Virology L4&5 Flashcards
EFFECT OF VIRAL INFECTIONS ON HOST CELLS
Mechanism of cell injury
Inhibition
§ Host cell ? ? synthesis
§ Host cell ? transcription
§ Host cell ? synthesis
Cytopathic effect
§ ? viral proteins
§ Interference with cell ? function e.g. uptake of Na etc.
Neoplastic transformation (transforming the cell into a ? cell;
characteristic of cancer cell: uncontrolled growth, thus virus can cause cancer (15% of cancer))
Drive host cell to apopotosis
Non-? changes (persistent)
(- need no specific changes in a cell but that allow resistance over time and then gradually lead to progressive immunodeficiency
That’s a key for immunodeficiency virus. Usually they don’t cause disease or clinical signs at the first moment of infection, but gradually progressive)
EFFECT OF VIRAL INFECTIONS ON HOST CELLS
Mechanism of cell injury
Inhibition
§ Host cell nucleic acid synthesis
§ Host cell RNA transcription
§ Host cell protein synthesis
Cytopathic effect
§ toxic viral proteins
§ Interference with cell membrane function e.g. uptake of Na etc.
Neoplastic transformation (transforming the cell into a cancer cell;
characteristic of cancer cell: uncontrolled growth, thus virus can cause cancer (15% of cancer))
Drive host cell to apopotosis
Non-cytocidal changes (persistent)
(- need no specific changes in a cell but that allow resistance over time and then gradually lead to progressive immunodeficiency
That’s a key for immunodeficiency virus. Usually they don’t cause disease or clinical signs at the first moment of infection, but gradually progressive)
example of Abnormal cell growth
- Thymic lymphoma in a Feline Leukemia Virus (retrovirus) positive cat
- Large skin lesions caused by Papilloma virus infection in giraffe (Kruger NP)
-> secondary infections also involved as there would be puss dripping form the skin leisions thus attracting bacteria and parasites so they usually don’t survive
-> It’s a benign, abnormal cell growth. But the extent with which this can have detrimental effects on the animals.
Cell damage and cell death
Abnormal cell growth
(cancer)
Cell damage/death
§ ?
§ Cell membrane ?
§ ? (cell suicide)
No apparent changes
§ Persistent
§ Latent (e.g. herpes)
§ Immuno-suppression
Cell damage and cell death and no apparent changes
Abnormal cell growth
(cancer)
Cell damage/death
§ lysis
§ Cell membrane altercation
§ apoptosis (cell suicide)
No apparent changes
§ Persistent
§ Latent (e.g. herpes)
§ Immuno-suppression
pic: our diagnostics rely on Can we see changes in the cells and those that reflect a viral infection? Because in histology I can’t see one single virus here in pic
What I will see is the change induced in the cell so changed cell membrane apoptosis lysis and so.
Viruses with specific host range and effect
BACTERIOPHAGES
§ Viruses that infect bacteria and can ? bacteria
§ Applied in ? therapy
ONCOLYTIC VIRUSES
§ Viruses that infect and kill ? cells through ?
§ Applied in ? therapy through stimulation of host ?-tumor ? response
Viruses with specific host range and effect
BACTERIOPHAGES
§ Viruses that infect bacteria and can kill bacteria
§ Applied in phage therapy
ONCOLYTIC VIRUSES
§ Viruses that infect and kill cancer cells through oncolysis
§ Applied in cancer therapy through stimulation of host anti-tumor immune response
(One of the advantages of using oncolytic viruses is that some of these oncolytic viruses are very specifically affecting one type of cancer cell.
So it does have no effect on other cells in your body.
As we all know, cancer (harsh) therapy comes with a lot of side effects.
Some of the herpes viruses and adenoviruses, they’re oncolytic viruses.)
Viral growth curve
§ Timing of a one-step growth cycle varies depending on virus and host
§ Bacterial viruses:20-60 min; animal viruses: 8-40h
(bacterial viruses, usually they have a much faster replication cycle, but that’s because they’re adapted to their host, which has a fast replication cycle as well.
it takes about 20 to 60 minutes for a bacterial virus bacteriophage
to complete its replication cycle and to have the release of new variants.
