Intro To Virology Lecture 1 and 2 Flashcards
virus description, replication scheme
Obligate intracellular parasite: a virus must enter a cell and use the existing metabolic pathways and macromolecules of cell, in order to replicate.
Extracellular virus or virions; unable to replicate outside of cell, antigenic proteins are components of viruses.
viruses replicate in a variety of host organisms, humans, animals, insects, fungi, bacteriophage, and plants.
replication of viruses can be lytic or productive infections result in the production of progeny.
Production of viral nucleic acids plus new viral proteins within an infected host cell combine to form progeny virus particles.
Extracellular virus: structural features
protein shell: capsid itself sometimes composed of subunits (capsomers), which are made of proteins. Other viruses have a protein capsid which is more like an amorphous mix of proteins. 1) Protects nucleic acid, 2) important for attachment of virus to cell surface receptors.
viral nucleic acid, RNA or DNA, not both, called viral genome.
+/- envelope, membranous outer structure of some viruses, surrounds capsid, accounts for either sensitivity of many enveloped viruses, protein spikes on envelope are often involved in virus attachment to cell receptors and are important antigens.
Classification of virus (5)
based on structural and biochemical characteristics, not disease characteristics.
size: poxvirus (largest) to parvovirus (smallest). Parvovirus are about the size of a ribosome.
shape of virus: Icosahedral (envelope), complex (poxvirus, retroviruses), helical envelope always represent for helical animal viruses.
number of capsomere: 252 capsomere for adenovirus.
Type of nucleic acid: only one type. Either RNA or DNA.
Classification of virus 2 (3)
Strandedness of nucleic acid: only one type per virus. Double stranded or single stranded DNA or RNA.
Size of nucleic acid: viral genome. Correlated with relative size of virus. Pox virus DNA (150 x 10^6) encodes 300 proteins; pox viruses are large and large double stranded DNA. Parvovirus DNA (1.5 x10^6) encodes 5 proeins; small virus and small DNA
Segmentation: 1 to 10 segments, depends on particular virus; influenza 8 RNA segments, but parainfluenza virus has a single genome.
Other criteria for classification: Genera, strains (7)
Ether sensitivity (enveloped virus usually sensitive, except for poxvirus)
heat, chemical, pH sensitivity, detergent susceptibility
antigenicity (capsid proteins and envelop spikes are viral antigens)
host cell specificity (cells i culture or cells within tissues in vivo are susceptible to infection by certain viruses only; tropism
type of morphological changes of infected cells (cytopathic effect, CPE)
disease in host organism (limited usefulness for classification, totally different viruses can cause diseases with same symptoms.
genetic relatedness: hybridization, similarity of nucleotide sequences between the nucleic acids of two viruses.
Confluent monolayer- stained
fibroblastic appearance of cells, elongated and spindle shaped.
viable cells absorb stain and acquire reddish appearance.
normal cells express contact inhibition (density dependent inhibition)
tumor derived or transformed cells grow in multilayers or (loose contact inhibition and have altered growth characteristics)
Viral infection of susceptible cells in tissue culture
Multiplicity of infection (MOI): # of infectious virus particles added per cell to initiate infection.
optimal conditions for infection:
1. Ions: Ca, Mg help virus to attach to receptor on cells.
- inhibitors of adsorption (attachment) - serum components, proteins, cross reacting antibodies; use serum-less media at time of infection.
- Confluent vs non confluent cells - certain viruses require cells at specific state of division or metabolism.
- Permissive vs non permissive cells influence lytic or productive infection. Permissive cells support replication to produce progeny virus. Non-permissive lack essential factors for complete viral replication, may be transformed by certain tumor virus.
Cytopathic effects (CPE) (3)
Rapid destruction of cells “lysis” (cytolytic), termination of host cell protein and nucleic acid synthesis.
slow cell death with continuous release of virus over a period of time.
enhanced cell proliferation with or without virus production, “transformed” cells, caused by DNA or RNA tumor viruses, cells do not die as a result of viral infection
cell maintenance with or without obvious effect on the cell; persistently infected cells, latenly infected cells, virus may no longer be evident but viral genome is present.
Quantitation of viruses and the detection of virus infected cells
Virus assay (infectious virus particles)- quantitaion of viruses
cytolytic viruses can form plaques (localized areas of dead cells in a cell monolayer) under agar; dead cells are not stained by neutral red, a single infectious virus particle can be initiate the formation of a single plaque, therefore on plaque equals on infectious virus particle.
plaque forming units (PFU) measure of infectious virus, 1 PFU = 1 infectious virus.
Virus with tumorigenic potential
can transform cells.
FOCI = proliferation of transformed cells in localized areas of a cell monolayer.
Focus forming unit (FFU) can be used to quantitate virus.
Virus assay (infectious and non infectious virus particles, no distinction) HA positive vs negative
Hemagglutination (HA); virus-erythrocyte lattice formation:
positive HA (+HA) = diffuse coating of erythrocytes on wells of plate, negative HA= pellet or button at the bottom of the wells which indicate no viral erythrocyte interactions and no lattice formation.
(some viruses have proteins on their surface which bind to receptors on the surface of specfic erythrocytes, virus protein is often called “hemagglutinin”.
detection of infected cells (4)
headsorption: RBCs attach to surface of infected cells via viral proteins (hemagglutinins) expressed on cell surfaces
immunofluorescence (If) and other staining procedures, “If” requries fluorescent Ab which specifically binds to viral proteins on infected cells, irradiate fluorescein with UV light, gives off visible light; cells stained with antibody- fluorescein look bright green under microscope
death of animals, lethal dose 50% (LD50)- 1 LD50 equals the amount of virus, which kills 50% of animals
infection of cells in cultures, tissue culture infectious dose 50% (TCID50), 50% of cell cultures show CPE at a particular dilution (amount) of virus; eg. 2 of 4 monolayers show CPE
Attachment
first step, virus attaches to the outer surface of the cell. Capsid or envelop proteins bind to the receptors on the surface of cells. Some cell types may lack receptors and therefore the virus will not attach and will not cause infection.
Factors influencing viral attachment to receptors (6)
- ratio of the quantity of virus particles: to infectious viruses (1:1 to 2000:1) Multiplicity of infection (MOI) number of virus infectious particles used to infect each cell.
- presence of appropriate receptors on cell surface. Specificity between virus structural protein and cell receptor, no receptor to no virus attachment to cell, receptors determine tissue or cell tropism.
- proper ion concentration Ca, Mg helps binding of virus to receptors
- lack of attachment inhibitors remove serum proteins from media
- attachment occurs over wide range of temperatures 4 to 37 C.
- Neutralizing antibodies bind to surface proteins of the virus and prevent the virus from bidning to the receptor block attachment.
Penteration
viropexis: phagocytic engulfment or endocytosis: cell cytoplasmic membrane engulfs virus that is attached to receptor. Virus is taken up in vacuoles, pH of endocytic vacuole is important for uncoating
fusion: second type of penetration, pertains to eveloped viruses, cell membranes fueses with viral envelope, dependent upon viral proteins which mediate fusion, many fusion proteins must be proteolytically cleaved to cause fusion.
occurs optimally at 37 C, minimally or not at all at 4 C.
