1. Virion attachment to host cells 2. Viral entry and uncoating 3. Expression of the replicase-transcriptase complex 4. Viral RNA synthesis 5. Assembly and release of virion The replication steps of the virus: 1. Engaging of the receptor and entry 2. Translation into the protein 3. Remodelling cell structure (double-membrane vescicle) 4. Membrane fusion and exit 5. Cut and release of the virus Virion attachment to host cells Known viral receptors engaged to enter into the cells: CD26 (MERS) ACE2 Angiotensin-Converting Enzyme 2 (SARS) Turin Pagina 43 How the Coronavirus infects the cells: The roles of spike proteins Each SARS-CoV-2 virion (virus particle) has an outer surface peppered with 24-40 spike proteins that reacts with specific receptors over the cell SARS-CoV-2 spikes are wildly flexible and hinge at three points That allows the spikes to flop around, sway and rotate, which could make it easier for them to scan the cell surface and for multiple spikes to bind to the RBD (Receptor Binding Domain) of the cell The roles of the Receptor Binding Domain The RBDs of SARS-CoV-2 spike proteins attach to the ACE2 receptor, which lies outside of most human throat and lung cells ACE2 receptor is also the docking point for SARS-CoV, but SARS-CoV-2 binds to ACE2 an estimated 2-4 times more strongly The a and d variants and the binding to RBD: ○ The Alpha variant, for example, includes ten changes in the spike protein sequence, which result in RBDs being more likely to stay in the ‘up’ position making it easier to enter into cells ○ The Delta variant hosts several mutations in the S1 subunit, including three in the RBD that increase its ability to bind to ACE2 and evade the immune system The entry of the virus into the cells SARS-CoV-2 uses either of two host protease enzymes to break in: TMPRSS2 the faster route in Endosomal pathway, relying on cathepsin L When virions enter cells by this route, however, antiviral proteins can trap them SARS-CoV-2 efficiently uses TMPRSS2 TMPRSS2 is an enzyme found in high amounts on the outside of respiratory cells The TMPRSS2 way of entry 1. TMPRSS2 cuts a site on the spike’s S2 subunit. That cut exposes a run of hydrophobic amino acids that rapidly buries itself in the Turin Pagina 44  1. TMPRSS2 cuts a site on the spike’s S2 subunit. That cut exposes a run of hydrophobic amino acids that rapidly buries itself in the membrane of the host cell 2. The extended spike folds back onto itself, like a zipper, forcing the viral and cell membranes to fuse. The virus then ejects its genome directly into the cell avoiding to be trapped in endosomes The virus’s speedy entry using TMPRSS2 explains why the malaria drug chloroquine didn’t work in clinical trials as a COVID-19 treatment. The in vitro studies supporting cloroquine use were carried out in cell models that rely exclusively on cathepsins for endosomal entry. When the virus transmits and replicates in the human airway, it doesn’t use endosomes. Chloroquine, which is an endosomal disrupting drug, is not effective in real life There is also a potential for the use of protease inhibitors as a promising therapeutic option to prevent a virus from using TMPRSS2, cathepsin L or other proteases to enter host cells. One TMPRSS2 inhibitor, camostat mesylate, which is approved in Japan to treat pancreatitis, blocked viral entry into lung cells, but the drug did not improve patients’ outcomes in an initial clinical trial Engaging of the receptor and entry Human angiotensin-converting enzyme 2 (ACE2), the receptor of Cov-SARS , is distributed primarily in the lower respiratory tract, rather than in the upper airway, thus explaining: Few upper way respiratory symptoms - ACE-2 is absent over the upper respiratory tract symptoms Peak viral shedding occurred late (≈10 days) in illness when individuals were already hospitalized The pathogenesis of the Coronavirus diseases: the life cycle inside the cells and the evasion of the immune response After the virus shoots its RNA genome into the cell, it takes over the cell’s machinery and mRNAs that code for a total of 26 known viral proteins are generated Coronaviruses take over that machinery in at least three ways, suppressing the translation of host mRNA in favour of its own None are exclusive to this virus, but the combination, speed and magnitude of the effects seem unique 1. The virus eliminates the competition: viral protein Nsp1, one of the first proteins translated when the virus arrives, recruits host proteins to systematically cuts all cellular mRNAs that don’t have a viral tag 2. NSP1 reduces overall protein translation in the cell by 70%. Nsp1 is again the main culprit, this time physically blocking the entry channel of ribosomes so mRNA can’t get inside. The little translation capacity that remains is dedicated to viral RNAs 3. NSP1, shuts down the cell’s alarm system preventing the cellular mRNA from getting out of the nucleus, including instructions for proteins meant to alert the immune system to infection. The protein seems to jam up exit channels in the nucleus, interferons in particular 4. Remodelling the interior and exterior of the cell to its needs: formation of syncytia and double-membrane vescicle (DVM) Feline