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132 Cards in this Set
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Give examples of viruses with ss/ds DNA.RNA.
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Describe how HCV was isolated originally?
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Describe the classification of DNA viruses.
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Describe in descending order the relative sizes and structures of different classes of DNA virus.
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Describe in descending order the relative size and structures of RNA viruses.
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How do viruses compare in size with eukaryotes, bacteria and proteins?
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What is a CPE?
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>Cytopathic effect are morphological changes within cells as the virus replicates.
- These changes are referred to as a cytopathic effect / CPE. - Different viruses produce different CPE - Possible to make a highly educated guess as to which virus is present within a particular culture showing a CPE. |
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How were viruses demonstrated indirectly?
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1. Infected tissue was ground into a fluid suspension
2. Filtered to retain bacteria etc. 3. Filtrate innoculated to animals and plants 4. Observe for disease 5. Repeat and pass in series |
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Give some examples of early virus discoveries.
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>1881 - Pasteur passes rabies to rabbits
>1892 - Iwanowski - Tobacco Mosaic Virus >1898 - Frosch - Foot and mouth disease >1902 - Reed - yellow fever >1905 - Ciuffo - Human warts >1908 - Ellermann/Bang - fowl leukaemia >1911 - Rous - chicken sarcoma >1917 - d'Herelle - bacteriophage |
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What prompted advances in virus detection?
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Chick embryos:
>Use of chick embryos still continues to manufacture flu vaccine >Incubated in amniotic fluid to grow the virus Virus genetic material: >Viruses have DNA or RNA not both >TMV 1935 - RNA >Vaccinia 1940 - DNA Tissue culture: >Also used for vaccines and diagnostic reagents >1949 - tissues can grow on flat surfaces (glass or plastic plates) >Innoculate with virus and observe CPE Electron microscopy: >Defines morphology by negative staining or thin section >EM can resolve up to 1nm whereas LM only up to 200nm Other molecular techniques: >E.g. similar to HCV diagnosis >1989: - Plasma from chimps ultracentrifuged - Nucleic acid extracted - Cloned into bacterial virus (bacteriophage) - Grow bacteriophage clones in bacteria - Screen 10^6 clones for virus protein with human anti-serum - DNA from the only +ve clone sequenced - Use sequenced DNA as probe for virus (turned out to be RNA virus) Extra note: - nitrocellulose blocks used to express proteins, probe with patient serum, pick positive plaques and sequence DNA |
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Describe the composition of infectious particles or virions?
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1. Genetic material - DNA or RNA NOT BOTH
2. Protein coat = capsid (subunits capsomeres) 3. Virus symmetry based on capsomeres (capsid either has icosahedral or helical symmetry) 4. Capsid + nucleic acid = nucleocapsid 5. Non structural protein = enzymes 6. Some families have an envelope |
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Describe four methods of virus classification.
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1. Genome:
- DNA/RNA - ss/ds - +/- sense RNA - Polycistronic or segmented 2. Replication strategy 3. Morphology - helical/icosahedral/complex +/- envelope 4. Size |
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What is a prion?
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>Term derived from 'proteinaceous infectious particles'.
>Prions are believed to be composed entirely of protein. >Normal host cellular gene encodes the prion protein, which exists as a cellular isoform, PrPc, or Mr 33-35kDa, and as an abnormal isoform PrPRes (resistant). >PrPc is completely digestible by proteinase K but PrPRes is digested only as far as a 27-30kDa product. >PrP 27-30 therefore accumulates in tissues containing PrPRes. |
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What are prion diseases and examples of them?
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>Referred to as slow infections, transmissible spongiform encephalopathies (TSE), and, in humans, transmissible dementias.
>Animal diseases include scrapie (sheep), and bovine spongiform encephalopathy (BSE, cows); human diseases include Kuru (cannibals) and Creutzfeld Jakob disease. |
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How do prions replicate?
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>Theory suggests that spontaneous conversion of PrPc into PrPRes is catalysed by the presence of PrP 27-30.
>Thus once PrPRes has appeared, there will be an inevitable accumulation of PrPRes and PrP 27-30 |
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Describe CJD.
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>Creutzfeld Jakob Disease is the commonest prion disease in humans.
