Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
73 Cards in this Set
- Front
- Back
- 3rd side (hint)
|
DNA Virus Replication:
Polyoma virus (EX: SV40 virus) |
-Non-enveloped, icosahedral, circular dsDNA viruses
- Early and late functions -Multiple use of the same DNA sequence (alternative splicing, overlapping reading frames) -Multifunctional protein (T antigen) -Small genome- so not surprising that virus codes for a very limited number of proteins -Host cell provides RNA synthesis machinery, RNA modification machinery, DNA synthesis machinery, histones for packaging DNA -Bidirectional mode of replication (theta replication) |
|
|
|
DNA Virus Replication:
Adenovirus |
-Non-enveloped, icosahedral viruses
-Genome about 7x size of polyoma virus genome -linear dsDNA, associated with viral coded, basic proteins in virion -Unlike polyoma (SV40) viruses, adenoviruses do not use cell histones to package virion DNA -code for their own DNA polymerase and DNA packaging proteins use host factors in addition to viral proteins for DNA replication use host RNA polymerase and RNA modification systems and so nucleic acid synthesis needs to be in the nucleus -DNA polymerase cannot initiate synthesis de novo, they need a primer, the virally coded terminal protein (TP). This primer is thus found covalently linked to the 5' end of all adenovirus DNA strands -DNA replication mechanism: a strand displacement mechanism (NO Okazaki fragments), both strands are synthesized in a continous fashion -E1A: immediate early gene |
|
|
|
DNA Virus Replication:
Parvovirus |
-Non-enveloped, icosahedral capsid virus
-DNA replication occurs in nucleus -Alternating hairpin priming and nick priming for DNA replication |
|
|
|
DNA Virus Replication:
Poxvirus |
-large genome, enveloped, dsDNA virus
-replicate in the cytoplasm -provides their own mRNA and DNA synthetic machinery -Linear duplex that has covalently closed hairpin termini -nick priming -Cruciform Holliday intermediate |
|
|
|
DNA Virus Replication:
Herpes Simplex Virus |
-Linear, enveloped, icosahedral dsDNA virus
-replicates in the nucleus -use host RNA polymerase -use host RNA modification enzymes -3 origin of replications (2 oriS and 1 oriL) -The existence of cis-acting replication origins -high level of recombination |
|
|
|
DNA Virus Replication:
Cytomegalovirus |
-Lytic phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a cis-acting element, the origin, within which initiation occurs
|
|
|
|
DNA Virus Replication:
Herpesvirus |
-replication involves a rolling circle intermediate
-parental linear viral DNA is circularized shortly after entry into the host cell and that replication takes place predominantly by a rolling-circle mechanism, generating linear concatemers of tandemly repeated viral genomes |
|
|
|
Plus-stranded RNA viruses
|
-In these viruses, the virion (genomic) RNA is the same sense as mRNA and so functions as mRNA. This mRNA can be translated immediately upon infection of the host cell
-has the same "sense" as mRNA -does not need to be transcribed in order to be translated -translation of the viral genome gives rise to all the proteins necessary for viral replication |
HINT:
-Polio -Coxsackievirus -Hepatitis C |
|
|
Negative-stranded RNA viruses
|
-The virion RNA is negative sense (complementary to mRNA) and must therefore be copied into the complementary plus-sense mRNA before proteins can be made. Thus, besides needing to code for an RNA-dependent RNA polymerase, these viruses also need to package it in the virion so that they can make mRNAs upon infecting the cell
-not the same sense as a mRNA, but rather its complement. Therefore, in order to make mRNA, the genome must be transcribed before any viral proteins can be made |
HINT:
-Influenza -measles virus -mumps virus |
|
|
Double stranded RNA viruses
|
-the virion (genomic) RNA is double stranded and so cannot function as mRNA. Thus these viruses also need to package an RNA polymerase to make their mRNA after infection of the host cell
|
HINT:
-rotavirus -reovirus |
|
|
Sequence Relationships across Polymerases
|
-Little sequence homologies between different polymerase