Corona Viruses (FCoV) FCoVs occur as 2 pathotypes: Feline enteric coronavirus(FECV), "ubiquitous enteric biotype" Feline infectious peritonitis virus (FIPV), the "virulent biotype that causes FIP in individual cats" FECV commonly causes mild or asymptomatic infection in domestic cats and felids Turin Pagina 45 FECV commonly causes mild or asymptomatic infection in domestic cats and felids In some cats --> mutation of the S-glycoprotein, ORF3b and 7b occurs causing FIP The virus gains the ability to replicate into the macrophages which: Transport the virus to the various districts Produce a wave of pro-inflammatory cytokines which induce apoptosis of T cells Feline Infectious Peritonis (FIP) The pathogenesis of SARS: Immune Complex-Mediated (Type III) Hypersensitivity Mediated by the formation of immune complexes Antibodies-virus in the circulation, that lose solubility in plasma and precipitate 1. Formation of immune complexes 2. Deposition of immune complexes 3. Tissue injury caused by Immune complexes that unleash complement reaction and induce in turn: a. Destruction of cells b. Chemotaxis of PMN and further destruction immune related destruction Human Corona virus Regarded as agents of upper respiratory tracts with low mortality (before 2003) although a role of HCoV in Multiple Sclerosis was postulated because: Mouse CoV causes demyelinating disease Corona Virus particles have been occasionally detected in CNS of patients with MS SARS-CoV-related diseases Causes the most severe disease of any HCoV It is mostly a respiratory disease (really?) At least three diseases: SARS-COV (2003), MERS-COV (2012), SARS-COV-19 (2019) Pathologic findings and symptoms include: Diffuse alveolar damages Pneumocyte desquamation Alveolar edema Acute lung injury Turin Pagina 46  Acute lung injury ARDS Acute Respiratory Distress Syndrome The ARDS 1. A violent wave of Pro-inflammatory cytokines (IL8/IL1/IL4/Tnfa) (Cytokine storm) from macrophages causes PMN to migrate into alveolar space 2. PMN release mediators which contribute to alveolar damage, accumulating edema in air spaces, AND hyaline change (associated with the accumulation of proteins in tissues) 3. Other cytokines include TGFbeta and PDGF, that stimulate fibroblasts growth and connective tissue deposition The infection of alveoli in the lung 1. Type I and type II pneumocytes make up the alveolar walls and resident alveolar macrophages and pulmonary surfactant exist in the airspace 2. In the acute phase of SARS-CoV infection, type I and type II pneumocytes are infected and secrete inflammatory cytokines, while surfactant levels decrease 3. During the late stage/tissue damage portion of viral infection, viral titres decrease, while airway debris, pulmonary oedema and hyaline membrane formation all hamper a proper respiration Definition of Pulmonary surfactant Pulmonary surfactant is a complex and highly surface active material composed of lipids and proteins which is found in the fluid lining the alveolar surface of the lungs Surfactant prevents alveolar collapse at low lung volume It is involved in the protection of the lungs from injuries and infections caused by inhaled particles and micro-organisms (immunological, non- biophysical functions) The reduction of pulmonary surfactant causes the alveoli to collapse Pathogenesis of SARS as compared to Hcov The virus is different from that of the HCoV The host immunity is responsible for most of the clinical signs The infection of macrophages is violent (not regulated inflammation) but in the event is not effective against virus and causes a Sub-optimal T cell response Delayed kinetic of virus clearance Evasion of immune response by SARS virus The efficacy of the innate immune response determines: The extent of the initial viral replication The load that the host must overcome to clear the infection Turin Pagina 47  The load that the host must overcome to clear the infection The strategy to counter the innate immunity include: 1. The dysregulation of the expression and signaling of IFN type I 2. Double strand RNA is buried into double membrane vescicle 3. Formation of syncytia The evasion of immune response is due to the activity of accessory proteins: effects on host immune defences Evasion of immune response 1. The dysregulation of IFN type I Virus enters into Macrophages and plasmacytoid cells Circled in red the SARS accessory proteins involved in the Inhibition of IFN activation 2. Double strand RNA is buried into double membrane vescicle CoV infection induces the formation of a reticulovesicular network of modified membranes that are thought to be the sites of virus replication called double-membrane vesicles (DMVs), that are interconnected and contiguous with the rough endoplasmic reticulum (RER) Viral double-stranded RNA is mostly localized to the interior of the DMVs and inner vesicles of the VPs, whereas replicase proteins (nsp3, nsp5 and nsp8) are present on the surrounding CM Turin Pagina 48  3. Formation of Syncytia Syncytium is a multinucleate cell which can result from multiple cell fusions (e.g. muscle cell) 1st step: some of the newly made viral spike proteins travel to the surface of the cell and poke out of the host-cell membrane 2nd