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Viral Pathogenesis

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Viral Pathogenesis

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    1. Samira Mubareka, MD Dept. Microbiology and Division of Infectious Diseases Sunnybrook HSC and Research Institute samira.mubareka@sunnybrook.ca

    2. Mandell Field’s Virology Clinical Virology (Hayden) Red Book TWIV: This Week In Virology (iTunes) http://www.twiv.tv/ Persiflager’s Infectious Diseases Puscast (iTunes)

    4. Advances in Virology Reverse genetics

    5. Advances in Virology New platforms: Mass spec High-throughput screening Confocal and immunofluorescence microscopy Flow cytommetry Tissue culture, VLPs Sequence databases, bioinformatics RNAi Genomics and novel animal models

    7. Fields’ (Skip Virgin): Sequential: more readily correlated with disease Stochastic events and bottlenecks Viral determinants vs. host selection Integrated effects of host genetic variations Immunogenetics Other considerations: Tissue tropism Viral inoculum (effect on incubation period & outcome; eg. ebola) Route of transmission Socioeconomic factors/poverty

    10. Figure 1. Zaire ebolavirus. (A) Transmission electron micrograph of virions. (B) Transmission electron micrograph of viral nucleocapsids in a cytoplasmic inclusion body within an infected hepatocyte. (C) Scanning electron micrograph of virions budding from the surface of an infected primary human umbilical-vein endothelial cell. (D) Arrangement of the seven filovirus genes along the single-stranded negative-sense RNA genome; IR=intergenic regions; GP=glycoprotein; NP=nucleoprotein; VP=viral protein; L=large protein (RNA-dependent RNA polymerase). (E) Structure of a filovirus virion. A-C reproduced with permission from T Geisbert (US Army Medical Research Institute of Infectious Diseases).Figure 1. Zaire ebolavirus. (A) Transmission electron micrograph of virions. (B) Transmission electron micrograph of viral nucleocapsids in a cytoplasmic inclusion body within an infected hepatocyte. (C) Scanning electron micrograph of virions budding from the surface of an infected primary human umbilical-vein endothelial cell. (D) Arrangement of the seven filovirus genes along the single-stranded negative-sense RNA genome; IR=intergenic regions; GP=glycoprotein; NP=nucleoprotein; VP=viral protein; L=large protein (RNA-dependent RNA polymerase). (E) Structure of a filovirus virion. A-C reproduced with permission from T Geisbert (US Army Medical Research Institute of Infectious Diseases).

    11. Extensive liver, spleen, adrenal, gonadal necrosis without infiltration: Hepatcellular necrosis with ghostlike cells Intracytoplasmic inclusion bodies Tissue damage correlates with viral Ag and NA Elevated LFTs; coagulation dysfunction?hemorrhage (GI bleeding) Adrenal involvement?hypotension

    12. Lymphoid depletion in lymphatic tissues with minimal infiltrate; lymphopenia (T cells and NKs) TNF-related apoptosis-inducing ligand (TRAIL) Fas death receptor pathways Pro-apoptotic NO Proinflammatory cytokines IL-6/8/10/12, IP-10, MCP-1, RANTES, TNF-a, reactive oxygen and nitrogen spp.?increased vascular permeability IP-10=IFN-inducible protein MCP-1=macrophage chemoattractant protein RANTES=regulated on activation normal T-cell expressed and secreted *IP-10=IFN-inducible protein MCP-1=macrophage chemoattractant protein RANTES=regulated on activation normal T-cell expressed and secreted *

    13. IFN-antagonism: VP35 (block IRF-3) & VP24 GP, secreted (putative): vascular injury & hemorrhagic diathesis Endovascular lesions not consistently seen in humans; intact vascular endothelium in animal models, though increased permeability noted Persistence: viral RNA identified in semen up to 100d post-infection