-But can find 5 (or 4) common motifs -One motif (GDD) is often an indicator of a polymerase -Suggest common overall topology |
|
|
|
Retroviruses
|
-their virion RNA, although plus-sense, does not function as mRNA immediately on infection since it is not released from the capsid into the cytoplasm
|
|
|
|
Virus
|
-acellular, submicroscopic, obligate intracellular parasites
-Virus particles (virions) do not move on their own -replicate but do not "grow" or undergo division -Has a nucleic acid genome but does not encode machinery for the generation of metabolic energy or for protein synthesis |
|
|
|
Unifying principles of virology
|
-all viral genomes are packaged inside particles that mediate their transmission from host to host
-the viral genome contains the information for initiating and completing an infectious cycle within a susceptible cell -All viruses are able to establish themselves in a host population so that virus survival is ensured |
|
|
|
Poxiviruses
|
-belongs to the Poxiviridae family
-enveloped -largest, most complex human viruses -Linear, double-stranded DNA-200kb pairs -Brick or oval-shaped with complex symmetry -entire poxivirus lifecycle occurs in the cytosol -can replicate in enucleated cells |
|
|
|
Smallpox vaccine
|
-modern vaccine consists of live vaccinia virus
-no antiviral drugs to smallpox. However, vaccine can be given up to 4 days post-exposure to lessen severity of illness or prevent it |
|
|
|
What factors faciliated eradication of smallpox?
|
-limited host range
-no animal reservoir -no carriers -single, stable serotype -Very effective, inexpensive, stable vaccine -infection induces long-term immunity -No sub-clinical transmission |
|
|
|
Cell culture:
Continuous cell lines |
-fragmented, duplicated chromosomes
-can overgrow each other -essentially immortal |
|
|
|
Cell culture:
Primary cells |
-normal chromosomes
-contact inhibited -have a finite lifetime measured in divisions -do not have differentiated cell characteristics |
|
|
|
Multiplicity of infection (M.O.I)
|
-Average ratio of infectious viral particles to target cells in a given infection
|
Side Notes:
If a culture is infected with an MOI of 5, this is 5 PFU for every one cell in the culture |
|
|
Plaque Forming Unit (PFU)
|
-a unit of infectious virus determined by the ability of the virus to forma plaque or area of lysed cells on a monolayer of susceptible cells
|
Side Notes:
If a culture is infected with an MOI of 5, this is 5 PFU for every one cell in the culture |
|
|
Cytopathic effect (CPE)
|
-referes to degenerative changes in cells, especially in tissue culture, and maybe associated with the multiplication of certain viruses
|
|
|
|
Plaque Assay
|
-Often used to determine the titer of a virus prep
-performed by applying a suitable dilution of virus preparation to a confluent or semiconfluent adherent monolayer of suceptible cells |
|
|
|
Virus Titers
|
-The amount of virus present in 1mL
-expressed in PFU/mL |
|
|
|
Particle to PFU ratio
|
-ratio of total viral particles to infectious particles in a specific virus stock
|
Side Notes:
A particle to PFU ratio of 100 or more is typical for many viruses, indicating that many non-infectious particles are made by the infected cell |
|
|
One-step growth curve:
Eclipse period |
-immediately after the start of infection when fewer PFU are detected than you started with
|
|
|
|
One-step growth curve:
Latent Period |
-the time before the first new extracellular viral particles appear
|
|
|
|
One-step growth curve:
Yield |
-the net amount of virus made from the infection
-Unit: PFU/mL |
Side Note:
Yield = End titer- start titer |
|
|
Koch's postulates
(as applied to viruses) |
-the researcher must show that the same virus exists in every case of the disease studied
-The researcher must be able to isolate the virus and grow it in pure culture -THe researcher must be able to add the virus to susceptible test animals and create the disase once again -The researcher must be able to then isolate the virus from the infected animal, grow a new culture, and repeat the entire process |
Side Note:
SARS: Identification of a human coronavirus as the causative agent of severe acquired respiratory syndrome (Koch's postulates fullfilled in 30days) -Diseases with multifactorial causes are less amenabe to Koch's postulates -Modern methods such as sequence-based evidence are important in the absence of a pure culture (postulate 2) and/or the absence of an animal model of infection (postulate 3 and 4) |
|
|
2 types of cellular receptors involved in virus entry:
Attachment factors vs Entry receptors |
-Attachment factors: Non-essential BUT can enhance entry
-Entry receptors: Essential (Ex: binding of some enveloped viruses to a required receptor triggers conformational change in viral glycoprotein that is needed for membrane fusion and entry) |
|
|
|
Attachment factors:
Heparant sulfate (glycosaminoglycan) |
-Attachment receptor for herpesviruses, togaviruses, and retroviruses
|
|
|
|
Attachment factors:
DCSIGN and DCSIGNR (C-type lectins) |
-Attachment receptor for HIV, West Nile, Ebola viruses
-these virues do not enter and infect dendritic cells (DCs) but rather they "hitch a ride" -Intact virus is transported with the DC and disseminated to its intended target cell |
|
|
|
Diversity of virus-receptor interactions
|
-A single virus can use diverse receptors (HSV)
-the same receptor can be used by more than one type of virus (CAR receptor is used by both Coxsackie B and adenovirus serotypes) -There are common families of molecules that serve as receptors for viruses (Ig-like molecules, integrins, etc.) |
|
|
|
Receptor-binding domain
|
-Many viruses have evolved to bind to an exposed part of the cell receptor, often the external tip or amino-terminus
|
|
|
|
Co-receptors
|
-Some viruses require sequential binding to two distinct receptors prior to entry
|
Side Notes:
Adenovirus uses CAR receptor and integrin co-receptor -HIV uses CD4 receptor and chemokine co-receptor |
|
|
pH-dependent endocytic entry:
Direct vs Indirect mechanisms |
Direct: Acid pH triggers conformational change in viral surface protein
Indirect: Acid pH activates host protease that inturn acts on surface protein (i.e. Cathepsin) |
|
|
|
Conformational change in fusion glycoprotein can be triggered by:
|
-Low pH
-Protease cleavage -Receptor-binding |
|
|
|
Amantadine
|
inhibits Influenza uncoating
|
|
|
|
Signal peptide
|
An N-terminal stretch of approximately 20-40 hydrophobic aa that ensures targeting to the ER
-cleaved off of the mature protein in teh ER by signal peptidase |
|
|
|
Fusion peptide
|
-A stretch of relatively hydrophobic aa within a membrane anchored fusion protein
|
|
|
|
Emerging Pathogen
|
-A new, re-emerging or drug resistance infection with increased incidence in humans over the past 20 years or whose incidence threatens to increase in teh future
|
|
|
|
Viral Hemmorhagic Fever
|
-A severe, multi-system syndrom involving damage to the vascular system and discruption of homeostasis
-Hemorrhage is a hallmark symptom, but rarely the cause of death -Caused by several distinct families of viruses including: Filoviruses, Bunyaviruses, Arenaviruses, and Flaviviruses -Many VHF virses are emerging pathogens that cause severe life-threatening disease |
Causes of emergence:
-changes in land use -changes in agriculture -changes in demogrpahics -societal changes |
|
|
Filoviruses
|
-Enveloped, filamentous, pleiomorphic
-Linear, ssRNA negative polarity -14-19kb -can cause severe illness in humans and non-human primates -biosafety level 4 agents -replication of RNA genome occurs in cytosol -Newly assembled, progeny virions are released via budding from the plasma membrane -infections appear to zoonotic -reservoir is unknown -Transmission is person-to-person via close contact or contact with bodily fluids -Airborne transmission is not likely -No vaccines -No specific antiviral drugs -Diagnosis can be diffiult, especially if there is only one case involved -Many symptoms are similar to other infectious diseases such as malaria and typhoid fever |
|
|
|
Ebola virus
|
-belongs to the Filoviruses family
-5 unknown subtypes -RNA virus w/ a high degree of sequence conservation (very unusual) |