Animal viruses could take a little bit longer, but that’s still very short if you think about our replication cycles in time.
So the viral growth curve has this special shape in which you can see that you have here the number of infectious units decreasing, dropping when the viruses enter our cells because they’re no longer in the extracellular matrix,
and then you’ll have an increase of the intracellular viruses the moment you have your assembly/maturation step
and then they’re finally released into the surrounding tissues. So outside of the cells. And then you see again an increase in extracellular virions (dotted line under release)
This is something that we measure in the lab and this is a curve.
So you’ll have viruses that will enter that will disappear from your extracellular matrix. and later appear again
*** The LATENT period is that specific period during which a virus will have its uncoating, replication, and assembly step.
In theory, if you look at one virus infecting one cell, you have no viruses outside of the cell.
*** Within that latent period, we defined the ECLIPSE period as the time during which the infectivity of the virus disappears.
And remember, the encoding step is that moment when it starts to lose its infectivity. Because the viral genetic material by itself cannot cause infection in animal viruses. So the eclipse spirit is that point when they are in step three, uncoating and step 4 replication cycle (eclipse, remember it with the lunar or solar eclipse and this disappearance, so disappearance of infectivity)
§ Eclipse: infectivity of the virus ? during ? and ? stage
§ Latent period: replication of viral ? acid and ?
§ Maturation period: assembly of viral ? and ? into mature virus particles. If at this time cells are broken, active virus can be detected
§ Eclipse: infectivity of the virus disappears during uncoating and replication stage
§ Latent period: replication of viral nucleic acid and protein
§ Maturation period: assembly of viral genome and protein into mature virus particles. If at this time cells
are broken, active virus can be detecte
Viremia = Spread of viruses via the ?
§ ? viremia: direct inoculation of virus in host’s bloodstream and no replication at site of entry (e.g. contaminated syringe, bite of arthropods)
§ ? viremia: viremia following virus replication in host
§ ? viremia: spreading into the blood from infected area
§ ? viremia: spreading to other organs/tissues
Viremia = Spread of viruses via the bloodstream
§ passive viremia: direct inoculation of virus in host’s bloodstream and no replication at site of entry (e.g. contaminated syringe, bite of arthropods)
(notes: So direct inoculation, you don’t have a first replication cycle somewhere in the body.
§ active viremia: viremia following virus replication in host
(here can retrieve viruses from blood, After they have replicated somewhere in the body. So first, a replicate, for example, in the skin to organ systems through blood)
from active viremia can lead to 2 below:
§ primary viremia: spreading into the blood from infected area
§ secondary viremia: spreading to other organs/tissues
So imagine here you have viruses that will infect your body’s surface epithelial cells.
Some viruses stay their papilloma virus as an example.
They cause these warts and they will have a localized replication in the epithelial cells.
Then we have the movement of certain virions through lymph nodes, lymph channels into the blood, and we have this primary viremia.
So the first spreads here after the initial replication, and it goes to other organ systems, or it could continue in the blood after replication here
And then we have a secondary viremia, secondary viremia bringing the viruses to, for example, respiratory tract or back to the skin or, for example, your neurological system.
And here an example of kidneys.
Each time you find a couple of examples of these viruses, don’t study this in a vacuum, we will come back to canine distemper, rinderpest.
- knowing the path of viruses is imp. for vets to know where to take the sample from the host.
(Luckily, as we will talk about in diagnostics, showing the virus is not the only thing we can do in diagnostics.