    14. Figure 3. A model of the pathogenesis of filoviral haemorrhagic fever, based on studies of Zaire ebolavirus infection. Infection causes lysis of monocytes/macrophages, dendritic cells, and hepatocytes and suppresses innate immune responses in these cells, aiding further dissemination. Direct injury to infected cells is accompanied by indirect effects that are mediated by proinflammatory and anti-inflammatory effector molecules, including interleukin 1ß (IL1ß), interleukin 6 (IL6), TNFa, interleukin 10 (IL10), and type I interferons (IFN). The severe illness results from the combined effects of widespread viral cytolysis and massive release of proinflammatory mediators. Proinflammatory cytokines and chemokines are also produced by activated endothelial cells, resulting in a feedback loop to the monocytes/macrophages. Lymphocyte apoptosis is also apparently brought about through effects of proinflammatory mediators; it may contribute to immunosuppression by weakening adaptive immune responses. The cell-surface expression of tissue factor by virus-infected monocytes/macrophages induces disseminated intravascular coagulation (DIC). MCP=monocyte chemoattractant protein; IL1RA=interleukin-1 receptor antagonist.Figure 3. A model of the pathogenesis of filoviral haemorrhagic fever, based on studies of Zaire ebolavirus infection. Infection causes lysis of monocytes/macrophages, dendritic cells, and hepatocytes and suppresses innate immune responses in these cells, aiding further dissemination. Direct injury to infected cells is accompanied by indirect effects that are mediated by proinflammatory and anti-inflammatory effector molecules, including interleukin 1ß (IL1ß), interleukin 6 (IL6), TNFa, interleukin 10 (IL10), and type I interferons (IFN). The severe illness results from the combined effects of widespread viral cytolysis and massive release of proinflammatory mediators. Proinflammatory cytokines and chemokines are also produced by activated endothelial cells, resulting in a feedback loop to the monocytes/macrophages. Lymphocyte apoptosis is also apparently brought about through effects of proinflammatory mediators; it may contribute to immunosuppression by weakening adaptive immune responses. The cell-surface expression of tissue factor by virus-infected monocytes/macrophages induces disseminated intravascular coagulation (DIC). MCP=monocyte chemoattractant protein; IL1RA=interleukin-1 receptor antagonist.

    19. Schematic diagram of the multiple functions of NS1 within infected cells. (a) Pre-transcriptional limitation of IFN-ß induction. (b) Inhibition of the antiviral properties of PKR and OAS/RNase L. (c) Post-transcriptional block to processing and nuclear export of all cellular mRNAs. (d) Enhancement of viral mRNA translation. (e) Activation of PI3K. Interactions with unknown consequences and/or localizations are detailed in the lower box.Schematic diagram of the multiple functions of NS1 within infected cells. (a) Pre-transcriptional limitation of IFN-ß induction. (b) Inhibition of the antiviral properties of PKR and OAS/RNase L. (c) Post-transcriptional block to processing and nuclear export of all cellular mRNAs. (d) Enhancement of viral mRNA translation. (e) Activation of PI3K. Interactions with unknown consequences and/or localizations are detailed in the lower box.

    24. Pathogenesis Bites: local replication in striated muscle Other routes: aerosol, parenteral, Tx, oral, vertical Viral GP binds host cell receptors in NMJ: post-synaptic nicotinic ACH receptor; hence fox is susceptible & possums are resistant CD56 Neurotrophin receptor p75 Retrograde motor neuron transport (50-100mm/d) via viral P protein-dynein interaction?transynaptic transfer of nucleocapsid (viral G protein)

    25. Pathogenesis Diffuse centrifugal spread (NOT viremia) Viral shedding in lacrimal & salivary glands Immune response: none until symptom onset Neutralizing antibody CD4+ T cells and B cells central; viral N protein epitopes

    26. Paralytic rabies Vampire bat; previously vaccinatedParalytic rabies Vampire bat; previously vaccinated

    30. Fig. 1. Hypothetical scheme of poliovirus pathogenesis based on experimental findings in humans, monkeys, chimpanzees, and CD155 transgenic mice. Ingested virus initially replicates in the oropharyngeal and intestinal mucosa. Virus replication at these sites reaches the blood through the lymph nodes, resulting in a primary viremia. Invasion of virus into the central nervous system may occur either directly from the blood, or by retrograde axonal transport when virus enters the neuromuscular junction. It is believed that invasion of the brain or spinal cord must be preceded by viral multiplication in extraneural tissues, which leads to a sustained viremia. These extraneural tissues may include skeletal muscle and brown fat. Virus is spread most frequently by the fecal–oral route. Shedding of virus from the nasopharynx may lead to transmission of infection by the respiratory route, which occurs in developed countries with high standards of sanitation. Fig. 1. Hypothetical scheme of poliovirus pathogenesis based on experimental findings in humans, monkeys, chimpanzees, and CD155 transgenic mice. Ingested virus initially replicates in the oropharyngeal and intestinal mucosa. Virus replication at these sites reaches the blood through the lymph nodes, resulting in a primary viremia. Invasion of virus into the central nervous system may occur either directly from the blood, or by retrograde axonal transport when virus enters the neuromuscular junction. It is believed that invasion of the brain or spinal cord must be preceded by viral multiplication in extraneural tissues, which leads to a sustained viremia. These extraneural tissues may include skeletal muscle and brown fat. Virus is spread most frequently by the fecal–oral route. Shedding of virus from the nasopharynx may lead to transmission of infection by the respiratory route, which occurs in developed countries with high standards of sanitation.

