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WEEK 27: Viral Infections: What are the types of hepatitis viruses? ✦ Hepatitis A (picornavirus) ✦ Hepatitis B (hepadnavirus) ✦ Hepatitis C (flaviviridae) ✦ Hepatitis D (deltavirus) ✦ Hepatitis E (hepevirus) When is HAV stable at? ✦ at low ph Where is inactivation of HAV rapid with? ✦ autoclaving ✦ ...

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WEEK 27: Viral Infections: 1. What are the types of hepatitis viruses? ✦ Hepatitis A (picornavirus) ✦ Hepatitis B (hepadnavirus) ✦ Hepatitis C (flaviviridae) ✦ Hepatitis D (deltavirus) ✦ Hepatitis E (hepevirus) 2. When is HAV stable at? ✦ at low ph 3. Where is inactivation of HAV rapid with? ✦ autoclaving ✦ UV radiation ✦ chlorine-based detergents. 4. Who are at high risk of contracting hepatitis D? ✦ chronically infected with HBV 5. How does virus get entry during HBV replication cycle. ✦ Nucleoporin 153 ✦ 6. Describe HBV replicative cycle? ✦ RNA dependent DNA Polymerase 7. 3 domains ✦ Terminal protein, RT and RNAse H ✦ Terminal domain = responsible for priming (-)- strand DNA synthesis. ✦ RT and DNAase H = required for DNA synthesis. HBV Polymerase acts a template specific.

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Aelisha Shakya

8. What is the current main target of anti-HBV chemotherapy? ✦ HBV Polymerase 9. What are the similarities between HIV-1 RT and HBV Polymerase? ✦ Both perform a key function for the virus and do not have a mammalian equivalent. ✦ The agents that operate against HIV-1 RT do show some activity against HBV RT. ✦ Both uses Mg 2+ions to facilitate the chemistry ✦ NRTIs for both required phosphorylation by the host cell’s kinase enzymes ○ phosphorylation occurs inside the cell 10.What are NRTIs? ✦ are analogues of endogenous 2’-deoxy-nucleosides/tides and ○ must be phosphorylated in order to be active. ✦ NRTIs are hydrophillic anad have limited permeability 11.Where is the high level of 2’- deoxycytidine kinase found? ✦ The relatively high levels of 2’- deoxycytidine kinase found in liver cells (lamivudine, 3-TC) 12.Which nucleosides show a high degree of specificity for the 2deoxycytidine kinase? ✦ Nucleosides with L-configuration show a high degree of specificity for the 2-deoxycytidine kinase. 13.Which HIV drug is not a direct obligate DNA chain terminator?What is it then? ✦ ETV is not a direct obligate DNA chain terminator. ✦ It is a pseudo-terminator. 14.How does ETV(Etavirine) work? ✦ Exocyclic alkene in ETV binds in a hydrophobic pocket in the active site of the DNA Polymerase complex

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Aelisha Shakya

✦ The binding forces the DNA at that point to move away from the ideal conformation and hence although a few more base are added, the conformation of the growing DNA no longer adequately fits the active site--- end of reaction. ETV and TFV have a higher barrier to generic resistance than other NRTIs. 15.What is HBx? ✦ Viral protein that is used by HBV for efficient infection and replication. ✦ Does not bind directly to DNA, ○ but regulated transcription by transcription factors and chromatin or ○ indirectly via activation of signal transduction pathways in the cytoplasm. Many of its effects on signal transduction lead to aberrant biochemistry. 16.What are the examples of Hepatitis C? ✦ Yellow Virus ✦ Dengue Virus ✦ West Nile Virus ✦ Japanese encephalitis Virus 17.How does Hepatitis C infect? ✦ Infect a range of cells via receptor mediated endocytosis, followed by intracellular membrane fusion. 18.What are the targeted proteins for HCV replication? What are their features? ✦ NS3 Protein ○ 70 kDa and is multifunctional ○ N-terminal serine protease domain ○ C-terminal RNA helicase/ NTPase domain ○ NS3A requires interaction with NS4 to operate

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○ Needs co-ordination with Zn 2+ ion for complete folding ✦ NS3-4A ○ Active site contains catalytic triad of His-57, Asp-81 and Ser-139 ○ Serves two main purpose ✓ maintaining the correct conformation==== prevents degradation) and ✓ facilitates recognition of RNA substrates ✦ NS5A protein ○ Vital for successful viral replication ○ Zinc-binding phosphoprotein essential for ✓ RNA replication and ✓ virion morphogenesis. ○ Effective against all 4 genotypes ○ Dasabuvir- new agent that targets the NS5B protein.