>Usually presents after the age of 60 years, as a progressive dementia, but cases in younger individuals arise by iatrogenic transmission (e.g. from contaminated neurosurgical instruments, or human pituitary-derived hormones). >Familial CJD (15% of all cases) is associated with mutations in the prion gene. |
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What are BSE and vCJD?
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>Epidemic of BSE emerged in the UK in the late 1980s.
>BSE agent thus entered the food chain and has resulted in the appearance of a new variant of CJD in humans. >The ultimate size of the resultant vCJD epidemic is as yet unknown. >Theoretically, person to person spread of vCJD may occur through contaminated surgical instruments and transfusion of contaminated blood. |
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What are Koch's postulates?
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To prove an agent causes a disease must:
1. Find in lesions 2. Isolate in pure culture 3. Inoculate pure culture and cause disease 4. Recover again from lesions of host |
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Describe the process of measuring virus titre by plaque assay.
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Agar prevents motion of virus.
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Describe the growth curve for non enveloped viruses.
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Describe the virus replication/infectious cycle.
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Infectious cycle includes:
>Attachment and entry of the virion >Decoding of genome information >Translation of viral mRNA by host ribosomes >Genome replication >Assembly of virus particles >Release of particles containing the genome |
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Describe the structure of the influenza virus. Describe its classification.
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>Genome:
- 8 segments of single stranded RNA, which encode 10 or possibly 11 proteins. >Influenzas: - A (widespread in nature, infecting a number of species including humans, swine, horses and birds) - B (human pathogen) - C (not a serious human pathogen) >Typing is on the basis of the nature of the internal viral proteins e.g. nucleocapsid proteins. >A virus subtyping: - Nature of two surface glycoproteins - Haemagglutinin (H or HA) - Neuraminidase (N or NA) NB. 15 distinct H and 9 N molecules - each differs by >/= 20% in amino acid sequence. Reference to influenza must indicate which subtype it is e.g. H1N1 or H3N2 etc. |
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Describe the attachment of the influenza virus.
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>Virus attachment protein (HA) on the surface of the virion binds to the Sialic Acid receptor on the cell surface.
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Describe 3 methods of virus entry.
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>Fusion - enveloped virus or
>Endocytosis - enveloped/non-enveloped >Followed by UNCOATING = release of viral genome from capsid and or the lipid envelope, making the genome accessible for replication. >Digestive endosome aids the release of the capsid. NB. Permissive cells are those which have the necessary machinery for viral genome replication. |
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What are ICAMs?
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>Intercellular adhesion molecules e.g. Poliovirus.
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Describe the key characteristics of the viral genome.
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>Its replication depends on virus (DNA or RNA) but ALWAYS depends on viral RNA.
>Early viral genes are transcribed pre-replication: - Regulate cell NA and protein synthesis - Regulate expression of viral genome - Code for viral enzymes required for replication of viral nucleic acid >Late viral genes are transcribed post replication. |
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How are viruses released from host cells?
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>Cell lysis / death
>Released from a viable cell |
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Draw/describe the Baltimore classification.
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NB. DNA viruses replicate in the nucleus, RNA is the endgame.
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What is the difference between positive sense mRNA and negative sense RNA?
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>Positive acts as mRNA (translated directly by ribosomes)
>Negative: - Complementary to mRNA - When copied acts as mRNA (by RNA dependent polymerase) |
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Describe/draw the structure of HIV.
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1. Genetic material - RNA
2. Protein: - Structural e.g. capsid, glycoproteins - Non-structural e.g. reverse transcriptase, integrase 3. Lipid envelope - bearing virus attachment protein GP 120 (with GP41 stalk) |
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Describe the HIV envelope and how HIV gains entry to the cell.
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1. Viral envelope protein is a trimer
- 3 GP120 subunits bind cell receptors - 3 GP41 subunits cause fusion 2. Envelope first attaches to CD4 molecule on T-lymphocyte cell membrane - Changes in GP 120 expose a binding site for the coreceptor (a chemokine receptor) 3. Coreceptor binds, leading to GP41 changes and membrane fusion. |
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Describe the HIV replication cycle.
