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Virology (MD PhD) (Subject) / Trkola (Lesson)

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• Fulfilling of postulates not always possible 1. Hepatitis C virus (HCV) 2. HHV-8 3. Human papilloma virus  --> Definition through molecular diagnostics 
• Influenza A structure - 3 surface proteins: HA, NA, M2 Ratio HA:NA = 5:1 - Hemagglutinin: Attachment und Fusion - Neuraminidase: Spaltet Glykosidverbindung der Sialinsäure. Notwendig damit Virus Zelle verlassen kann.  - Capsid/nucleocapsid is elongated with helical symmetry. - Segmented ssRNA(-) linear genome, encapsidated by nucleoprotein (NP). - Contains 8 RNA segments coding for 10 - 11 proteins. 
• Giant viruses - Contain proof reading enzymes - Contain metabolizing enzymes - Contain ubiquitin pathway components - Contain tRNAs 
• Enveloped viruses with high lipophily - HCV - Herpes viruses - Retroviruses (HIV)
• Enveloped viruses with low lipophily - HBV - Poxvirus
• Non-enveloped viruses with high hydrophily - Picornaviruses (Enterovirus, Coxsackie viruses, Hepatitis A virus) - Parvoviruses 
• Non-enveloped viruses with low hydrophily - Adenoviruses - Noroviruses (most common cause of viral gastroenteritis in all ages) - Rotaviruses (most common cause of severe diarrhoea among infants)
• Norovirus - Family Caliciviridae  - positive strand, ssRNA - genomic RNA is infectious! - naked - Not all disinfection strategies work! - High environmental resistance (-20°bis +60°C) - diverse genotypes --> no longlasting immunity  - No therapy - in Switzerland ca. 400’000 people are infected every year 
• Norovirus Symptoms - Heavy vomiting, diarrhea, abdominal cramps, elevated temperature, muscle cramps, headache - Outbreaks in hospitals are huge problem - Incubation time: 12 - 48 h - Length of illness: 12 – 72 h
• Norovirus Transmission -  fecal-oral 60%  -  Vomit: droplets -  Stool: contact - Highly infectious: 10 - 100 Viruses establish infection - High Virus shedding for 48h (107 virus particles per ml stool!) - Virus shedding continues up to 2 weeks after cessation of symptoms! 
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• Capsid forms - helical: Influenza, Measles, VSV (Vesicular Stomatitis Virus) - icosahedral: Hepatitis B virus - rod: Baculovirus - conic: HIV
• Family: Orthomyxoviridae Genus: - Influenza virus A: birds, swine, human, seal, various mammals 16 HA subtypes (H1-16), 9 NA subtypes (N1-9) - Influenza virus B: human, no subtypes - Influenza virus C: swine, human; no subtypes - Thogotovirus: ticks - Isavirus: infectious salmon anemia virus (ISAV)   
• Family: Filoviridae Genus:- Cuevavirus - Marburg Virus - Ebolavirus (-) RNA, enveloped 
• Human DNA viruses Class I (ds DNA)- Adenoviridae- Herpesviridae- Poxviridae Class II (ss DNA)- Parvoviridae- Anelloviridiae Class VII (ds DNA reverse transcribing viruses)- Hepadnaviridae
• Human RNA viruses Class III (ds RNA)- Reoviridae- Picobirnaviridae Class IV (+ ssRNA)- Picornaviridae- Astroviridae  Class V (- ss RNA)- Orthomyxoviridae- Arenaviridae Class VI (ss RNA reverse transcribing viruses)- Retroviridae
• Attachment: Enveloped viruses Use viral encoded envelope glycoproteins to bind to cellular receptor 
• HIV-1 structure - Envelope spike: Trimer bestehend aus drei gp120/gp41 Heterodimeren  - Enzyme (RT, IN, PR) - Matrix (p17) - genetic material (+ssRNA) - capsid (p24) - lipid-bilayer (host cell derived) 
• Attachment: Non-enveloped viruses - Capsid attaches directly to cell receptor - Adsorption occurs through structural properties of capsid (canyons oder loops) eg Picornavirus (Rhinovirus) - Adsorption through fibers eg Adenovirus 
• Non-specific attachment factors - Mediate concentration of virus particle on host cell surface - Improve entry efficiency but are not mandatory for entry! –  Attachment interactions are often unspecific • Charge-charge interactions, heparan sulfates, carbohydrates – Specific interactions of attachment factors • eg: ICAM-1/LFA-1 binding improves HIV attachment and entry 