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Aelisha Shakya

WEEK 31: Infection in CF: 1. How does CF result? ✧ from a mutation in a gene that encodes for a chloride channel called the cystic fibrosis transmembrane conductance regulator (CFTR). ✧ thickened secretion in organs with epithelial cell lining ○ Lungs, GI, vas deferens, sweat glands, pancreas 2. What is role of CFTR? ✧ water and salt movement across cell membranes 3. Describe the pulmonary effects of CF? ✧ defective ion transport causes the liquid depletion in airway surface resulting in impaired mucociliary clearance. ✧ airways become clogged with thick sticky mucus which impairs the clearance of microorganisms ○ Recurrent infection ○ Staphylococcus aureus and pseudomonas aeruginosa ✧ Thus abnormal inflammatory response ✧ Bronchial damage, bronchiectasis and respiratory failure. 4. Is pulmonary damage due to CF reversible or irreversible? ✧ progressively irreversible 5. Which is the most common problem of CF? ✧ respiratory symptoms 6. What monitorings are required for respiratory symptoms?

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✧ frequent monitoring of cough swab and sputum culture ✧ regular monitoring of lung function with spirometry and oxygen saturation. 7. What is the mainstay therapy for respiratory problems? ✧ Antibiotics 8. What is the treatment of S.aureus? 9. What is the treatment of severe exacerbations caused byS.aureus? 10.What are the treatment for MRSA (Methicillin Resistant Staphylococcus Aureus)? 11.What are the treatments for haemophylus influenzae? 12.What are the treatments for Pseudomonas aeruginosa? TREATMENT FACTORS FOR FOLLOWING PATHOGENS S.aureus isolated or Severe exacerbations by minor exacerbations S.aureus ● Flucloxacillin orally

● IV Flucloxacillin By slow IV injection over 34 minutes or infusion ● IV Vancomycin Infusion related events if given too quickly, S/E: nephrotoxicity, ototoxicity, neutropenia, thrombocytopenia

MRSA

Treat according to sensitive ● Fusidic acid + Rifampicin/Trimethoprim ● Oral linezolid (FBC required) Acute exacerbation ● IV Vancomycin or ● Teicoplanin

● Inhaled Vancomycin

Haemophilus influenzae

Pseudomonas aeruginosa

If sensitive : Amoxicillin orally

Aggressive Tx as chronic colonisation associated with a more decline in lung function and x ray findings

Severe infections:

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Chloramphenicol Cefuroxime IV

Variation in regimens: ● SIX weeks of ciprofloxacin with between three and six months colistin ( polymyxin antibiotic) ● THREE months of both ciprofloxacin and colistin ● Inhaled tobramycin if intolerant to ciprofloxacin and colistin or early growth of pseudomonas aeruginosa. Patients who are chronically colonise with P.aeuroginosa: Nebulised antibiotics shown to reduce the rate f deteriation of respiratory function and the number of IV courses needed ● Colistin first line

13.What are the other effects of CF? ✧ Pancreatic insufficiency ✧ Increased viscosity of intestinal mucus result in ductal obstruction ✧ Maldigestion and malabsorption ✧ CF related diabetes ✧ Congenital bilateral absence of the vas deferens (CBAVD) ✧ Obstruction of the Wolffian ducts during foetal development ○ Most males are infertile 14.What are the factors needed to be investigated for CF diagnosis? ✧ Newborn screening test ○ Measurement of immunoreactive typsinogen (IRT) levels in blood ○ Pancreas-derived enzyme is elevated in CF ○ Genetic mutation analysis ○ Diagnosis within 5 working days ✧ Sweat Test ○ Measures the concentration of chloride that is excreted in sweat ○ Elevated in CF

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Aelisha Shakya

Week 25: HIV 2 Anti-HIV Chemotherapy 1. What is virus? What are also they known for? 2. What are the components of virus? ● genome consisting of either RNA or DNA ● viral proteins (non- structural proteins including enzymes) ● protective coat 3. What is protein coat composed of ? 4. What is envelope? 5. What is capsid? ● protein shell of a virus ● structural proteins that posses the capacity to self-assemble without the help of enzymes into a ball which is identical in shape and size to a mature virus particle. 6. What are virions? ● insertion of the core(interior material of the virus), that results in reassembly into mature virus particles. 7. What is capsomeres? 8. How many identical capsomeres per virion does rhinoviruses have? 9. How many capsomeres do adenoviruses have which are not all identical? 10.Where is the other structural form of for the protein coat found in and why does it have helical symmetry? ● found in where the capsids are assembled in the shape of rod in which the genome is packed in a helix with the structural protein. ● every protein of the structural protein is therefore in an identical environment and the particle has helical symmetry. 11.Give an example of naked icosahedral virus? 12.Give an example of enveloped icosahedral virus?