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>Example of Provirus
- Retroviral DNA is integrated into the host cell genome = provirus. - Template for retroviral RNA and genomic RNA - Enables latency: won't be expressed until DNA is transcribed. >Integrase is unique to reverse transcriptase viruses, it integrates viral DNA into the human chromosome. |
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What is the difference between a Productive infection and a Latent phase?
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Productive infection:
>Complete infectious cycle with release of visions from the infected (permissive) cell. Latent phase: >Infected cell is transiently non-permissive i.e. proviral DNA is present but not transcribed. |
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Describe an example of a reversivirus.
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Hep B virus:
>DNA virus but has an unusual complex replication cycle involving an RNA intermediate. >Uses reverse transcriptase >Group 7 in Baltimore classification. |
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In which three easy do viruses exhibit genetic diversity?
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1. Point mutation:
>RNA viruses often have a much higher error rate as RNA polymerases have NO PROOFREADING activity. 2. Recombination: >Exchange of nucleic acid sequences - part but not all of the full length genome - between different but closely related viruses. >Occurs during viral replication by polymerase strand switching - copy choice (HIV does this). 3. Reassortment >The exchange of entire RNA molecules between genetically related viruses with segmented genomes. >E.g. Influenza virus |
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What is meant by quasi-species (viruses) and what are its implications?
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>Complex mixture of molecular variants of an RNA virus e.g. HIV.
>Cloud of viruses with one 'dominant' species (changeable) >Contributes to superinfection. |
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How does influenza diversify genetically?
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>Point mutations allow evasion of the immune response.
>Reassortment allows crossover of animal / human genes. |
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What is the difference between the entry and release of enveloped and non-enveloped viruses?
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Non enveloped viruses:
>Assembly of the capsid and viral genome occurs in the nucleus or cytoplasm depending on the virus and disintegration of the dying cell is needed for the release of the virus. Enveloped viruses: >Nucleocapsids attach to viral proteins inserted in the cell membrane and the new vision buds from the still viable cell. |
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To which receptor in which cell does Epstein barr attach/infect?
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Complement receptor 2; B lymphocytes
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To which receptor in which cell does Influenza attach/infect?
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Sialic acid; respiratory epithelia
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To which receptor in which cell does Rhinovirus attach/infect?
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ICAM 1; respiratory epithelia
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To which receptor in which cell does HIV attach/infect?
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CD4 (+coreceptor - cellular chemokine receptor); T lymphocytes and macrophages
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To which receptor in which cell does Rabies attach/infect?
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Acetylcholine receptor; nerve cells
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How can viruses be counted?
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>Through infecting eggs with viruses and then counting the pocks that appear on the chorioallantoic membrane (cumbersome)
>Other method is to measure concentration by plaque assay using tissue culture. |
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Which Baltimore group are adenovirus and other dsDNA viruses in?
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Group 1
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Which Baltimore group is Parvovirus B19 and other ssDNA viruses in?
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Group 2
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Which Baltimore group are dsRNA viruses e.g. Rotavirus in?
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Group 3
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Which Baltimore group are influenza or measles viruses in?
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Group 5:
ss negative sense RNA (segmented and non segmented respectively) >Virus mRNA is transcribed from the parental genome. |
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Which class of viruses fall into Group 6 of the Baltimore classification?
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>Retroviruses with ssRNA e.g. HIV.
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Which viruses fall into group 7 of the Baltimore classification?
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dsDNA retroviruses (reversivirus), e.g. HBV.
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Describe in which ways viruses may be spread from person to person.
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Define vertical transmission.
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Spread of virus from parent to embryo e.g. CMV.
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Define horizontal transmission.
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Between individuals, via respiratory droplets, sexual contact etc. E.g. Rhinovirus, HIV.
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Define iatrogenic infection.
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Acquired by patient during treatment.
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Define nosocomial infection.
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Acquired by patient/carer in hospital.
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Define zoonosis.
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Disease of animals transmitted to humans (e.g. toxoplasma).
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Describe two different zoonoses.
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Identify this pathology and its cause/infectious cycle.
