Virology wk 1

  1. difference between virulence and pathogenesis
    Virulence is quantifiable (LD50, etc)
  2. cytokines for fever
    IL-1, TNF-alpha, IL-6
  3. lymphadenopathy (swollen LN) cause
    lymphocyte mobilization and proliferation (caused by viral antigen, etc)
  4. transmissibility
    • ability to spread between members of the same species
    • could cause dead-end infection
  5. 6 stages of viral replication
    • Enter host cell (fusion/endocytosis)
    • traffic to site of replication (nucleus or cytoplasm)
    • replicate genome and express viral proteins
    • assemble new virus particles
    • traffic to site of exit
    • release of new virions (budding, exocytosis, lysis)
  6. direct fusion
    • how some enveloped viruses enter a cell (herpes, retrovirus)
    • envelope fuses with plasma membrane of host cell, leaving nucleocapsid to enter
  7. receptor-mediated endocytosis
    • Most common way for viruses to enter cells (adeno, parvo, influenza)
    • Cell phagocytizes/pinches off vesicle with virus inside, envelope fuses with vesicle to release nucleocapsid
  8. direct entry
    • way some unenveloped viruses enter cells (picorna)
    • bind, recruit more receptors, conformation change in capsid to expose fusion pore and inject nucleic acid into cell
  9. Viral exit strategies
    Exocytosis (lysogenic, enveloped virions), vs lysis (non-envelope)
  10. DNA virus replication occurs in the
    Nucleus (except with pox virus)
  11. host dependency of viruses
    • always depend on host cell ribosome
    • some viruses make own enzymes and are less dependent on hosts, but there are more viral targets for drugs.
  12. DNA viruses are more _________ than RNA viruses
    stable (more mistake checks and fixes)
  13. DNA viruses replicate in the _________, RNA viruses replicate in the __________
    • nucleus
    • cytosol 
    • Pox is a DNA that replicates in cytosol.  
    • Influenza also an exception
  14. DNA viruses are mostly ________, RNA viruses are mostly _________
    double stranded, single stranded
  15. parvovirus
    • Non-enveloped, icosahedral capsid ss DNA virus, small ("parvum" = small) (26nm, 2 genes)
    • Receptor: Transferrin (cat/dog) or sialic acids
    • Entry: Receptor-mediated endocytosis
    • Exit: cell lysis
    • can ONLY replicate in ACTIVELY dividing cells, very HOST dependent
  16. Parvovirus replication
    • ssDNA to dsDNA by HOST enzymes
    • ds DNA attracts HOST transcription factors and RNA polymerase, makes viral protein (NS1)
    • NS1 binds to dsDNA letting HOST DNApolymerase make new ssDNA copies
  17. Adenovirus
    • non-enveloped, icosahedral capsid dsDNA virus, medium (90nm, 30,000 bases, 22-40 genes)
    • Early and late genes
    • specialized fiber attached to capsule to reach through mucus layers and attach (respiratory disease)
    • Receptor: Integrins (RGD), epithelial cells, canine parvovirus
    • Entry: receptor-mediated endocytosis
    • Exit: cell lysis
  18. Adenovirus replication
    • endocytosis, endosomal acidification, capsid releases nuclear material into nuclear pore.  
    • early phase genes (host RNA polymerase) sent to cytoplasma, translated to early viral proteins for DNA synthesis (viral DNA polymerase) and to interfere with host
    • Late phase genes using viral DNA polymerase make proteins that are exported and turn into viral proteins and structural packaging
  19. Herpesvirus
    • enveloped, icosahedral dsDNA genome (large - 120-250nm, 170,000 bases, 100-200 genes) with tegument (VP16 and virion host shutoff protein).  
    • Immediate, early and late genes.  
    • Receptor: viral glycoproteins
    • Entry: direct fusion
    • Exit: exocytosis
    • can establish latency ("herpein" to creep) in neurons or immune cells (activity but no infectious virus made), then reactivates (ANY TIME.  Viral vs clinical latency
    • immediate early, early and late
  20. viral "footprint"
    viral glycoproteins left on cell membrane after direct fusion (herpes), can signal host immune
  21. Herpesvirus replication
    • direct fusion of envelope, release VP16 an nucleocapsid, transport to nucleus, inject dsDNA in nuclear pore
    • immediate early protein transcription initiated by VP16, products initiate expression of early proteins (host RNA polymerase).  Early proteins are viral DNA polymerase, DNA binding proteins, viral enzymes etc, initiate expression of late proteins.  
    • late proteins are mostly structural.  Some stay in cytosol to surround nucleocapsid, some return to nucleus to assemble.
    • inner nuclear membrane makes envelope they use (perinuclear space) to get through outer nuclear membrane.Late proteins that stay in cytosol gather around nucleocapsidgolgi gives vesicle/envelope and mature glycoproteins.  Exocytosis gives second envelope.
