Lecture 1 TYJ Flashcards
What are viruses?
- 20 to 300 nm have to be visualized using an electron microscope
- cannot multiply on their own and cannot generate energy
- have to multiply within host while utilizing host cellular machineries
-infect almost every species on earth including animals, plants and bacteria - harm host cells = parasite
Two important characteristics of virus (genetic information) viral genome
1) genetic information (viral genome) and size
viral genome is either DNA or RNA
single stranded or double stranded
circular or linear
continuous or segmented
Two important characteristics of virus (morphology of viruses)
2) morphology of viruses
nucleocapsid: protein shell + viral genome
the viral genome is packaged in the capsid for protection
capsid is made up for capsomere (morphological subunit of viruses), repeated copies of it
3 types of capsids: helical, icosahedral and complex
envelope vs naked virus
envelop is a lipid bilayer adopted from the host cell, the surface proteins aka spike proteins aka glycoproteins are synthesized by viral progeny
when mature virion buds off from host cell membrane they can adopt the envelope, they can also adopt their envelop intracellularly from ER, golgi and nuclear membrane
enveloped virus protects the virus from degeneration by chemical and enzymatic agents but they are unstable in the environment
once the envelop virus loses the envelop, they can loose their infectivity as the spike proteins and the fusion peptides on the surface dictates its infectivity
Genetic economy dictates that
the viral genome is too small to encode all the machineries needed in its life cycle
hence it needs to enter into the host cells and use its machinery to replicate its genome and synthesis viral proteins (amplification)
viral life cycle (5 important steps)
1) attachment and entry
2) decoat its capsid to release viral genome either in the cytoplasm or into the nucleus for transcription and replication
3) protein synthesis using host enzymes and ribosomes, tRNAs
4) re-assemble and packaging of the viral genome into capsid
5) budding at plasma membrane via exocytosis
what are structural proteins and non structural proteins
structural proteins = proteins involved in packaging of the viral genome and re-assemble the viral progeny
non structural proteins = proteins involved in replication and amplification of viral genome and present during infection
identification of viral components in virus particles
genotypic methods: PCR and nucleotide sequencing
immunological methods: ELISA, serological testing and antigen testing kits (ART)
Genotypic methods
if viral genome is DNA then PCR can be conducted directly
if viral genome is RNA then have to use reverse transcriptase to reverse transcribe the RNA into cDNA and then PCR can be completed
requires prior knowledge of viral gene of interest sequence in order to design sequence specific primers (limitations)
immunological methods
antigen detection kits (eg: COVID test kits)
- requires lab made antibodies (primary and secondary antibodies conjugated with an indicator)
- primary antibodies recognizes structural proteins eg: nucleocapsid instead of spike protein because they can easily mutate
- eg: sandwich ELISA: primary antibodies present on microtire plates then inoculate patient sample (has antigen), primary antibodies bind to antigen followed by secondary antibodies binding to the antigen
methods to diagnose viruses (virus culture)
- primary virus culture, introduce host cells for the culturation
- continuous cell line, cells grow indefinitely due to mutation or is a cancerous cell
- culturing viruses in an embryonic egg
why do we have to culture viruses?
virus load too low requires amplification so that they can be used for diagnosis and vaccine development
what type of samples can be taken from infected person
blood serum, skin, nasal swab
what samples to take depends on tissue tropism
what is cytopathic effects (CPE) and how to view them
they can be viewed using electron microscope via positive and negative staining to visualize the morphology of the virus (eg: its envelop and its spike, its viral genome and its cytoplasmic proteins)
CPE is observed after the virus has infected its target host cells eg when T4 bacteriophage infects e.coli cells resulting in cell lysis and formation of plaque
What is immunofluorescence assay (IFA)
IFA uses antibodies conjugated with fluorescence agent that binds to viral proteins within host cells.
after an infection by viral agents, primary antibodies is able to enter into the host cell and bind to viral proteins within the infected cells, the secondary antibodies will then bind to the primary antibodies resulting in fluorescence
Serological method as a immunological method
Instead of generating lab made antibodies, serological testing assay involves lab made viral proteins