step: the infected cell fuses to neighbouring cells expressing ACE2, developing into massive individual respiratory cells filled with up to 20 nuclei The advantages for the virus is unknown Evasion of immune response and pathogenesis of SARS The kinetics of virus clearance is delayed --> The persistence of virus induces an excessive T/B cells and cytokine response --> The persistence of virus induces an excessive T/B cells and cytokine response that causes immunopathological diseases The post-covid 19 syndrome The term “long COVID” is commonly used to describe signs and symptoms that continue or develop after acute COVID-19, that include persistent fatigue, breathlessness, brain fog, and depression could debilitate many millions of people globally. Very little is still known about the condition Potential mechanisms contributing to the pathophysiology of post-acute COVID-19 include: virus-specific pathophysiologic changes immunologic aberrations and inflammatory damage in response to the acute infection expected sequelae of post-critical illness Other Coronaviruses: the MERS Emerging Corona virus diseases MERS (Middle East Respiratory Syndrome) Initially reported in September 2012 A beta-CoV isolated from a patient who died of pneumonia Very low human-human transmission (so far except for nosocomial infection) The emergence of MERS-CoV from dromedary camels Is facilitated by the presence of a similar receptor DPP4 (CD26) Animal models for COVID-19 The main issue to the infection of mouse cells with SARS-CoV-2 is the lack of appropriate receptors to initiate viral infection SARS-CoV-2 uses the cellular surface protein angiotensin-converting enzyme 2 (ACE2) to bind and enter cells mouse ACE2 does not effectively bind the viral spike protein Two strategies have been developed to solve this problem: Turin Pagina 49 Two strategies have been developed to solve this problem: Virus adaptation to mouse ACE-2 Expression of human ACE-2 in genetically modified mice Mouse: Young inbred mice (BALB/c, C57BL6, 129S) support viral replication but fail to show clinical signs of disease Older inbred mice (BALB/c), knockout mice (STAT 1−/−, Rag 1−/−, CD1−/−, Beige) and transgenic mice (K18-hACE2, A70-hACE2) develop generalized illness, robust viral growth and pronounced lung pathology consistent with pneumonia and acute lung injury. The K18-hACE2 transgenic mice develop central nervous system disease, which is not a feature in humans. Inbred mice are not naturally susceptible to infection Transduced mice (Ad5-hDPP4) develop clinical signs and support replication of virus with interstitial pneumonia and viral antigen found in the lungs Transgenic mice (hCD26/DPP4) develop robust respiratory and generalized illness with high viral titers and extensive inflammation in the lungs. Lethality was also observed in this model Virus adaptation to mouse ACE2 (modification of spike protein to increase the binding to ACE2) Background: the population of SARS-COV-2 is not homogeneous but it is made by a swarm of quasispecies, containing mutation in several proteins including the Spike protein By sequential infecting passages of mouse lung tissues with SARSCOV-2 it is possible to select variants that are pathogenic for mouse Useful to study pathogenesis, therapy (antiviral molecules), vaccines Expression of human ACE2 in genetically modified mice Background: mice are modified to express human ACE-2 There are three models (January 2021) different in the promoter: Krt8 promoter for epithelial cells Universal promoter Endogenous mouse Ace2 promoter ACE-2 All the genetically modified mice are sensible to SARS, but some develop encephalitis Several differences resulting in a pathogenic range of mild to lethal disease Animals that are probably not suitable for in vivo models: Pigs: In silico data suggested that swine ACE2 should bind the spike protein of SARS-CoV-2 In vivo data indicate that pig is not susceptible to infection with SARS-CoV-2 (no clinical signs and no clear evidence of virus replication Pigs can be sensitive to other SARS-CoV and beta bat coronavirus, causing swine acute diarrhoea syndrome coronavirus (SADS-CoV) Chicken and ducks In silico data suggested that chicken is potentially susceptible to SARS-CoV2 In vivo data indicate that neither chicken or ducks are susceptible to infection. Non Human primates Rhesus macaques, cynomolgus macaques, African green monkeys and common marmosets are susceptible to infection Clinical signs, viral replication and pathology depend on the species Rhesus macaques develop a transient infection with moderate viral replication and pathology in the lung Common marmosets have a more severe response to the virus with higher viral titres and severe pathology in the lungs Lethality is also observed in this mode Hamster and Ferret Clinical illness (measured by a decrease in activity on the exercise wheel) is accompanied by viral replication and pronounced histopathological changes such as inflammation, pneumonitis and consolidation in the lungs Hamsters do not support replication Clinical illness (fever and sneezing), is accompanied by viral replication and histologic changes in the lungs Ferrets do not support replication Turin Pagina 50