    31. Fig. 2. Immunofluorescence analysis of CD155 expression in human tonsil (A), Peyer's patches (B), appendix (C), and rectum (D). Frozen tissue sections were stained with monoclonal antibody D171 specific for CD155 (red). To localize crypt epithelium, tonsil section was double-labeled with monoclonal antibody specific for cytokeratin (green; A). To visualize goblet cells, Peyer's patch section was double-labeled with lectin, Ulex europaeus agglutinin1 (green; B). Images were captured with use of 20X objective lens. CE, crypt epithelium; G, germinal center; I, interfollicular region; L, lumen. White arrow in panel B indicates follicle associated epithelium overlying dome. No background staining was detected with mouse IgG used as control (data not shown). Identical staining was observed with a second CD155-specific monoclonal antibody, clone 18. Nuclei were stained with 4,6-diamidino-2 phenylindole (blue). From Iwasaki et al., 2002; with permission of the authors. Fig. 4. A causative link between site of muscle inoculation and replication within the spinal cord. A 6-week old CD155tg mouse was injected with 106 plaque forming units of pliovirus type 1 (Mahoney) into the left gastrocnemius muscle. Thirty-three hours post infection, at the first sign of muscle weakness, the animal was sacrificed, and frozen sections of the lumbar spinal cord were prepared. Poliovirus was detected with rabbit polyclonal antiserum against PV1 capsid proteins and visualized with goat anti-rabbit Cy3 conjugated secondary antibodies. Virus is found exclusively within motor neurons of the left anterior horn. Incidentally it is the same motor neurons that innervate the left (injected) hind leg, arguing in favor of a direct transport mechanism via the sciatic nerve. Note: the outline of the spinal cord has been retraced for viualization purposes. Fig. 2. Immunofluorescence analysis of CD155 expression in human tonsil (A), Peyer's patches (B), appendix (C), and rectum (D). Frozen tissue sections were stained with monoclonal antibody D171 specific for CD155 (red). To localize crypt epithelium, tonsil section was double-labeled with monoclonal antibody specific for cytokeratin (green; A). To visualize goblet cells, Peyer's patch section was double-labeled with lectin, Ulex europaeus agglutinin1 (green; B). Images were captured with use of 20X objective lens. CE, crypt epithelium; G, germinal center; I, interfollicular region; L, lumen. White arrow in panel B indicates follicle associated epithelium overlying dome. No background staining was detected with mouse IgG used as control (data not shown). Identical staining was observed with a second CD155-specific monoclonal antibody, clone 18. Nuclei were stained with 4,6-diamidino-2 phenylindole (blue). From Iwasaki et al., 2002; with permission of the authors. Fig. 4. A causative link between site of muscle inoculation and replication within the spinal cord. A 6-week old CD155tg mouse was injected with 106 plaque forming units of pliovirus type 1 (Mahoney) into the left gastrocnemius muscle. Thirty-three hours post infection, at the first sign of muscle weakness, the animal was sacrificed, and frozen sections of the lumbar spinal cord were prepared. Poliovirus was detected with rabbit polyclonal antiserum against PV1 capsid proteins and visualized with goat anti-rabbit Cy3 conjugated secondary antibodies. Virus is found exclusively within motor neurons of the left anterior horn. Incidentally it is the same motor neurons that innervate the left (injected) hind leg, arguing in favor of a direct transport mechanism via the sciatic nerve. Note: the outline of the spinal cord has been retraced for viualization purposes.