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>Warts
>Caused by papilloma virus - dsDNA virus - most important in terms of human infection - >80 types >Infect squamous epithelial cells on both keratinised and mucosal surfaces giving rise to a papilloma, more commonly known as a wart. >Clinical presentation may be with: - cutaneous warts (HPV 1-4) - mucosal papillomas (6-11) - malignant change (16 or 18 assoc. with uterine cancer; 5 or 8 maybe associated with squamous cell carcinoma of skin). |
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Describe this pathology, its cause and infection.
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>Orolabial herpes - extensive ulceration in the oral cavity (cold sore)
>Enveloped dsDNA virus with icosahedral capsid. >Acquired through exposure to contaminated saliva or genital tract secretions. >All herpesviruses exhibit latency, primary infection is followed by lifelong carriage (latent site = nerve cell body). >Treated with acyclovir. |
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Which virus can be transmitted by this creature?
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Rabies virus
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What are the main routes of viral transmission by direct contact? Give examples for each route.
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i) Skin:
Papillomavirus - warts Herpes simplex virus - oral/saliva Rabies - animal bite ii) Tissues, blood: HIV & hepatitis iii) Sexual contact: Genital herpes, genital warts, cervical cancer HIV, Hepatitis B iv) Congenital/transplacental: Rubella - german measles Cytomegalovirus (CMV) v) Perinatal: Birth canal - HIV, hepatitis B Breast milk – HIV vi) Respiratory secretions – large droplet spray onto conjunctiva, mucous membranes of eye, nose, mouth |
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Which virus can cause this pathology? Describe it.
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>Rubella virus
- +ve ssRNA virus - transmitted via respiratory droplets, can cross the placenta: causes CNS, eye and heart abnormalities - IgM antibody response is diagnosis - no treatment |
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Describe different routes for faecooral infection.
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What are the main routes of viral transmission by indirect contact? Give examples for each route.
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i) Inhalation
Aerosols or droplet nuclei - influenza, chickenpox, measles ii) Vehicle-borne * Fomite = object able to harbour infectious agent & transmit Respiratory secretions, smallpox iii) Vehicle-borne - Faecal/oral route Drinking water Food - manure flies utensils hands iv) Vehicle-borne – Blood Blood Transfusion, Needlestick, Organ transplant v) Vector-borne – arthropod bite (insect, tick) Animals - natural host/reservoir Arbovirus*- replicates in arthropod vector * Ar(thropod)bo(rne)virus Man - infected incidentally e.g. jungle yellow fever tick-borne encephalitis Man to man e.g urban yellow fever dengue |
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Which factors determine transmission?
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>Virus titre
>Infectious period - Short: Influenza - Medium: Hepatitis A - Long HIV, herpes >Critical community size e g measles v. HIV >External survival - Resistant to drying, temperature, UV, pH (Hepatitis A virus) >Evasion of host immunity - Multiplication on: skin / mucous membrane (Papilloma virus Respiratory virus) - Antigenic variation: e,g, Influenza virus, HIV |
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How can virus transmission be prevented?
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1. General hygiene, hand washing, barrier nursing/disinfection/autoclaves.
2. Protected sex. 3. Food & water hygiene. 4. Arthropod vector control 5. Protection of susceptible hosts* by: Antiviral drugs Immunoglobulin prophylaxis (short term) Vaccination programmes (long term) * Needlestick, newborn of HIV or HBV mother |
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What are the requirements for virus infection in a multicellular host?
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1. A portal of entry
2. Target tissue in which to replicate 3. Ability to evade the immune system 4. Portal of exit 5. Means of transmission |
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What is meant by virus tropism?
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The ability of a virus to replicate in a particular cell type; requires specific cellular receptors to which the virus attaches and gains entry to the cell.
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What are examples of localised infection and disease?
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E.g. Epithelial infections:
>Respiratory - Influenza - RSV >GI - Rotavirus - Norovirus >GU - Papillomavirus >Skin - Papillomavirus |
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Why do some viruses only infect epithelia?
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>Limited virus tropism
>Optimal local temperature >Polarised virus assembly e.g. influenza >Role of host defences e.g. IFN |
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Describe typical portals of virus entry (9) using examples of pathogenic viruses.
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MUCOSAL:
>Conjunctiva: - Adenovirus >Oropharynx: - HSV >Respiratory tract: - RSV, Influenza >Alimentary tract: - Poliovirus, Norovirus >Genital tract: - HBV, HIV SKIN: >Papillomavirus BLOOD: >HIV, Hepatitis B Virus >Biting arthropods - yellow fever virus PLACENTA: >Congenital rubella >CMV |
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Why do some viruses exhibit apical polarity?