  22. concatemeric DNA
    Long strands of repeating viral DNA without 5' caps, get cut into appropriate sized pieces to go into capsids.
  23. Poxvirus
    • enveloped brick-shaped capsid dsDNA, HUGE (300nm, 300,000 bases, 200 genes)
    • Receptor: variety
    • Entry: direct fusion
    • Exit: budding (envelope) vs lysis (no envelope)
    • REPLICATES IN CYTOPLASM (replication like Herpes), so no access to host RNA polymerase.  Have viral DNA-dependent RNA polymerase present in virion and released at binding.
  24. Retrovirus
    • enveloped positive sense RNA virus (100nm, 10,000 bases.  Gag (capsid), Pol (reverse transcriptase, integrase), Env (envelope)
    • retroviruses have multiple protein shells
    • Receptor: CD4 and co-receptors (CCR5, CXCR4)
    • Entry: direct fusion (usually) some endocytosis
    • Exit: budding (immature - viral protease cuts to make mature virions)
  25. replication of Retrovirus
    • DNA intermediate necessary
    • Reverse transcription turns ssRNA into dsDNA via RNA-dependent DNA polymerase via reverse transcriptase, and integrase incorporates the dsDNA provirus into host DNA.
  26. how herpesvirus avoid immune system
    • viral proteins bind chemokines (gG)
    • viral proteins mimick cytokines (vIL-10)
    • downregulation of MHCI and MHCII
  27. Three kinds of herpesviruses
    • Alpha: most diseases, short replication cycle, latency in neurons
    • Beta: important in immunosuppressed, slow replication cycle, latency in monocytes
    • Gamma: chronic infections (asymptomatic tumors), variable replication cycle, latency in lymphocytes
  28. alpha herpesvirus
    • causes most diseases
    • productive infection, usually epithelial (primary from mucosal surfaces)
    • short replication cycle
    • latent infection in sensory neurons (some exceptions like EHV-1 and MDV use leukocytes)
  29. beta herpesvirus
    • Humans: mostly only immunosuppressed
    • animals: bats, macaques, elephants (EEHV = elephant endothelioropic herpes virus)
    • productive infection in variable cells
    • slow replication cycle
    • Latent in monocytic precursors in bone marrow
    • causes cytomegaloviruses due to syncytia/giant cells (multinucleated)
  30. gamma herpesvirus
    • humans: asymptomatic chronic infections to tumors (sarcoma, lymphoma)
    • animals: many, few important (MCF, EHV, maybe feline gammaherpesvirus)
    • productive infection in lymphoid cells (+/- epithelial and fibroblast)
    • variable replication
    • genes induce viral and cellular transformation
  31. canine herpesvirus type I
    • alpha, common asymptomatic infection
    • Primary of respiratory/corneal, long term of viral shedding from vagina or respiratory, latency in trigeminal and sacral ganglia
    • Most important in <2wk pups (chilled, vasculitis causes DIC) and immunosuppressed older (chemo, latency).  high mortality in pups
    • Clinical signs age-dependent
    • neonates: crying, anorexia, dyspnea, rapid death, 80-100% mortality
    • older pups: milder symptoms, fever
    • adults: usu asymptomatic.  Repro disorders (abortion, infertility, fetal mummification), respiratory, ocular disease (primary conjunctivitis, recurrent keratitis)
  32. clinical signs of canine herpesvirus type 1
  33. Feline herpesvirus type I
    • alphaherpesvirus, most cats infected.  
    • causes feline viral rhinotracheitis, oral and nasal spread (aerosol).  Often coinfects with calicivirus.  part of feline respiratory disease complex
    • Latency in trigeminal ganglia
    • primary infection in mucosal/epithelial, cell lysis, ulceration, inflammation, sneezing, clear discharge.  Possible abortion in queens
  34. suid herpesvirus type I
    • alphaherpesvirus of swine, aka pseudorabies virus (PRV) or Aujeszky's
    • most important swine disease (economics)
    • pigs primarily, but also ruminants, cats, dogs, wild mammals *usually fatal in non-pig hosts
    • age-dependent signs
    • piglets: fever, anorexia, neuro (trembling, paddling), rapid death
    • Older piglets: same signs with vomiting, respiratory problems, lower mortality
    • adult pigs: mild or no symptoms, some repro, resp or neuro problems
    • feral: often asymptomatic
    • Cows: bellowing, mad itch, death
    • horses: mad itch, death (VERY rare)
    • dogs: excessive lacrimation, howling, seizures, death
    • cats: seizures, sudden death
    • rodents: seizures, death
    • humans: no clinical signs
  35. Gallid herpesvirus type I
    • alpha of poultry (infectious laryngotracheitis (ILT))
    • control with biosecurity
    • most common in 4-18month in open range or backyard
    • morbidity 100%, mortality 10-70%.  HIGHLY contagious, affects pheasants too
    • primary respiratory epithelium, erosion ulceration and hemorrhage in trachea.
