Samira Mubareka, MD

Dept. Microbiology and Division of Infectious Diseases Sunnybrook HSC and Research Institute

samira.mubareka@sunnybrook.ca

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)

DNA RNA

AcuteAdenovirusParvovirusPoxvirusHHV6/7

LatencyHSV, VZV, CMVPolyomaviruses

OncogenicEBV, HHV8HBVPapillomaviruses

Arthropod-borne/zoonoticArenavirusesAlphavirusesBunyavirusesFlavivirusesRhabdoviruses

P2P

RetrovirusesHIV I/IIHTLV I/II

RespOrthomyxovirusesParamyxovirusesFiloviruses

GIPicornavirusesCalicivirusesHCV

Reverse genetics

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

Nature Reviews Microbiology 5, 453-463 (June 2007)

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

• Entry via: •contact /MM (incl sexual)•Parenteral• oral (organ infectivity=109 pfu/g)

•Target cells: monos, macrophages, DCs GP binds host cell receptor (DC-SIGN?) hepatocytes, splenocytes, fibroblasts, adrenal cortical cells

• Viremia: 106-108 pfu/ml; direct EM+RNA polymerase

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

dysfunctionhemorrhage (GI bleeding)• Adrenal involvementhypotension

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-α,

reactive oxygen and nitrogen spp.increased vascular permeability

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

Horimoto, T. Nature Microbiology Reviews 2005;3:351-600

Subtyping:16 HA9 NA

A/Toronto/R8557/2009(H1N1)

Virus typeGeographic origin

Virus subtype

Strain #Year

K. Shinya, Nature, 2006;440:435-436

Nasal mucosa

Paranasal sinuses

Bronchus

Bronchiole

Alveolus

Krug, RM, Lamb, RA Orthomyxoviridae: The Viruses and Their Replication 2001 Fields Virology. 4th edition, editors: Knipe DM, Howley PM,. Philadelphia: Lippincott Williams & Wilkins.

NA

Hale, et al. J Gen Virol 2008;89:2359-2376

1918 HA (Ian Wilson,Scripps)Connenello & Palese. Cell Host & Microbe, 2007;2(4):207-209

www.clinical-virology.org/gallery/images/em/rabies2.jpg

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 interactiontransynaptic transfer of nucleocapsid (viral G protein)

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

Furious rabies (brainstem, CN, limbic)

Paralytic rabies(medulla, spinal cord)

PV capsid interacts with host cell receptor CD155

Virology. 2006 Jan 5;344(1):9-16

100 TCID50infection

Resistance to acid

Fecal shedding=6 weeks

Replication in tonsils/Peyer’s patches (1-3d)

Deep cervical/mesentericnodes with minor viremia

RES (LN/BM/liver/speen)With major viremia

CNS invasion(across BBB & retrograde)

Cutter’s vaccine incident & provocation poliomyelitis

Virus Res. 2005 Aug;111(2):175-93

Immunofluorescence analysis of CD155

tonsil Peyer’s

appendix rectum

PV1 capsid proteins in L ant horn of spinal cord 33h post- gastoc

Infection of CD155tg mouse

Virus Res. 2005 Aug;111(2):175-93

PV incorporated into endosomes at NMJ

after viremia and muscle seeding or

GIvagal nerve

Neurovirulence

• Sabin strains• 5’ UTR mutations destabilize secondary structure• VP 1,3,4 (PV1)• VP 1 (PV2)• VP 1,3 (PV3)

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

Nature Reviews Microbiology 5, 453-463 (June 2007)

Viral E2 binds putative host cell receptor CD81

Primary replication in liver within 2d of infectionviremia

vRNA isolated from PBMCs, LN, spleen, brain, adrenals, BM, pancreas, thyroid

Quasispecies result of stochastic events

http://www.nature.com/onc/journal/v25/n51/fig_tab/1209941f4.html

K/O in HCV-permissive cells

K/O

in

HC

V-

per

mis

siv

e ce

lls

NS5 & E2 interfere in JAK/STAT

pathway

Nature Reviews Immunology 5, 215-229 (March 2005)

T cells key: hosts who have robust CD4+ response clear virus

Elevated AST temporally relates to CD8+ cytotoxic activity and fall in VL

Inflammation & Fibrosis Periportal lymphocytic

infiltration, hepatocellular necrosis

Mediated by NK and T cells as well as hepatic stellate cells (fibrogenic, synthesis of collagen)

Steatosis Core protein & genotype 3 are

key viral determinants Block assembly & release of

VLDL, interfere with PPARα Mitochondrial injury

Hepatocellular carcinoma HCV does not integrate into the host genome HCC due to ongoing fibrosis, oxidative stress, etc… Core protein, S3, NS5A/B may be oncogenic by

modulating cell proliferation

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