Lecture 3 Flashcards
What are are the req’s for BSL-1?
microbes are not known to consistently cause disease in healthy adults, and present minimal potential hazard to lab personnel and the environment.
Ex. nonpathogenic strain of E. coli
What are are the req’s for BSL-2?
microbes pose moderate hazards to lab personnel and the environment. Microbes are typically indigenous and assoc’d w/ disease of varying severity.
Ex. Staphylococcus aureus
What are are the req’s for BSL-3?
microbes can be either indigenous or exotic, and can cause serious of potentially lethal disease through respiratory transmission.
Ex. Mycobacterium tuberculosis
What are are the req’s for BSL-4?
microbes are dangerous and exotic, pose a high risk of aerosol-transmitted infections. Infections caused by these microbes are frequently fatal and without treatment of vaccines.
Ex. Ebola virus, Marburg virus
What are the types of biosafety cabinets?
There are three main classes of biological safety cabinets (BSC). Each type of cabinet provides protection through different configurations of HEPA filtered laminar air flow into/within the cabinets as well as HEPA filtered exhaust air.
Class I BSC: not used often – tend to be used to enclose equipment or procedures which have the potential to generate aerosols.
Class II BSC: most common type – used w/ BSL 1, 2, 3 agents
Class III BSC: designed for work w/ BSL 4 agents – provide maximum protection.
Describe the uses of animals with specific examples for Virus propagation:
i. Viruses that cannot be grown in other systems
1. Ex. some rotaviruses
2. Use of “germ free” animals inside a sterile chamber directly from birth
a. Inoculate/infect the animal with the agent under study now referred to as a “gnotobiotic animal” (since you know exactly what agent is within the animal)
Describe the uses of animals with specific examples for Virus surveillance:
i. sentinels for virus surveillance
1. Ex. WNV, other insect-borne viruses
a. Sentinel chickens are placed at several sites in the surveillance area – chickens are bled once a week and serum tested for WNV and SLE virus-specific IgM Ab
Describe the uses of animals with specific examples for Diagnostics including discovering new viruses:
i. Diagnostics when tissue culture sensitivity may be an issue
1. Ex. rabies virus
a. If an individual is bitten by an animal that is suspected to have rabies, the brain is removed and an impression smear is performed, then stained using IFA. (-) result from IFA staining does not mean that the animal is free of rabies, however – you need to take the testing further and perform animal inoculation (mouse inoculation test) to look for the presence of cytoplasmic inclusion bodies (Negri bodies) in Perkinje cells.
i. A suckling mouse in inoculated with 0.05 mL of a clarified suspension of brain material.
ii. Observe the mouse for CS/death.
1. If the mouse dies w/I 24 hrs, probably not due to rabies virus (instead, due to trauma of brain inoculation)
2. When the mouse dev’ps neuro signs of dies, remove the brain and do an impression smear.
a. If (-), conclude that it is probably not rabies virus.
b. If (+), confirm via serological means.
i. (take brain material from the mouse, make a clarified suspension and use it as the Ag in a serological test)
Describe the uses of animals with specific examples for pathogenesis studies:
i. The monkey model for SARS virus studies
1. A wildlife species was determined to pass the virus to humans as they were held in markets for food purposes. Sometimes an animal (in this case, monkey) has to be used for pathogenesis studies.
Describe the uses of animals with specific examples for vaccine development:
i. Challenge studies
1. Ex. use of ferrets to test for influenza virus vaccine efficacy
a. Ferrets are highly susceptible to both human and avian influenza viruses, therefore pathogenesis and transmission studies can both be performed in the ferret model. Numerous CS of human infection w/ influenza viruses are present in the ferret. Ferrets are also well suited to vaccine efficacy studies
What are the sites of the embryonating egg that are used for virus inoculation?
amniotic cavity
chorioallantoic membrane CAM
yolk sac
Describe why the amniotic cavity (sac) of the EE is the preferred inoculation site for isolating wild type influenza virus from chickens
The amniotic cavity of the EE is the preferred site for isolating influenza viruses because during development of the embryo, the amniotic fluid is inspirated and expirated by the embryo. Any virus present within the fluid will have access to cells from all embryologic origins (epidermal, mesodermal, endodermal).
Describe indications of virus infection of the EE with specific examples.
x
Describe how EE can be inoculated
EE are inoculated around 7-10 days of age of the embryo. The egg is held up to a light source (“candled”) to determine whether there are distinct BV and presence of a dark eye – these indicate health of the embryo. The egg is sanitized and a small hole is punched through the shell. A needle is inserted directly into the egg’s allantoic cavity through the shell membrane and the egg is inoculated with approximately 0.1mL of sample. The needle is removed and the hole is sealed with wax, glue, or tape. The egg is placed in an incubator for a period of time (duration depending on the virus), then removed and harvested.
Describe the optimum site of the EE used for propagating/isolating rickettsial and chlamydia agents
Rickettsial and chlamydia agents are optimally propagated in the yolk sac. After ~12 days of incubation, the yolk sac membrane is harvested and stained, revealing clusters of elementary bodies in the cytoplasm of cells lining the stalk of the yolk sac.
