Viruses 2
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- Created by: amazingemilyjones
- Created on: 14-04-19 19:41
Viruses 2
Viruses 2
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HIV
Diagnosis Specimen for Viral Diagnosis
- Direct and indirect ways
- Symptoms
- Virus isolation (direct) - immunofluorescence
- Viral antigens/virus - haemagglutination inhibition test, neutralisation, complement fixation test
- Antivirus antibodies - immunofluorescence, ELISA
- Molecular biology - PCR
- Biological specimen for viral diagnosis
- Nasopharynx swab, nasopharynx aspirate, throat swab, lesion swab, eye swab, rectal swab, blood, saliva, tissue biopsy, cerbral spinal fluid (CSF), urine, stool
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Virus Cultivation and Multiplication
- Viruses have to be cultured in the presence of the host cell
- This often makes research/diagnosis difficult
- Plaque assay
- Grow a lawn of host cells on dish
- Add virus
- Virus replicate and lyse host cells
- Viral plaques: cytopathic effect
- Count the number of plaques
- Results of assay expressed as plaque forming units (pfu)
- Count of infectivity not number of viruses
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Immunodiagnostics
- Detection of host antibodies against virus
- Viral haemagglutination
- Some viruses (influenze, mumps and measles) have the property of haemagglutination (causing red blood cells to stick together)
- Presence of anti-virus antibodies in patient serum can inhibit reaction
- Haemagglutination Inhibition Assay (HI test) - Influenza
- Measure how well antibodies bind to influenza viruses
- Red blood cells (turkey, guinea pigs) in a solution will sink to the bottom of the assay well and form a red dot at the bottom
- When an influenza virus is added to the red blood cell solution, the virus' haemagglutinin surface proteins bind to multiple red blood cells and keep red blood cells suspended
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Immunodiagnostics
- Antibodies can be used to inhibit, detect and quantify virus
- Detection of an antiviral antibody using Enzyme-Linked Immunosorbent Assay (ELISA) - uses antibodies and colour change to identify a substance
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Molecular Biology and Diagnostic Virology
- Modern molecular techniques of both protein chemistry and DNA/RNA biochemistry
- Improve the specificity of virus diagnostic procedures
- Methods include:
- Viral DNA hybridisation with labelled probes
- Sequencing of portions of the viral genome
- Polymerase chain reaction (PCR)
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Antiviral Chemotherapy
- Targets
- Virus specific enzymes
- Life cycle process
- Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
- Reverse transcriptase is the number one target for drug therapy
- Most highly active antiretroviral therapy (HAART) therapies contain a reverse transcriptase inhibitor component
- Well-known NRTIs for HIV treatment include zidovudine (AZT) and tenofovir but also abacavir, didanosine (DDL), emtricitabine (FTC), lamivudine (3TC), stavudine (d4T)
- AZT: Zidovudine (Azidothymidine): by mouth 500-600mg daily divided in 2-3 doses
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NRTIs: AZT
- DNA is synthesised from four deoxynucleotide triphosphates
- AZT is a nucleoside analogue - it resembles the nucleotides that are the building blocks of DNA
- When AZT gets used to build DNA, it prevents any more from being made - it blocks further extension of the growing DNA chain
- When reverse transcriptase is creating new DNA it 'accidentally' inserts a molecule of zidovudine (or tenfovir) rather than a nucleoside
- This terminates the DNA chain and no more can be appended to it. Other nucleoside analogues which inhibit reverse transcriptase are ddI (Didanosine) and d4T (Stavudine)
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Non-Nucleoside Reverse Transcriptase Inhibitors (N
- NNRTIs also targets the HIV reverse transcriptase
- NNRTIs attach to the reverse transcriptase and affect the activity of the enzyme by restricting its mobility and making it unable to function
- Antivirals include efavirenz, etravirine, neviparine, rilpivirine
- Efavirenz (Sustiva): 600mg once daily
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Protease Inhibitors
- Viral proteinsase (protease): an enzyme that cleaves peptide/polypeptide/protein chains
- Protease cleaves the polypeptide into functional proteins - mature virus
- HIV protease inhibitors binds to enzyme active site
- HIV virus unable to process proteins
- Atazanavir (Reyataz): 300mg daily
- Darunavir, fosamprenavir, indinavir, Lopinavir, ritonavir, saquinavir, tipranavir
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Integrase Inhibitor
- Viral integrase: an enzyme that allows the transfer of HIV cDNA to cellular DNA
- Dolutegravir (Trivicay): 50mg once daily
- Raltegravir (Isentress): 400mg twice daily
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Entry Inhibitor
- Fusion inhibitor: an enzyme that prevent HIV envelope fusion
- Enfuvirtide (Fuzeon): 90mg twice daily (subcutaneous injection)
- Receptor antagonist: entry inhibitor; a negative allosteric modulator of the CCR5 receptor
- C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines (chemotactic cytokines)
- Maraviroc (Celsentri): 300mg twice daily
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Inhibitor of Viral DNA Polymerase
- Viral DNA polymerase: enzyme that creates DNA molecules by assembling nucleotides - essential for viral DNA synthesis
- Acilovir is a guanosine analogue
- Aciclovir is converted by viral thymidine kinase to aciclovir monophosphate which is then converted by host cell kinases to aciclovir triphosphate (ACV-TP)
- ACV-TP competitively inhibits and inactivates HSV-specific DNA polymerases
- Aciclovir (Zovirax): 200mg 5 times daily for 5 days
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Neuraminidase Inhibitors
- Neuraminidase inhibitor: prevent the release of the virions (slow progression of infection)
- Oseltamivir is a competitive inhibitor of the activity of the viral neuraminidase
- It is a prodrug as it needs to be hydrolysed in the liver to be active (oseltaminivir carboxylate)
- Oseltamivir (Tamiflu): 75mg once daily for 10 days
- must be taken within 48 hours that symptoms arise
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Types of Vaccine
- Inactivated vaccines
- Poliovirus, influenze, hepatitis A virus, rabies
- Attenuated vaccines
- yellow fever, measles, rubella, mumps (MMR)
- more durable immunological responses and preferred type for healthy adults
- may not be safe for use in immunocompromised individual
- Protein subunit vaccines
- Hepatitis B virus; papillomavirus, infleunza (haemagglutinin, neuraminidase)
- a viral fragment can elicit an immune response
- protein subunit are usually produced in a bacteria/yeast (genetic engineering)
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Poliovirus
- Polio (poliomyelitis) mainly affects children under 5 years of age
- One in 200 infections leads to irreversible paralysis. Among those paralysed, 5-10% die when their breathing muscles become immobilised
- Polio cases have decreased by >99% since 1988, from an estimated 350,000 cases then to 416 reported cases in 2013
- In 2014, only 3 countries (Afghanistan, Nigeria and Pakistan) remain polio-endemic, down from more than 125 in 1988
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