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>Limited to the apical surface due to the site of production and release.
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Describe the papillomavirus infection cycle.
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ENTRY:
>Virus infects primitive basal keratinocyte at low concentration - 10 copies per cell - enters via fissue DISASSEMBLY & VIRAL TRANSCRIPTION: >Genome in form of episomal plasmid (can replicate independently of chromosomal DNA) - Genome taken up in this form (type of latency) VIRAL REPLICATION: >Early stages of squamous cell differentiation little virus proteins are made - Expression increases as keratinocytes differentiate - Viral genomes = 000s per cell >Desquamating cells are ready for infection of naive individuals CYCLE = 4-6 WEEKS BASAL LAYER: >Linked to cell cycle >Replication as an episome >No virions SQUAMOUS LAYER: >No host DNA synthesis >Virus replication >Virions produced |
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Describe the pathway using examples for viral sub epithelial invasion.
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E.g.
>Neuronal spread e.g. rabies, HSV, VZV - NB. Distance from site affects incubation period >Directly to blood e.g. yellow fever >Via lymphatics to blood e.g. polio / measles |
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How does virus infect blood (primary infection)?
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E.g. Free virus: Polio, hep B or Cell associated e.g. measles, HIV
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Compare localised viral infection and disease with systemic viral infections.
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LOCALISED:
>Respiratory / Intestinal - Short incubation period e.g. norovirus 48hrs >Skin - Longer incubation period >Entry, infection and disease at the same site SYSTEMIC: >Longer incubation period e.g. HBV weeks to months >Disease distant from portal of entry (both examples incubate for weeks) - Poliovirus enters via gut but disease in CNS - Measles virus - enters via respiratory tract, disease generalised |
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What are examples of virus exit portals?
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1. Respiratory tract - RSV, rhinovirus, measles (most infections diseases)
2. Oropharynx - HSV 3. Alimentary tract - Poliovirus, norovirus 4. Genital tract - HSV, HIV, HBV 5. Blood - HIV, HBV, yellow fever |
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What factors affect virus transmission?
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>Amount of shed virus
>Duration of shedding >Ability of virus to survive in the environment - e.g. HIV enveloped, damaged easily outside of host |
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What characteristics might a persistent virus possess?
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>Where virus persists for long periods (months/years) after primary infection:
- Latency: HSV, VZV, HIV - Chronicity: HBV, HCV |
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What are the consequences of virus infection for a multicellular host?
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>Cell death leading to tissue injury
- E.g. poliovirus damages the CNS - Influenza virus damages the respiratory tract >Excessive immune response - e.g. HBV in adults - Acute EBV infection in glandular fever / infectious mononucleosis >Malignant neoplasm aka 'cancer' - e.g. EBV, HBV, some papillomaviruses |
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How does disease manifestation in viral infection and why?
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>Due to host status
>Susceptibility to virus can be determined by genetics of the host. EBV: - Child silent primary but adolescent / adult IM RUBELLA (GERMAN MEASLES): - Mother - rash - Foetus - congenital infection (cataracts etc.) HSV: - Immunocompetent - cold sores - Immunocompromised - pneumonia (opportunistic infection) - Genetically determined specific immunodeficiency - herpes simplex encephalitis |
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Describe the action of oncogenic viruses.
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>Cancer results from the growth of cells in which mutations have accumulated
>These mutation affect cell proliferation (transformation) >Viruses contribute to these changes in at least 20% of cancers |
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Describe the infection of infectious mononucleosis.
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>Epstein Barr Virus
>Infects oral epithelial cells, B cells >Immortalises B cells with production of heterophile Ab >T-cells (atypical lymphocytes) control B cell infection but also contribute to symptoms - cytokines contribute also |
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Describe the iceberg concept of infection.
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How does virus invade tissue?
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>From blood vessel by:
a) Replication in endothelial cells b) Transcytosis (endocytosis, trancytosis, exocytosis) c) In leukocytes e.g. HIV d) Through fenestrated capillaries NB. Aided by inflammation |
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Describe the spread of secondary systemic viral infection.