What is organ culture?
refers to cross sections/portions of organs maintained in cell culture medium.
what are tissue explants?
are fragments of tissue that are created by cutting up a specific organ. These fragments of infected tissue are placed on a stainless steel platform inside a cell culture plate that has a cell monolayer used to detect the virus. The cells surrounding the explants are observed for the presence of CPE
Describe uses for explants and organ culture?
When cells are by themselves (e.g. in cell culture), there are a certain number of specific genes that are active/being expressed- some receptors are present and others are absent. When in the presence of other cells, there is much cell interaction occurring and more receptors may be present. When studying the effects of an agent on an organ, we want to use the entire organ so that we can get accurate results / “the big picture”
How are explants useful in studying herpesvirus latency, use PRV?
Herpesviruses are characterized by having the ability to establish latent infections (infections of cells that do not go to completion). The virus infects the host and replicates in the cells (specifically neurons). These DNA viruses require the nucleus for replication – once in the nucleus of the neuron, the replication stops. The viral genome persists in the nucleus in an epizomal form (not incorporated into the host cell DNA, but instead exists in a free circular form) that can last for weeks, months, or years. Sometime later there is an event that triggers reactivation of the latent infection and causes it to start up again and go to completion/generate progeny (events include physical trauma, emotional trauma, stress, or co-infection with another agent)
When testing an experimental vaccine for pseudorabies virus, an important question was whether the vaccine would latently infect the pigs- if a latent infection occurred, this would be undesirable. To test this, researchers vaccinated a group of pigs with the experimental vaccine, waited a few months, then stressed them out by putting them in a truck and driving around for a few hours. They followed some of the pigs serologically to see if there was a reactivation of serum Ab titer. They examined the other pigs by post-mortem removal of the trigeminal ganglion, which they cut up into small explants. They were put into culture with a cell monolayer that was observed for presence of CPE. The result was that the physical trauma of cutting up the ganglion tissues was enough to reactivate the virus, and CPE observed in the cells was determined to be a result of pseudorabies virus.
Describe the components of growth and maintenance media
Growth and maintenance media contain water, balanced salt solution (BSS), a buffering system, and serum that contains GFs that are critical to cell growth and replication. Fetal bovine serum (FBS) is preferred over neonatal serum because it does not have maternal Ab. The difference between growth and maintenance media is in the amount of serum they contain. Growth serum has a high amount of serum, and maintenance serum has a lower amount of serum. This becomes important during growing of a cell population: initially, growth medium is used to support high amounts of cell replication. As the islands of cells come together, contact inhibition occurs and a cell monolayer forms. At this point, the cells need to slow down their metabolism or else they will generate a lot of waste products and this will destroy the cells. The growth medium is replaced with maintenance medium to achieve this slower metabolism rate
Define, give the characteristics and uses, and describe how Primary cells are produced:
i. Cell cultures that are derived directly from the host. The cells are collected, disaggregated, and grown in cell culture. These cultures are referred to as being heterogeneous because the tissues contain many kinds of cells. Fibroblasts predominate in primary cell lines because they form the framework that holds everything else together – specialized epithelial cells are the minority, even though there are many different kinds of epithelial cells.
1. cells have normal # of chromosomes and a finite # of replications
2. primary cell lines are useful for isolating new viruses – they allow you to expose all cells of a certain organ to the virus at once when trying to observe and determine infective characteristics of the virus
Define, give the characteristics and uses, and describe how Diploid cells are produced:
i. Diploid cell strains are homogenous cell populations that are generated from a single cell which is cloned from a heterogeneous cell population. This is accomplished by using a cell sorter.
1. Cells have normal # of chromosomes and are capable of a finite # of replications.
2. Useful in vaccine production – ex. human rabies virus vaccine
Define, give the characteristics and uses, and describe how continuous cells are produced:
i. Emerge from primary (heterogeneous) cell population through spontaneous or induced mutation. Mutations can be induced by chemical agents, physical agents (radiation), or genetic manipulation.
1. Population is homogenous
2. Cells have an abnormal # of chromosomes and are capable of an infinite # of replications (essentially a cancer cell)
3. Generally easy to propagate – good candidate for use in diagnostic labs
4. Epithelial cell origin
What is subpassaging?
Sub-passaging is the process of creating a subculture. A subculture is a new culture made from an original culture by transferring some or all cell types to fresh growth medium (which becomes necessary once a population of cells uses up all of the nutrients within the current supply of medium). This is a common method used to prolong the life and/or expand the number or cells in the culture.
What is the basic procedure of subpassaging?
a) Remove old media from cell culture flask.
b) Add buffer solution to rinse cells: does not contain any nutrient, but is used to wash off any residue from old media, since process used to detach cells may be affected by any media present.
c) Use trypsin (digestive enzyme) to disrupt proteins that stick the cells to the sides of the flask:
d) Put the flask in a 37C incubator for 3-4 minutes to allow trypsin to work at its optimal temperature. Don’t leave the cells in the trypsin for too long, or else you will remove the cell’s surface signaling proteins.
e) To arrest the trypsinization, add fresh culture medium - contains abundant protein to absorb the trypsin.
f) Remove all liquid from the flask and transfer to a centrifuge tube for low speed centrifugation.
g) Dump the supernatant (culture medium) and resuspend the cell pellet in fresh medium.
h) Take a portion of the resuspended cell culture and transfer it to a new flask with fresh medium.
i) Incubate at 37C to achieve adequate cell growth