We can look for antibody development, we can look at responses for the immune system. So we don’t always need to show a virus in order to diagnose an viral infection)
iMP SLIDE
§ Acute infection: ? onset of disease and symptoms (e.g. ? is common cold, Rotavirus, Influenza virus) and later it decreases as well
acute infection can lead to:
§ Latent infection: virus remains in ? host cell for long periods (e.g. Herpes simplex virus)
§ Chronic infection: often with ? shedders that have constant or intermitted shedding, can go ? (e.g. BVD) bovine viral diarrhea
§ Persistent infection: after initial viral infection proliferation is ? but viral genome not fully ? (e.g. FIV)
§ Persistent slow infection: characterized by a ? incubation period followed by ? disease (e.g. Jaagsiekte Sheep retrovirus, Lentiviruses)
§ Acute infection: rapid onset of disease and symptoms (e.g. Rhinovirus, Rotavirus, Influenza virus)
§ Latent infection: virus remains in asymptomatic host cell for long periods (e.g. Herpes simplex virus)
§ Chronic infection: often with persistent shedders that have constant or intermitted shedding, can go unnoticed (e.g. BVD)
§ Persistent infection: after initial viral infection proliferation is ceased but viral genome not fully eradicated (e.g. FIV)
§ Persistent slow infection: characterized by a long incubation period followed by progressive disease (e.g. Jaagsiekte Sheep retrovirus, Lentiviruses)
Patterns of infection in a host
§ Threshold level of virus is required to activate adaptive immune response
But remember, you need to have an exposure to a certain level of antigens before you have actually the proper activation of your immune system, before you potentially have to build up of a memory immune response.
But we the more we know also about what is that threshold or that exposure, the more we can look into the development of vaccines.
poll everywhere:
budding is the process of merging the virus envelope with the host cell lipid bilayer membrane FALSE!! budding is the process of exit and this statement is fusion process!
basic unit of. a viral capsid: capsomere
The infectious period of a viral infection always begins after a clinical onset? FALSE ! In fact, many start before you see clinical signs again e.g. flu You’ll be spreading the flu before you show clinical signs of the flu that’s well known and the same accounts for many other diseases.
The first animal virus discovered and proof of viral disease in animals in 1898 was..
FOOT AND MOUTH VIRUS
(tobacco mosaic virus: first PLANT virus that could be seen
rabies virus: Louis Pasteur experimented with, but he didn’t know it was a virus.
KNOW THE GENERAL ORDER OF THIINGS! e.g. We needed the filter before we use the electron microscope for visualization.
Yeah, VIRUSES are obligate intracellular parasites because they lack an active metabolism (and energy so mooch off of hosts)
envelope of virus:
the envelope protects, of course, your genetic material,
It can help in entry to the host cells, and it can assist a virus to evade immune systems as well.
And so all of these are functions from your envelope
ON EXAMS THERE WILL BE PICS!!!
So know how a virus looks like, know how a viral replication cycle looks like.
but a very specific clinical sign from a viral disease that I expect you to not get.
Why perform a diagnosis of viral infection?
- viral disease diagnosis
- screening of blood donors (safety eg. hep B)
- proper management of diseases
- early detection of epidemics
next slide:
- direct methods include: virus isolation, genome detection and antigen detection
- indirect methods include: serology IgM and serology IgG
(serology: the term usually refers to the diagnostic identification of antibodies in the serum
serology hard to use to diagnose if disease or not; when u can take sample virus isolation, genome infection (looking for antigens - using PCRs); better to use methods which show it ave virus and not e.g. indications like elevated body temp).
SAMPLING
- depending on the disease, ? type, ? species and ?
Blood
§ ?
§ Virus (if animal is in ? stage)
§ Virus in blood ?
?/bronchial washes
?
? fluid
Biopsy/necropsy tissues
§ For culture -> ?
§ For antigen -> ? fixed ok
(tissue needs to be absolutely fresh (shold be first thing taken), if wait for cople of hours then no use
formalin fixed: use them w other tools)
SAMPLING
- depending on the disease, virus type, host species and diagnostic test
Blood
§ antibodies
§ Virus (if the animal is in viremic stage)
§ Virus in blood cells
sputum/bronchial washes
feces
cerebrospinal fluid
Biopsy/necropsy tissues
§ For culture -> fresh
§ For antigen -> formalin fixed ok
Lab diagnosis (see pic, more lab diagnosis there)
Virus culture and isolation: cytopathic effect (EM, histopathology), TCID50 assays, Fluorescent Ab staining
-living animals
-cell cultures
-embryonated chicken eggs
(virus culture and isolation: diff. than bacterial infections
living animals not used anymore, still used today tho in some experiments but encouraged to use cell cultures etc.)