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Describe the three types of persistent viral infection.
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>True latency e.g. HSV, VZV:
- Lurk in neurones - Latent or productive viral infection - Antiviral immune response present / normal >Incomplete latency e.g. HIV - Latent in some cells, active in others - Antiviral immune response present but virus evades >Chronic infection e.g. hepatitis B virus (HBV), HCV - Productive infection - Antiviral immune response, present but ineffective |
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Describe the difference between productive and latent viral infections.
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>Productive: virus replication with production of virions
>Latent: Non-productive infection potentially active state but no obvious effect on cell function |
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Describe primary infection with VZV.
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INCUBATION:
>Inoculation of respiratory mucosa >Viral replication in regional nodes --> virus infected cells into capillaries >Primary viremia (replication in liver / spleen) ACUTE INFECTION: >Secondary viremia: mononuclear cell; transport to skin and mucous membranes >Virus release into respiratory secretions >Replication in epidermal cells - virus in dorsal root ganglia >VZV specific immunity --> resolution of replication |
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Identify this pathology and its cause.
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>Reactivation of latent VZV
>Virus resides in DRG, emerges into skin via innervatory nerves >Normally midline dermatomes affected but trigeminal ganglion can become infected hence this presentation |
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Where does HSV reside in cold sores and genital sores respectively?
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>Cold sores: trigeminal ganglion
>Genital sores: sacral ganglion |
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What are the reasons for diagnosing viral infection?
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>Management - antiviral drugs / prognosis
- Timely diagnosis is important for treatment and decision-making - e.g. preventing the vertical transmission of HIV >Prophylaxis - preventing the spread of infection >Surveillance: - outbreak detection - immunisation campaigns |
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What are the two main ways of diagnosing viruses?
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>Via the host Ab response
- diagnose infection (IgG/IgM) early in the humoral response - determine the susceptibility of IgG >Via detection of the virus, Ag or nucleic acid |
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Define serology.
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>Study of Ag-Ab reactions in vivo
>Measurement of Ab or Ag in serum or plasma |
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Which Ab may be detected to diagnose acute infection?
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>IgG, BUT this requires acute and convalescent sera at least 10 days apart
>IgM in single serum gives rapid diagnosis and is suitable for congenital infections |
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Which test should be used to diagnose persistent infection?
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>IgG e.g. to HHV, cytomegalovirus, varicella zoster
>IgG to HIV |
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Which Ab response should be tested to determine susceptibility to infection?
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>Test for rubella IgG in pregnant women
>Test for IgG to Hep B surface Ag after immunisation |
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How might virus specific Ab be tested?
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>Solution:
- Neutralisation (inhibition of viral activity, this is very labour intensive - used in vaccine testing) - Latex agglutination >Solid phase: - Immunofluorescence - Enzyme-linked immunoassay (ELISA, EIA) |
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Explain the latex agglutination test.
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>Sample is mixed with latex beads coated with a specific antibody or antigen.
>If the suspected substance is present, the latex beads will clump together (agglutinate). |
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When detecting virus or viral particles what samples may be used?
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>Blood (e.g. HBsAg)
>Urine (e.g. CMV) >Faeces (e.g. HAV, Rotavirus) >Nose and throat epithelia (RSV, rhinovirus) >Nasopharyngeal aspirate (NPA) >Vesicle fluid (Varicella) |
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What are the advantages of viral detection over host response tests?
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>Immunosuppressed patients may not make Ab
>Problem of passively acquired Ab in newborn e.g. CMV >Window period - virus infection precedes Ab |
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Which factors may be tested early after primary HIV infection?
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>IgG and p24 Ag
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What are the challenge of detecting CPE in tissue culture?
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>Can take weeks for CPE to appear.
>Some viruses cannot be cultured e.g. HCV. >Most rapid option is the direct detection of virus. |
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How is electron microscopy used in viral diagnosis?
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>Can be used to directly identify virus via morphological characteristics.
>Negative staining with electron dense material e.g. phosphotungstic acid. >Used for diagnosis of viral gastroenteritis, by examination of faecal samples. NB. An expensive tool and requires technical expertise. |
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Describe the polymerase chain reaction and it's application in virological diagnosis.
