virus replicaiton

attachment

• what is the receptor usually

• what does the virus receptor interaction determine

• what do some viruses do

• how is the virus prepared for entry

• viral surfce proteins bind to receptors on cell surface

• receptor is cellular protein or glycan that a viral protein or glycoprotein has evolved to use

• virus receptor interaction determines specificty of viruses for cells and tissues

  • tropism

• soem viruses use mutiple receptors or coreceptors

• spike cleaved by enxymes prepares for entry

entry

• how is the virus released from endosome

• endocytosis

  • virus is released from endosome by pH change

  • fusion of viral envelope with endosomal membrane

• some enveloped virus fuse directly with the plasma membrane

uncoating

• release of viral nucleic acid from viral capsid

• some viruss, nucelic acid is still in a nucleoprotein complex

• some virses the capsid is only partially disintegrated

transcription of DNA viruses

• mRNA transcription in the nucleus using cellular RNA polymerase

• transport of mRNA to ribosomes in cytoplasm for translation

RNA viruses- transcription

• cells do not possess enzyme for transcription of RNA from RNA template

• RNA viruses need to use their own enzymes to make meesenger RNA (RdRp)

• most RNA viruses remain in cytoplasm for replication

• must avoid RNA degedation

early proteins

• non strutural eg polymerase to replicate

• and protein to shut down the hosts immune system

late proteins

• structural

• eg capsid and envelope glycoproteins

where is genome replication of dna and rna viruses

dna- in the nucleus

rna- in the cytoplasm

exceptions

• POX- dna makes viral factories in the ctoplasm (a dna dependent RNA polymerase)

• influenza- replicates in the nucleus

assembly

• ackaging of new genomes with viral proteins

• genomes needs a packaging signal

• concentrate viral components

  • eg negri bdy in rabies

• complex

  • influenza needs to package 8 segments

release

1. budding (enveloped viruses aquireenvelope through host lipid bilayer)

2. exocytosis

3. lysis- most unenveloped

culture of viruses in chicken egg

fertlilised eggs

sterile inoculation (eg growth of influenza in allantoic)

cell culture

• easy to handle and easy to scale up

• treatment with trypsin eaches cells from plastic

• cells are trasnferred to new flasks= passaging

• once cells growing well they may be used for virus culture or isolation

primary cell culture

• what is it

• how much passages can it survive before differentation

• cells freshly prepared from tissue sample

• can survive for about 10-15 passages before differentiation prevents further cell division

continuous cell lines

• what are they

• what are they often derived from

• what are they widely used for

• features

• immortalised cells that continue to grow

• often derived from tumours

• widely used for virus culture

• loss of receptors, major chromosomal abnormalitites

organoids

• 3d cell cultures derived from stem cells

  • increased realism as multiple cell types

  • can be grown for month

  • can model diff donors

  • expensive/difficult

• eg intestinal organoids used to grow norovirus

• human airway epithelial cells

isolation

• sample eg nasal swab, faeces, tissue from postmortem. freeze for long term storage

• inoculate cell cultures and wait for changes- cytopathic effect

cytopathic effets

• what are they

• exmples

• visible changes to cells following virus infection

  • cells round up and detach from flask

  • holes appear in cell layer

• syncytia formation: cells start to fuse creating very large cells with several nuclei and eventually cell death

• inclusion bodies (masses of viral proteins within hte cell)

non cytopathogenic

• no visible signs of replication but viral particles prodcued

• use staining or qPCR

infectiivty assays

to quantfy number of infectious virus particles produced eg plaque assay

plaque assay

Quantify plaque forming units (pfu)/ml:

10-fold serial dilutions of virus stocks/sample  Addition of agar overlay over inoculated cell cultures  Virions infect cells – give rise to plaques

Count plaques – calculate pfu/ml = virus titre