    • Latency in trigeminal ganglion and in trachea
    • reactivated with stress
    • nasal discharge, sneezing, coughing, gasping, labored breathing, conjunctivitis, depression, reduction in egg production
    • sloughing of epithelium and clotted blood form diphtheritic membranes occluding bronchi, syrinx, trachea
  36. gallid herpesvirus type II
    • alpha of chickens, aka Marek's disease (MDV)
    • latency in lymphocytes (like a gamma)
    • Turkeys have their own (HVT) that is used as a modified live vaccine given pre-hatch or at 1 day. Other birds, mammals not susceptible
    • spread by inhalation, can cause paralysis or neurologic disease (transient or permanent ataxia as lymphomas press on nerves), depression, reduction in eggs, atherosclerosis, early mortality
    • irregular pupil margin, enlarked feather follicles, neoplasms (lymphomas)
  37. "borrowed life"
    • obligate intracellular parasites (need host to replicate)
    • can't capture or store free energy, or move independently outside host
    • incredibly adaptive
  38. capsid
    protein shell surrounding nucleic acid
  39. nucleocapsid
    capsid plus genome *mostly helical nucleocapsids of enveloped viruses
  40. envelope (viral membrane)
    • lipid bilayer enclosing viral capsid/nucleocapsid.  
    • Viral glycoproteins or spikes are inserted in envelope for host specificity
  41. virion
    complete infectious virus particle
  42. tegument
    unstructured layer of proteins found between envelope and capsid of herpesvirus
  43. spike (glycoprotein)
    trimer of glycosylated viral proteins inserted in envelope to bind to cell surface receptors and mediate virus attachment and entry
  44. viral strain or isolate
    a genetic variant below species level
  45. viral serotype
    based on presence of unique surface antigen recognized by antibody or serum (differences)
  46. viral serogroup
    viruses of different serotypes that can be grouped based on shared surface antigen (share one marker, but not all)
  47. viral pathotype or biotype
    classification based on disease/pathological outcome
  48. difficulties in diagnosing viral infection
    • too small to see in a microscope
    • cannot be grown in cell-free media
    • not all can be grown in cultured cells
    • may have limited time frame in some samples
    • fragile, easily inactivated (drying, detergents)
  49. diagnostic techniques for virus: electron microscopy
    • thin-section or negative stain
    • can see general shape, distribution of morphological subunits
    • can sometimes ID family
  50. diagnostic techniques for virus: viral isolation
    • only test for INFECTIOUS virus.  very sensitive, definitive if +, can take tays to weeks, must be culturable, needs experienced lab, not all viruses cause cytopathic effect (like rounding or detachment of cells or syncytium)
  51. virus titer
    • measure of infectivity
    • concentration of INFECTIOUS virus
  52. diagnostic techniques for virus: Plaque assay
    • various dilutions, innoculate, wait, remove innoculation, overlay (so can't wander), fix/stain, count
    • 9 plaques at 10^-5 dilution with 0.1mL (9x10^6 due to 0.1) = 9x10^6 Plaque Forming Units (PFU)/mL
  53. diagnostic techniques for virus: direct or indirect immunofluorescence assay
    • direct: testing for antigen by adding fluorescent antibody
    • indirect: testing for antibodies by adding fluorescent antibody FOR antibody.
  54. diagnostic techniques for virus: Enzyme-linked immunosorbant assay (ELISA)
    • enzyme converts substrate into color
    • fast, sensitive, in-house with kits, + means virus is present.  Does not measure infectious virus or infectivity
    • can be direct, indirect or sandwich/capture
  55. diagnostic techniques for virus: PCR
    • Polymerase chain reaction for DNA viruses
    • reverse-transcription polymerase chain reaction for RNA viruses
    • fast, sensitive, makes reagents to detect any virus
    • does not measure infectivity, specialized equipment, contamination causes false +
  56. diagnostic techniques for virus:  hemagglutination assay (HA)
    • viruses can bind and cross-link sugar molecules on RBC surface, form a lattice and don't settle out
    • hemoagglutination titer is the lowest dilution where agglutination still occurs (no dot).  If not crosslinked, sinks to bottom to make a dot.
  57. diagnostic techniques for virus:viral antibody assay
    • checks for correct vaccination
    • ELISA, hemoagglutination inhibition assays, serum neutralization (virus mixed with patient serum, positive titer is dilution that reduces plaques by 50%).
Card Set
Virology wk 1
IV Virology week I