    32. Fig. 5. Proposed model of CNS invasion by poliovirus. (1) Virions near or at the neuromuscular junction are taken up at the presynaptic membrane of a motor axon by CD155 mediated endocytosis. This may or may nor be preceded by virus replication in adjacent muscles. Muscle injury or inflammatory responses due to the PV infection may facilitate this process by upregulation of CD155 expression. The virus/receptor complex by interaction with Tctex-1 is targeted to the microtubular network of the axon. (2) The intact 160S virions complexed to the dynein motor by virtue of CD155 interaction with Tctex-1 are transported along microtubules by fast retrograde axonal transport. (3) Arriving at the motor neuron's cell body, the change from axoplasm microenvironment to cytoplasm environment may triggers virus uncoating and escape of viral genomic RNA from the endocytic vesicle. Virus replication ensues, thereby killing the motor neuron. Paralysis of the muscle fiber formerly innervated by this motor neurons follows. Spread to neighboring spinal neurons may occur laterally and kill those neurons directly and independently of retrograde axonal transport, or by trans-neuronal spread between neuron, in a retrograde transport dependent fashion (from Mueller et al., 2002; with permission of the authors).Fig. 5. Proposed model of CNS invasion by poliovirus. (1) Virions near or at the neuromuscular junction are taken up at the presynaptic membrane of a motor axon by CD155 mediated endocytosis. This may or may nor be preceded by virus replication in adjacent muscles. Muscle injury or inflammatory responses due to the PV infection may facilitate this process by upregulation of CD155 expression. The virus/receptor complex by interaction with Tctex-1 is targeted to the microtubular network of the axon. (2) The intact 160S virions complexed to the dynein motor by virtue of CD155 interaction with Tctex-1 are transported along microtubules by fast retrograde axonal transport. (3) Arriving at the motor neuron's cell body, the change from axoplasm microenvironment to cytoplasm environment may triggers virus uncoating and escape of viral genomic RNA from the endocytic vesicle. Virus replication ensues, thereby killing the motor neuron. Paralysis of the muscle fiber formerly innervated by this motor neurons follows. Spread to neighboring spinal neurons may occur laterally and kill those neurons directly and independently of retrograde axonal transport, or by trans-neuronal spread between neuron, in a retrograde transport dependent fashion (from Mueller et al., 2002; with permission of the authors).

    33. Innate immunity (type 1 IFNs) Humoral immunity (not sterilizing) Persistent enteroviral infections in Ig-deficient states Neutralizing epitope in capsid protein VP1 and 2C protease contain T cell epitopes

    37. TLR3-dependent and RIG-I-dependent signaling to the innate immune response: specific cleavage of signaling adapters by HCV NS3–4A protease. Engagement of endosome-associated TLR3 by dsRNA recruits the TRIF adaptor, resulting in the activation of TBK-1 and IKK kinases that phosphorylate IRF-3 and IRF-7. TRIF also signals NF-B activation via the IKK/ complex, which phosphorylates IB, resulting in the release of the NF-B DNA-binding subunits. The RIG-1 pathway activates NF-B and IRF-3/7, following the recognition of incoming viral ribonucleoprotein complexes. RIG-I, through C-terminal RNA helicase domain, interacts with viral dsRNA and through the CARD domains interacts with the MAVS/IPS/VISA/Cardif adaptor. MAVS contains a transmembrane domain (TM) that localizes this adaptor to the mitochondria. NS3–4A protease activity of HCV cleaves the C-terminal domain of MAVS at Cys-508, disrupts RIG-I signaling to IFN activation and establishes persistent infection. NS3–4A also targets the TRIF adaptor molecule in the TLR3-dependent pathway (modified from Hiscott et al., 2006).TLR3-dependent and RIG-I-dependent signaling to the innate immune response: specific cleavage of signaling adapters by HCV NS3–4A protease. Engagement of endosome-associated TLR3 by dsRNA recruits the TRIF adaptor, resulting in the activation of TBK-1 and IKK kinases that phosphorylate IRF-3 and IRF-7. TRIF also signals NF-B activation via the IKK/ complex, which phosphorylates IB, resulting in the release of the NF-B DNA-binding subunits. The RIG-1 pathway activates NF-B and IRF-3/7, following the recognition of incoming viral ribonucleoprotein complexes. RIG-I, through C-terminal RNA helicase domain, interacts with viral dsRNA and through the CARD domains interacts with the MAVS/IPS/VISA/Cardif adaptor. MAVS contains a transmembrane domain (TM) that localizes this adaptor to the mitochondria. NS3–4A protease activity of HCV cleaves the C-terminal domain of MAVS at Cys-508, disrupts RIG-I signaling to IFN activation and establishes persistent infection. NS3–4A also targets the TRIF adaptor molecule in the TLR3-dependent pathway (modified from Hiscott et al., 2006).