Virus culture and isolation
§ Viruses need host cells to replicate -> NO GROWTH ON ? MEDIA
§ Test whereby samples are placed with ? cell type. If cells show expected changes (cytopathic
effects), then the culture is considered positive or negative?
CELL LINES
§ Primary cell lines: derived from tissues, die after few ? (turnover, not useful for long run)
§ Diploid cell lines: developed from human embryos, grow for #? generations
§ Continuous cell lines: transformed (cancerous) mortal or immortal? cell lines (e.g. HeLa cell lines, Vero cells, …)
- NEED living cells, used living animals before for it but now using cell cultures
(HeLa (her cells that are continuously used), cervical line of hers used;
continous: most useful one as keep multiplying bc they have these cancer aspects which are transformed immortal cell lines and are no longer in Petri dishes but they will be present as number 3 below in pic;
transformed don’t grow in monolayer)
Virus culture and isolation
§ Viruses need host cells to replicate -> NO GROWTH ON artifical MEDIA
§ Test whereby samples are placed with susceptible cell type. If cells show expected changes (cytopathic
effects), then the culture is considered positive
CELL LINES
§ Primary cell lines: derived from tissues, die after few generations (turnover, not useful for long run)
§ Diploid cell lines: developed from human embryos, grow for 100 generations
§ Continuous cell lines: transformed (cancerous) immortal cell lines (e.g. HeLa cell lines, Vero cells, …)
It’s believed that more HeLa cells have been grown and cultured around the world than
were originally in Henrietta Lacks’ body
Virus culture and isolation
Embryonated Chicken Eggs (ECE)
§ Cells (embryo + membranes) that support ? growth
§ Used for virus ?, ? & ? of vaccines
§ Not all viruses will grow in tissues of ECE, but many can be adapted to
ADVANTAGES
- Readily available, ?
- Easy to maintain
- ‘?’ from natural diseases
- ? immune system: no ? against virus
- Different ? routes
DISADVANTAGES
- use only ? eggs
- Site of ? differs depending on virus type
Virus culture and isolation
Embryonated Chicken Eggs (ECE)
§ Cells (embryo + membranes) that support virus growth
§ Used for virus isolation, identification & production of vaccines
§ Not all viruses will grow in tissues of ECE, but many can be adapted to
ADVANTAGES
- Readily available, cheap
- Easy to maintain
- ‘sheltered’ from natural diseases
- immature immune system: no defense against virus
- Different inoculation routes
DISADVANTAGES
- use only SPF eggs
- Site of inoculation differs depending on virus type
Virus inoculated in chicken embryos of 7-12 days old
Virus culture and isolation
?: method using bright light behind the egg to study growth & development of embryo inside
Detection of viral infection in ECE?
§ Mortality, deformity or haemorrhages in embryo
§ ? on the membrane (e.g. pocks)
§ Oedema of developing membranes
§ * ? bodies in embryo tissues
§ Presence of viral ? in egg fluids
Haemagluttination assays (antibody and antigen interaction; antigen should be present in sample in engg?)
Virus culture and isolation
CANDLING: method using bright light behind the egg to study growth & development of embryo inside
Detection of viral infection in ECE?
§ Mortality, deformity or haemorrhages in embryo
§ Lesions on the membrane (e.g. pocks)
§ Oedema of developing membranes
§ * inclusion bodies in embryo tissues
§ Presence of viral antigens in egg fluids
Virus culture and isolation
Determinants of infectivity and virulence of a virus:
TCID50 = Tissue culture infectious dose: number of ? required to cause infection in 50% of the cell culture (thus the smaller the number the MORE virulent it is!)
LD50 = 50% lethal dose: number of microbes or amount of toxin required to cause ? ? infections in 50% of the animals infected
ID50 = 50% infectious dose: number of ? required to cause infection in 50% of the animals
Virus culture and isolation
Determinants of infectivity and virulence of a virus:
TCID50 = Tissue culture infectious dose: number of viruses required to cause infection in 50% of the cell culture (thus the smaller the number the MORE virulent it is!)
LD50 = 50% lethal dose: number of microbes or amount of toxin required to cause terminal acute infections in 50% of the animals infected
ID50 = 50% infectious dose: number of microbes required to cause infection in 50% of the animals