|
|
|
Enfuvirtide (T-20)
|
-an antiviral drug that works at the level of viral entry
-successful as part of combination therapy for HIV - a 36 aa peptide that binds to HIV gp41 and prevents conformational change and fusion |
|
|
|
Hepadnaviruses
|
-family of viruses which can cause liver infection in humans and animals
-Enveloped, DNA viruses -replicate through an RNA intermediate |
|
|
|
Retroviruses
|
-an enveloped, RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome
-The DNA is then incorporated into the host's gemone by an integrase enzyme -The virus thereafter replicates as part of teh host cell's DNA |
|
|
|
Accessory Proteins
|
-May faciliate recognition of initiation site
-May stimulate polymerase activity -May unwind the product so that additional rounds of synthesis can occur |
Side Notes:
RNA polymerase doesn't act alone. Other proteins may direct the polymerase to the corret intracellular site |
|
|
Polio Virus
|
-single ORF
-is an mRNA because it can be translated into protein in an in vitro translation system -However, it is different from cellular mRNA's -Polyadenylated at 3' end -Part of the genome, not added by cellular enzymes |
|
|
|
Sindbis Virus
|
-genome contains a cryptic ORF
-mRNA from this is formed following a transcription step |
|
|
|
Coronovirus
|
-Capped and poly-A
-at least 5 separate translation reading frames -at least 6 subgenomic mRNA's |
|
|
|
Bunyavirus
|
-Tri-partite
-cytoplasmic -Cap snatching -subgenomic RNAs -Each segment shows a different expression strategy |
|
|
|
Ambisense
|
-refers to the small genomic segment of some bunyaviruses and adrenaviruses
-Coding information on both + and - strands -can't serve directly as a mRNA, but an mRNA of the same polarity is made |
|
|
|
Reovirus
|
-very stable dsRNA
-10 genomic segments -mRNA capped but not polyA -segments of different sizes -segments are capped on the + strand but 5'triphosphate on - strand; neither polyA -Each segment makes one protein |
|
|
|
Antivirals
|
-THe major targets of currently used antivirals are viral replication enzymes, proteases, and entry and exit pathway
-Inhibitors of nucleic acid synthesis -Protease inhibitors -Entry and fusion inhibitors -Integrase inhibitors -Miscellaneous anti-viral agents |
|
|
|
Inhibitors of nucleic acid synthesis-nucleoside analogues
Zidovudine (ZDV or AZT) |
-the analogue of nucleoside and converted to triphosphate analogue of dNTP by cellular kinases
-inhibits HIV reserve transcriptase -anti-HIV medication -HIV can rapidly develop resistance to it -These drugs are neuropathic (or cause neuropathy) |
|
|
|
Inhibitors of nucleic acid synthesis-nucleoside analogues
Zidovudine (ZDV or AZT) |
-analogue of thymidine and converted to triphosphate analogue of dTTP by cellular kinases
-HIV reverse transcriptase inhibitors -These drugs are neuropathic (or cause neuropathy) -Others in the same group: Didanosine (ddl), Zalcitabine (ddC), and Stavudine (d4T) |
|
|
|
Inhibitors of nucleic acid synthesis-nucleoside analogues
Lamivudine (3TC) Emtricitabine (FTC) |
-analogues of cytosine and converted to triphosphate analogues of dCTP by cellular kinases
-inhibit reverse transcriptase and hepatitis B viral DNA replication -Useful for HIV/Hepatitis B co-infection and no dose-limiting side effects -Combivir, is a combination of the two antiretroviral drugs: lamivudine (Epivir) and zidovudine (Retrovir) |
|
|
|
Inhibitors of nucleic acid synthesis-nucleoside analogues
Abacavir (ABC) |
--use with other antiretroviral drugs in the treatment of HIV infection in adults and children 3 months of age or older
-Epzicom is a combination of two antiretroviral drugs: abacavir sulfate (Ziagen) and lamivudine (Epivir) -Trizivir includes three antiretroviral drugs: abacavir sulfate (Ziagen), lamivudine (Epivir), and zidovudine (Retrovir) |
|
|
|
Inhibitor of nucleic acid synthesis-non-nucleoside reverse trnascriptase inhibitors (NNRITs)
|
-Four drugs have been approved by FDA: Nevirapine (NVP), Etravirine (ETV), Delavirdine (DLV), and Efavirenz (EFV)