• Entry Receptors - cellular receptors  - Induction of conformational changes in virus particle - Induction of signaling pathways - Induction of membrane fusion - Induction of internalization by endocytosis  - influence cell and host tropism  HIV requires 2 receptors for entry (CD4 and CCR5 cytokine coreceptor bind to the gp120/41 trimer)
• HIV receptors 1. CD4 2. CCR5- GPCR- most important coreceptor of HIV - R5 entry receptor- homocygotic mutation protects from R5 virus infection  3. CXCR4- GPCR- X4 entry receptor
• HIV cellular tropism: in vivo characteristics - Transmitted viruses are predominantly R5  - At later stages of the disease R5X4 and X4 viruses can emerge (estimated <50%) 
• Influenza receptor - is sialic acid - Sialic acid covalently linked to cell surface glycoproteins and glycolipids  Influenza A and B- Sialic acid (N-acetylneuraminic acid) is linked to galactose - Virus tropism is determined by linkage: avian viruses α(2, 3), human viruses α(2, 6) Influenza C- 9-O-acetyl-N-Neuraminic acid  - Interactions between hemagglutinin (HA) and sialic acid are of low affinity - multiple recptor interactions required for entry 
• Sialic acid Glycosidic linkage: α2-6 (Pharynx)- human: yes- pigs: yes- birds: no Glycosidic linkage: α2-3 (Lungs)- birds: yes- pigs: yes- humans: low Hemagglutinin determines host spectrum of influenza type
• Measles virus entry - utilizes different receptors during the infectious cycle  1. Measles virus enters the airways and infects macrophages and dendritic cells.SLAM (CD150) is the receptor for entry into macrophages and DC  2. The infected cells spread rapidly to the primary lymphatic organs thymus and spleen  3. The infection spreads to the epithelium of the airways by ways of infected immune cellsNectin-4 (adherens junction protein) is the receptor for entry into epithelial cells  4. Progeny viral particles are released in the trachea and expulsed by coughing and sneezing (aerosol-transmitted)
• Viral fusion proteins Class I- alpha-helical trimer - fusion peptide- "six helix bundle” eg Orthomxyoviridae (Influenza HA), Retroviridae (HIV) Class II- beta-Sheet - dimer- fusion loopeg E Dimer, Flaviviruses  Class III- central α-helical core trimer - Fusions domain has 2 fusion loops on tip of β-sheeteg Herpesviridae, Rhabdoviridae (VSV)
• Proof-reading capacity - RNA viruses (RNA replication, reverse transcription): One error per 10^3-10^6 bases added- Quasispecies - DNA viruses (viral and cellular DNA-Polymerases): One error per 10^7-10^10 bases added - DNA polymerases are 1000x better
• Recombination Can occur during superinfection of a host cell by related viruses - Retroviruses: Recombination by template switch  - Recombination by cleavage and ligation 
• Determining genotype can be important HIV-1: Genotypinging of RT and protease genes necessary to determine drug resistance  HCV: Antiviral therapy has different efficacy on different genotypes (eg Interferon treatment of genotypes 1 and 4 less effective as for genotypes 2 and 3) 
• Reassortment - Reassortment can occur during superinfection of a host cell with viruses that have a segmented genome (influenza, rotaviruses) - Complete genome segments are swapped and „new“ assorted viruses evolve Influenza: Antigenic Shift = Reassortment Antigenic Drift = Mutation 
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• Virus Tropism Factors which determine tropism 1. Presence of entry receptor (cell is susceptible)  2. Presence of all cellular functions, required for virus replication (cell is permissive) 3. Factors of innate defense (eg IFN system) and intrinsic host specific restriction factors (eg HIV: TRIM5α, Tetherin und APOBEC3G) are not present or can be shut down by virus 
• Virus induced cell death - Virus induces apoptosis: HIV, Adenovirus - Virus counteracts apoptosis: Adenovirus - Virus induces necrosis: Ebola 
• Epidemiologic characteristics or respiratory viruses - low relative humidities (as produced by indoor heating) and cold temperatures as features of winter favor virus spread Frequency:- Rhinovirus- Coronavirus- Influenza- Adenovirus- RSV