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Aelisha Shakya

13.Give an example of enveloped helical virus? 14.What is the term given where the genome may exist as a single molecule or several pieces of nucleic acid whether it is made up of DNA or RNA? 15.What is the term given for any strands of viral DNA and RNA which are complementary to the mRNA and who have the same sequence? The viral genome may be present in the form of a positive or negative strand. 16.What is the use of positive strand of RNA virus? 17.Define pinocytosis: ● association of the virus with the receptors on the cell causes the cell membrane to draw around the virus particle which results in an endocytotic vacuole. 18.How are viral proteins are derived using the host’s cellular machinery? ● HIV does not use its positive strand RNA directly for translation, ● but instead inserts its proviral DNA into the host’s chromosome, ● thus viral proteins are derived using the host’s cellular machinery. 19.What is the transcription process under the control of? 20.What is the whole process of transcription catalysed by? 21.What makes this enzyme makes an excellent target for antiviral chemotherapy? 22.What are roles of regulatory factors presence in transcription of virus? 23.What is one factor that HIV uses during transcription? What is its role? 24.What is a maturation in transcription in virus replication? ● The process where the virus assemble new viral particles once the virus has produced all its required components. 25.What are the process involved in maturation? ● assembly from individual protein molecules ● assembly from a polyprotein precursor ● chaperone-assisted assembly 26.What is the final phase of the maturation? 27.What is budding? ● fusion of a vesicle bearing viral glycoproteins which then creates a new membrane. 28.SLIDE 17 and 18 29.List the viral replication cycle.

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Aelisha Shakya

Stage 1 : Attachment of a host cell Stage 2 : Penetration of the host cell membrane Stage 3: Uncoating the virus Stage 4: Replication of the genetic material Stage 5: Maturation of progeny virus particles Stage 6: Release of progeny virus particles

Stage 1: ● All viruses contains proteins on their surface whether as a part of the capsid or as spikes embedded in the viral envelope ● These surface proteins recognise specific receptor proteins on the surface of the target cell. ● This recognition event is similar to other protein-protein interactions which involves a stereospecific network of hydrogen bonds and lipophilic associations.

Stage 2 and 3 : ● Involve : Enveloped Viruses ● Involve pinocytosis ● As the pH within the vacuole drops, the viral surface protein change shape triggering fusion within the cell membrane and eventually release of the core material. ● Also involve Non-Enveloped Viruses: ● Here the viral coat protein interacts with the cell-membrane bound proteins to open up a pore through which the viral core material can pass into the interior of the cell. Stage 4 :

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● Involves translation: ○ synthesis of new viral proteins ○ divided into early and late protein synthesis ● Early : very rapid production of key functional proteins that virus requires for replication. ● Late: production of the structural proteins required for assembling new virions. ● Viral proteins are derived using the host’s cellular machinery. ● Involves transcription: ○ well understood for most viruses. ○ is under the control of the RNA-directed RNA polymerase ○ process catalysed by enzyme reverse transcriptase ○ presence of regulatory factors ○ presence of TAT

Stage 5: ● maturation ● process of maturation ● final phase of maturation for all enveloped viruses This is a complex process whereby viral glycoproteins are inserted into the host’s membrane- the process which employs the usual protein secretory pathway of the cell. ● ultimately budding

Stage 6: -budding HIV challenge : ● HIV replicates very rapidly throughout the whole course of the infection ● An individual who has been infected with HIV for 10 years has viral genomes present in the body that are 3000 generations away from the initial virus that initiated infection

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● HIV has an extraordinary mutation rate.

Week 25: HIV 4 Nucleic Acid Structure and function 1. What are nucleotides? ● Phosphate ester of nucleotides. ● Nucleoside : Base + sugar only. No phosphate group. 2. 3 components found in all nucleotides? 3. How is nucleoside structure composed of? Where are the major components found in DNA and RNA? 4. Which nitrogen heterocyclic base does not exhibit tautomerism? 5. What is the sugar component, pentose in RNA and DNA? 6. How many chiral centres can be found in D-ribose? 7. What is the name of an enzyme that controls the phosphorylation of the various nucleosides? Give one role of that enzyme? NRTI metabolism is influenced by the cell type and whether the cell is activated or resting 8. How does kinases act as a therapeutic agent? ● Some viruses including HIV, HBV and influenza DO NOT encode a nucleotide kinase of their own and instead rely upon the host’s own cellular kinases. ● Other viruses, particularly members of the herpes group, encode their own nucleoside phosphorylating enzymes. Analogues of the natural substances for nucleic acid processing enzymes can be considered as potential antiviral compounds. Can also consider changing :

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■ part of the sugar : hydroxymethyl moiety ■ Sugar component and the base 9. Which aspects of the nucleotide structure are very important to its molecular recognition?What are the roles of the aspects? 10.What are the roles of these recognition features? ● These are used by phosphorylation enzymes, which convert nucleosides into nucleotides. ● These are used for maintaining the fidelity of the replication of a genetic material. 11.What is important in maintaining the fidelity of the replication of the genetic material? ● recognition due to specific hydrogen bonds between bases is perhaps the single most important interaction. Choice of Base: Slide 27-29 12.What is the key feature of many of the anti-HIV compounds? ● Dideoxy furanose ---------------- No hydroxyls ● Didehydro furanose -------------- Unsaturated bond 13.IMPORTANT : SLIDE 34 14.What is the importance of the heterocyclic base?

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14...