-they bind directly and noncompetitively to HIV reverse transcriptase and block DNA polymerase activity -THe most common side effects: rash and liver toxicity -Resistance emerges rapidly when it is used alone |
|
|
|
HIV protease inhibitors
|
-Peptidomimetic substrate analogues of HIV and bind competitively to the active site of HIV protease
-inhibits HIV protease and prevent the cleavage of viral polyproteins precusors and the formation of mature viruses -very toxic drugs w/ common side effects include headache, vomiting, diarrhea, dyslipidemia, insulin resistance, lipodystrophy, and cardiovascular disease -Drugs: Indinavir, lopinavir, nelfinavir, ritonavir, and saqunavir & (non-peptidic protease inhibitors) Darunavir and tipranavir |
|
|
|
Entry and fusion inhibitors: Maraviroc
|
-an entry inhibitor
-blocks the chemokine receptor CCR5 -can't block T-tropic virus (CXCR4 as co-receptor) |
|
|
|
Entry and fusion inhibitors:
Enfuvirtide (T20) |
-a 36 aa synthetic peptide
-disrupts the HIV-1 molecular machinery at the final stage of fusion with the target cells -administered by subcutaneous injection -The most common adverse side effects include: injection site reactions, peripheral neuropathy, depression, cough, and infections |
|
|
|
Untegrase inhibitors:
Raltegravir |
-targets HIV integrase which integrates the viral genetic material into human chromosomes
-Raltegravir: only approved for use in individual whose infection has proven resistance to other HAART drugs -most commonly side effects: diarrhea, nausea, and headache |
|
|
|
Highly Active Anti-retroviral Therapy (HAART)
|
-Cocktail of agents (three or four different drugs)
-to prevent the development of drug resistance -Effective in reducing HIV plasma concentrations -Not eliminating latent proviral DNA |
|
|
|
Anti-Herpersviruses
|
-Nucleoside and nucleotide analogues
-Inhibit DNA replication -Require phosphorylation by a viral enzyme as well as cellular kinases for activation |
|
|
|
Inhibitors of nucleic acid synthesis-nucleoside analogues
|
-This class of drugs not only inhibits the synthesis of nucleosides but also inhibits the polymerases that incorporate nucleosides into RNA and DNA
-In addition, some of these drugs are incorporated into nucleic acids and block their elongation -Acyclovir- the first effective anti-HSV agent and has been used for the treatment of serious HSV infections for the past 25 years |
|
|
|
M2 Protein inhibitors
|
-only inhibiting Influenza A by inhibiting viral genome uncoating in the host cell
-interferes with a viral protein M2 (an ion channel) which is required for the viral particle to become "uncoated" once it is taken inside the cell by endocytosis |
|
|
|
M2 Protein inhibitors:
Amantadine |
-M2 protein inhibitors BUT has been associated iwht several CNS side effects
|
|
|
|
M2 Protein inhibitors:
Rimantadine |
-inhibits influenza's viral replication, possibly by preventing the uncoating of the virus's protective shells
-Resistance to rimantadine can occur as a result of an amino acid substitution at certain locations in transmembrane region of the virus M2 protein. This prevents binding of the antiviral agent to the channel -can produce gastrointestinal and CNS adverse effect -produces fewer side effects than other anti-viral influenza treatments -safer for elderly |
|
|
|
Limitation of the current antivirals
|
-Narrow antiviral spectrum
-ineffectiveness against the latent virus -development of drug-resistant mutants -toxic side effects |
|
|
|
Antiviral resistance vs clinical resistance
|
Antiviral resistance: the decrease in susceptibility to an antiviral drug
Clinical resistance: viral infection fails to respond to therapy. This may or may not be due to the presence of a drug-resistant virus. It also depends on other factors such as the patient's immunologic status and the pharmacokinetics of the drug for that individual patient |
|
|
|
Phenotypic Assay vs Genotypic assay
|
Phenotypic Assay: in vitro susceptibility assays that measure the inhibitory effect of antiviral agents on the entire virus population in a patient isolate
Genotypic Assays: analyze viral nucleic acid to detect specific mutations that cause antiviral drug resistance |
|