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>PCR is a method of replicating DNA sequences in vitro using a thermostable DNA polymerase.
>Synthetic oligonucleotide primers hybridise to target DNA sequences and allow the enzyme to copy the template strand. >Once the target has been replicated, the resulting dsDNA is heated to denature into single strands >With cooling, the short primers hybridise again and the enzyme repeats the synthesis >Each transit of the DNA polymerase leads to the doubling of the amount of target sequence >With repeated cycles of amplification, there is an exponential increase of target sequences leading to the generation of vast numbers of amplicons (virus-specific DNA) which can be directly visualised by gel electrophoresis or by other means. >PCR can detect small numbers of viral specific genomes and has revolutionised molecular diagnostics. >Because the target sequences are amplified this type of assay is called 'target amplification' >If the virus contains an RNA genome the sequences are initially reverse transcribed into cDNA (using the enzyme reverse transcriptase) which is in turn subject to PCR amplification. |
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What is real time PCR?
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>A type of PCR in which amplicon accumulating tin the PCR tube is measured as the reaction proceeds
>Generation of amplicons hydrolyses fluorescent probes present in the reaction mix generating photon emission - up to four probes may be used at a time in a single amplification which allows for simultaneous detection of several species of nucleic acid >Also called Taqman assay and light cycler assay |
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What are the clinical applications of the detection of viral nucleic acid?
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>Detection and quantification of HIV, hepatitis B and C virus nucleic acid in blood, e.g. viral load, used for monitoring antiviral therapy
>Nucleic acid sequencing to determine viral susceptibility to antiviral drugs (genotypic resistance testing) |
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Why are enveloped viruses more susceptible to their environments?
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>Due to need for envelope to infect host cells which is damaged outside of a host
- explains why enveloped viruses are more reliant on direct contact for transmission |
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What is meant by incomplete latency?
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>Where a virus is infectious in some cells and silent in others.
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Draw a graph of Ab response in congenital rubella infection, including mother and foetus.
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Draw a graph of IgG and p24 Ag in primary HIV infection.
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Describe the indirect ELISA process.
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Draw a graph of immune response after HAV infection.
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What might this virus be?
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>Rotavirus
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What might this virus be?
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>VZV
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Describe the CPE of HIV.
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What are the different therapeutic strategies for virus infection?
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Inhibition at:
- Attachment - Penetration or fusion - Entry/uncoating - Viral DNA or RNA polymerases - Viral Reverse Transcriptase - Assembly/maturation (protease inhibition) - Release (neuraminidase inhibitors) |
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What is Acyclovir's mode of action?
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INHIBITION OF VIRAL DNA POLYMERASE:
>Acycloguanosine, a deoxyguanosine analogue acts as follows: - Acyclovir | (Herpesvirus TK) - Acycloguanosine-P | (Cellular kinases) - Acycloguanosine-PPP >Acycloguanosine-PPP inhibits herpesvirus DNA polymerase thus blocking synthesis of viral DNA >Acyclovir triphosphate both inhibits and acts as a substrate of the viral DNA polymerase - Competes with GTP and is incorporated into DNA leading to chain termination (Acyclovir lacks 3-OH group required for chain elongation) - Acyclovir resistant virus strains occur if there is a mutation in HHV TK or DNA Pol. |
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What is DNA's structure and how is it constructed?
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>Backbone of DNA consists of deoxyriboses linked by phosphodiester bridges
>3-OH of sugar moiety of one deoxyribonucleotide is joined to the 5-OH of the adjacent sugar by phosphodiester bonds >The variable part of DNA is its sequence of bases - Two purines are adenine (A) and guanine (G) - Two pyrimidines are thymine (T) and cytosine (C) >Nucleoside = base + deoxyribose (or ribose) e.g. adenosine, guanosine, cytidine, thymidine >Nucleosides can be phosphorylated by specific kinases int he cell on the sugar's primary alcohol group (-CH2-OH) referred to as 5', producing nucleotides (nucleoside monophosphate) which are the building blocks of DNA and RNA - Further phosphorylation leads to formation of diphosphates and triphosphates >DNA polymerase synthesises a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds - When a nucleotide is added to a growing DNA strand, two of the phosphates (Pyrophosphate) are removed and a phosphodiester bond is created so that the nucleotide is attached to the growing chain |
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How may HIV attachment be inhibited?