• NK cells - Both contact-dependent and soluble signals (IFN-α, IL-2, IL-12) induce activation of NK cells - receive signals from activating (NKG2D recognizes stress-induced ligands on cell surface) and inhibitory (MHC I) receptors.  - Release of soluble factors which inhibit viral replication (chemokines, IFN-γ) - Direct killing - Antibody dependent cellular cytoxicity (have Fc receptor for antibody tail)
• Dendritic cells - Pattern recognition - Release of soluble factors - Antigen presentation  Interaction of dendritic cells with immune cells is not always of advantage: - Infection by virus (eg HIV, cis-infection) - Trans infection of HIV 
• Macrophages - Pattern recognition - Antigen presentation - Phagocytes - ADCC Subversion by viruses- Direct infection - Long-lived reservoir - Dissemination in body 
• Dengue virus (DENV) - Flavivirus, ss RNA(+) - mosquito-borne virus infection (Aedes) - estimated 50 to 100 million infections per year - four serotypes (sequence variation of 30 to 35%) - Antibody-dependent enhancement  - infection with one serotype does not provide protection from infection with the other serotypes - secondary or sequential infections are and more likely associated with serious complications (dengue haemorrhagic fever) 
• HIV: viral enzymes - Reverse transcriptase - Integrase - Protease (needed to exit cell) - distributed inside the matrix (some inside capsid)
• HIV: Accessory proteins - vpu, vpr (viral protein u, r) - tat (trans-activator of transcription): essential - vif (viral infectivity factor) - rev (regulator of virion): essential, allows transport of unspliced mRNA out of the nucleus - nef (negative regulatory factor) - distributed inside matrix
• HIV: Genome ssRNA - positive strand - 2 copies
• HIV: Replication - Baltimore Class VI (+) RNA -- (reverse transcriptase) --> (-) DNA --> dsDNA --> Integration, Transcription, Replication
• HIV-1 replication complex - Two genomic viral RNAs are packaged in the virion. - The two viral RNAs from a dimeric complex - The viral RNA is coated by about 1500 nucleocapsid (NC) protein molecules in the viral core particle - tRNALys3 serves as primer when annealed to the viral PBS (primer binding site) RNA - The primer tRNALys3 is bound to the reverse transcriptase (RT) 
• HIV: preintegration complex - contains LEDGF/p75, which allows nuclear import - promotes chromatin association 
• ESCRT machinery - used for HIV to egress
• APOBEC3G - intrinsic antiviral factor - Causes excessive cytidine (C) to uridine (U) editing of negative sense reverse transcripts in newly infected cells. - APOBEC3G/F impedes the translocation of reverse transcriptase along template RNA.  - Vif counteracts APOBEC3G/F by targeting it for ubiquitylation
• Tetherin/BST-2 - intrinsic antiviral factor - blocks HIV release  - expression induced by IFN  - Vpu counteracts action of Tetherin 
• TRIM5 - intrinsic antiviral factor - capsid-targeting restriction factor  1. TRIM5 engages with the viral capsid, inducing premature uncoating (prevents reverse transcription) 2. Functions as PPR and activates cellular innate immune responses 
• Viral setpoint - Level of viremia, which is reached after acute phase - determines disease progression  - Steady state level = viral set point Determinants:- Host factors: genetic factors of the host (eg HLA type, CCR5 genotype), innate and adaptive immunity - Viral factors: replicative capacity, HIV subtype, coreceptor usage, genetic diversity, drug resistance 
• Retroelements - endogenous components of eukaryotic genomes that are able to amplify to new locations in the genome through an RNA intermediate - require reverse transcription for replicative transposition  - Retroelements have in part retroviral origin (eg Integrated forms of infectious retroviruses, proviral genomes which were integrated upon infection) Insertion of Alu, L1 or SVA in important genes causes diseases:- Hemophilia - Breast cancer -cystic fibrosis
• Alphaherpesvirinae - HSV-1, HSV-2, VZV - short replication cycle, rapid spread in culture 

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