    38. Innate immune response. Studies of experimentally hepatitis C virus (HCV)-infected chimpanzees show that intrahepatic type I interferon (IFN) responses do not correlate with the outcome of infection, indicating that HCV is not sensitive to type I IFN responses in vivo (for further details, see main text). In vitro studies show that natural killer (NK) cells of healthy individuals can be inhibited by high concentrations of the HCV envelope protein 2 (E2) and that NK cells of HCV-infected individuals are altered in their cytokine production and their capacity to activate dendritic cells (DCs) in vitro. b | Adaptive immune response. Viral escape from immune responses through mutations in antibody and T-cell epitopes has been shown for both HCV-infected humans76, 94, 95, 96, 97, 98 and chimpanzees92, 93. Humoral immune responses appear late during infection or not at all, and they do not protect against re-infection9, 11, 12, 13, 85, 86. HCV-specific T cells are less differentiated than virus-specific T cells raised to other pathogens103, and they seem to be impaired in their effector functions55. Potential mechanisms include reduced T-cell priming, with a potentially altered DC function104, 105, 106, 107, and inhibition of macrophage and/or DC and T-cell function through binding of the HCV core protein to the receptor for the complement component C1q (C1qR)99, 100, 101. Furthermore, peripheral CD4+CD25+ T cells (TReg cells)112, 113 and intrahepatic interleukin-10 (IL-10)-producing CD8+ T cells111, which both have regulatory functions, have recently been detected in patients with chronic hepatitis C, and their role in the outcome of infection needs to be further analysed. Finally, despite early and high HCV titres, HCV-specific T cells are not detectable in the liver within 1 month of experimental infection of chimpanzees, which might indicate impaired trafficking to the site of infection15. CXCR, CXC-chemokine receptor; FOXP3, forkhead box P3; NKG2A, NK group 2, member A; TCR, T-cell receptor; TGF-, transforming growth factor-.Innate immune response. Studies of experimentally hepatitis C virus (HCV)-infected chimpanzees show that intrahepatic type I interferon (IFN) responses do not correlate with the outcome of infection, indicating that HCV is not sensitive to type I IFN responses in vivo (for further details, see main text). In vitro studies show that natural killer (NK) cells of healthy individuals can be inhibited by high concentrations of the HCV envelope protein 2 (E2) and that NK cells of HCV-infected individuals are altered in their cytokine production and their capacity to activate dendritic cells (DCs) in vitro. b | Adaptive immune response. Viral escape from immune responses through mutations in antibody and T-cell epitopes has been shown for both HCV-infected humans76, 94, 95, 96, 97, 98 and chimpanzees92, 93. Humoral immune responses appear late during infection or not at all, and they do not protect against re-infection9, 11, 12, 13, 85, 86. HCV-specific T cells are less differentiated than virus-specific T cells raised to other pathogens103, and they seem to be impaired in their effector functions55. Potential mechanisms include reduced T-cell priming, with a potentially altered DC function104, 105, 106, 107, and inhibition of macrophage and/or DC and T-cell function through binding of the HCV core protein to the receptor for the complement component C1q (C1qR)99, 100, 101. Furthermore, peripheral CD4+CD25+ T cells (TReg cells)112, 113 and intrahepatic interleukin-10 (IL-10)-producing CD8+ T cells111, which both have regulatory functions, have recently been detected in patients with chronic hepatitis C, and their role in the outcome of infection needs to be further analysed. Finally, despite early and high HCV titres, HCV-specific T cells are not detectable in the liver within 1 month of experimental infection of chimpanzees, which might indicate impaired trafficking to the site of infection15. CXCR, CXC-chemokine receptor; FOXP3, forkhead box P3; NKG2A, NK group 2, member A; TCR, T-cell receptor; TGF-, transforming growth factor-.

    39. Elevated AST temporally relates to CD8+ cytotoxic activity and fall in VL portion of one classic hepatic lobule.  The portal tract shown in the upper right hand corner is expanded by a chronic inflammatory infiltrate of lymphocytes.  Clusters of lymphocytes and hyperplastic Kupffer cells are scattered throughout the hepatic lobule as well.  There is also focal apoptosis of individual hepatocytes (arrows).  The constellation of findings of chronic inflammation, cell death, and regeneration has been termed by pathologists: "lobular disarray".portion of one classic hepatic lobule.  The portal tract shown in the upper right hand corner is expanded by a chronic inflammatory infiltrate of lymphocytes.  Clusters of lymphocytes and hyperplastic Kupffer cells are scattered throughout the hepatic lobule as well.  There is also focal apoptosis of individual hepatocytes (arrows).  The constellation of findings of chronic inflammation, cell death, and regeneration has been termed by pathologists: "lobular disarray".

    40. extensive fibrosis and progression to macronodular cirrhosis, as evidenced by the large regenerative nodule at the center rightextensive fibrosis and progression to macronodular cirrhosis, as evidenced by the large regenerative nodule at the center right

    42. Lymphoproliferative disorders Mixed cryoglobulinemia (polyclonal IgG & IgM, types II and III respectively) due to clonal B cell expansion Small vessel vasculitis Peripheral neuropathy MPGN NHL *Porphyria cutanea tarda, lichen planus Sjogren’s syndrome Chronic polyarthritis *underluing genetic predisposition*underluing genetic predisposition

    46. ParvoB19ParvoB19

    47. Moluscum contagiosumMoluscum contagiosum

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