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>HIV binds to CD4 receptors on the surface of T cells, but binding to a co-receptor, CCR5 co-receptor is also required before the virus can enter the cell
>MARAVIROC (Celsentri) prevents binding to CCR5 co-receptors |
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Which type of viruses may be treated with nucleoside analogues i.e. acyclovir?
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>Most viruses which use DNA polymerase
- VZV - HHV - CMV >Other analogues include: - Ganciclovir (CMV >Foscarnet is a pyrophosphate analogue that inhibits herpesvirus DNA polymerase |
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How may HIV fusion be inhibited?
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>Peptide inhibitors such as enfuvirtide bind to the gp41 subunit of the HIV envelope glycoprotein
- prevent the conformational change required for viral fusion and entry into the cell |
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Why and how may HIV RT be inhibited?
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>Retroviruses e.g. HIV require RT to synthesis DNA from their RNA genome (some RT inhibitors also act against HBV - a DNA virus - with a complex replication cycle involving RT)
>NRTIs i.e. AZT inhibits this - MoA similar to Acyclovir although phosphorylation carried out by host kinases >NNRTIs e.g. Nevirapine - bind to RT near to the polymerase active site which becomes distorted - consequent inhibition of enzyme activity (allosteric inhibition) |
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How may HIV proteases be inhibited?
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>As virion matures, HIV protease cleaves a polyprotein to produce virus structural proteins
- Saquinavir mimics protease peptide substrate - Virus cannot mature and is not infectious |
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What is HAART?
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>HAART is designed to decrease the possibility of mutations
- Mutants of HIV are easily selected otherwise >At least three drugs used together: - NRTI, NNRTI and protease inhibitor - sub-maximal doses to minimise toxicity - efficacy and resistance can be monitored by measuring the amount of viral RNA in plasma (viral load) >HAART cannot eradicate latent HIV NB. QUAD is a 4 in one pill containing: - 2 NRTIs - Integrase inhibitor - Cobicistat (inhibitor of CYP450) |
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How is HIV integration inhibited?
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>Integration of viral genome is essential for
- maintenance of viral genome - genome expression and replication >HIV integrate responsible for integrating viral DNA into host cell - formation of provirus - incorporates DNA strands into host chromosome through strand transfer >Integrase inhibition prevents strand transfer :. formation of proviral DNA |
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How is interferon used in viral therapy?
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>IFN = cytokines which render cells refractory to a wide range of viral infections
- Used for HBV treatment - Combined with ribavarin (nucleoside analogue) for HCV |
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How is Influenza virus release from infected cells inhibited?
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>Newly formed influenza virus buds out of the infected cell
>Haemagglutinin on viral cell surface naturally binds to sialic acid on the cell surface >Neuraminidase (influenza enzyme) is essential for release since it removes sialic acid from the surface of the infected cell - Inhibitors block release because the haemagglutinin remains bound to sialic acid on the cell surface - E.g. Oseltamivir |
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How is Influenza virus uncoating and penetration inhibited?
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>Influenza haemagglutinin (virus attachment protein) binds to sialic acid (receptor) on the cell surface
- Taken up by vesicles / endosomes >In acid environment of endosome, protons enter virus via M2 protein ion transport channel - Allows uncoating of viral nucleic acid >Amantadine blocks the M2 protein ion transport channel, inhibiting uncoating |
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What are the key HIV inhibition strategies?
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What are the problems with HAART?
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>Toxicity
>Viral load rebounds if stopped >Resistance may develop if doses missed >Can't eradicate latent virus |
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Give an example of a therapy which inhibits each possible stage of virus replication.
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>Entry - HIV co-receptor and fusion inhibitors e.g. Maraviroc
>Uncoating - Amantadine >Nucleic acid synthesis - Nucleoside Analogues, NRTI and NNRTIs e.g. Acyclovir, AZT, Nevirapine >Integration - HIV integrase inhibitors e.g. Raltegravir >Maturation - HIV protease inhibitors - Saquinavir >Release - Neuraminidase inhibitors e.g. Oseltamivir |
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