4. Viral Infection and Diagnosis

In Nucleic Acid Hybridization, what quantity can give you a good value of how much viral load is in the sample?

Hybridization quantity

- Cell bursts open - May be the result of inclusion bodies (because they get really large and accumulate) - Loss of membrane integrity (occurs after a viral infection because of the budding process which the viral particle acquires the cytoplasmic membrane) - OR specific viral function of the virus to gain release from the cell

LYSIS

T/F: Primary cell cultures are prepared from freshly isolated animal tissue, these cells cannot grow indefinitely and either die out or mutate into cells that grow continuously

TRUE

T/F: If your sample has very little amount of nucleic acid of viral protein, it will amplify it to a quantity at which it will give you a ratio of how much viral load is in the sample.

TRUE

The damage caused by viruses on host cells is called ____________

Cytopathic effect: virus induced damage

Detects the presence of Antigens Direct Elisa OR Indirect Elisa ?

Direct Elisa ALSO known as sandwich ELISA because the primary antibody is getting sandwhiched between he substrate and antigen Only has a primary antibody conjugate

Provides a RAPID presumptive/initial diagnosis

Direct examination

Most active viral infections ultimately kill the cell. WHY does this happen?

Due to accumulated damage

When antibodies detected the presence of anti viral antibodies this is called ____________ assay Direct of indirect?

INDIRECT assay (serological method) IMPORTANT to note that this stage is already in the LATER stage of viral replication because the host has already recognized that there is a viral invasion and that they have developed antibodies against it.

The virus is injected to the brain, blood, muscle, body cavity, skin, OR footpads of an animal Live animal, bird embryo, tissue cell?

LIVE animal innoculation

What membrane is the protein transferred onto during western blot?

Nitrocellulose membrane

DIFFERENCES between PCR and Nucleic Acid Hybridization In ________ you do not use radioactive labeling

PCR

Major advantages of this specific method 1. Minute amounts of nucleic acids are required 2. You can amplify DNA or RNA (reverse PCR) 3. You can amplify directly from tissue sample - does NOT require isolation 4. You can sequence amplified DNA and exactly identify the infectious agent

PCR

DIFFERENCES between PCR and Nucleic Acid Hybridization IN _______ you AMPLIFY the DNA and check for sequence IN _________ you check for the radioactivity which is shown AFTER the hybridization

PCR Nucleic acid Hybridization

DIFFERENCES between PCR and Nucleic Acid Hybridization IN __________ there are primers IN _________ there are probes

PCR Nucleic acid hybdridization

What are the molecular methods used to diagnose viral infections?

PCR Nucleic Acid Hybridization

When animal cells grow in monoculture and become infected what is seen?

PLAQUE plaques are areas where the virus has infected you can see them because there is a lot cell lysis occuring in that region

Fusion of infected cell with neighboring cells (infected OR non infected)

Syncytia Many cells fuse together and you get LARGE multinucleate cells This is one of the cytopathic effect that can help use in the diagnosis of a viral infection. STILL CANT CONFIRM but you can use it to realize something is occurring in the patient.

What cytopathic effects is seen in mumps and measles virus?

Syncytium

Quiet process, does NOT elicit inflammation response.

Apoptosis

How can we diagnose a viral infection?

- Clinical symptoms - Season of the year travel history - LABORATORY diagnosis

What are the five types of cytopathic effects?

- Alteration in Shape or Size - Presence of inclusion bodies - Lysis - Apoptosis - Syncytia

What are the signs of viral growth in bird embryo innoculation?

1. Death of embryo 2. Defective embryo development 3. Localized damage resulting in opaque spots called POCKS

What are the steps for PCR?

1. Denaturing 2. Annealing 3. Extension

Diagnosis of viral infections is done by (4 ways)

1. Direct examination 2. Serological methods 3. Molecular methods 4. Virus cultivation

What are the purposes for using Virus cultivation?

1. Isolations and identification from clinical samples 2. Preparation of vaccines 3. Reasearch

Where can we cultivate viruses?

1. LIVE animals 2. Bird embryos (chicken, duck, and turkey eggs are the most common for inoculation) 3. Tissue culture (animal cell cultures grown in sterile chambers using special media —> cell grows a monolayer)

What is the BASIC PRINCIPLE OF TECHNIQUE of Nucleic Acid Hybridization?

A strand of complementary nucleic acid will bind (hybridize) with the viral nucleic acid

When antibodies detect the presence of antigen (virus) this is called a __________ assay Direct or indirect?

DIRECT assay (Serological method) This is PREFERRED because it will give you how much viral load is present.

Rapid diagnosis of _____ infection by indirect immunoflourescent staining of infected cells?

EBV

What are the serologic methods which produce colorimetric assays in which an enzyme attached to the SECONDARY Ab causes a color change when substrate is added?

ELISA and Western blot It also gives you the account of intensity of color which shows you how much is present so it is both qualitative and quantitative

1. Place and coat tube with antigen 2. Fill with diluted serum 3. Antibodies from serum will attach to antigen. WASH out tube and remove unwanted antibodies 4. Solution of animal antibody against human antibody is added THIS antibody is covalently conjugated to an enzyme. 5. Well is washed again to remove the unbound enzyme conjugated antibodies 6. SOLUTION of enzyme colorgenic substrate is added and this will develop color

ELISA steps (Got these from a youtube video) https://www.youtube.com/watch?v=RRbuz3VQ100

T/F In PCR you cannot directly amplify DNA from tissue sample - you must isolate it.

FALSE Isolation is not needed!

T/F Direct examination is sufficient when diagnosing viral infections.

FALSE Often a more concrete specific diagnosis is required because many viruses cause the same CPE (Cytopathic effect) - because these CPE can be a result of something else like maybe cancer - very important to confirm presumptive diagnosis! - Many viruses within a family cannot be distinguished without a more specific means (must distinguish to give proper treatment)

T/F: Electron microscopy examination of similar viruses does not need further means of identification

FALSE Similar viruses cannot be differentiated, so more specific means of ID is required. Used for Preliminary diagnosis

T/F Apoptosis elicits an inflammatory response.

FALSE NO inflammation Apoptosis is normal for all cells. But when it occurs at a fast rate and unnaturally then it becomes a concern.

T/F: Individual virions seen in the immunofluorescence proces

FALSE, NO they are not seen but if light is emitted than the antigen is present!

T/F: PCR only applies to nucleic acid

FALSE, also happening in the case of atoms. it is just a chain reaction which happens in a polymeric way. There is extension of whatever molecule you begin with. PCR is not a method, much rather a concept

T/F: Continuous cell lines can grow indefinitely without any additional nutrients

FALSE, continuous cell lines can grow indefinitely, as long as they are given fresh nutrients

This method combines light microscopy and serology by utilizing fluorescent tags attached to antibodies to detect viral antigens (direct) or anti-viral antibodies (indirect)

Immunofluorescence Microscopy In powerpoint under direct examination In packed falls under serological methods Either way it includes BOTH

Fluorescent tags are attached to commercially available antibodies. These antibodies are specific for the specific antigen present on the viral particle and bind to the viral particle and then the flour illuminates or displays fluorescent which is detected by computer

Immunofluorescence process

What are negri bodies and in what kind of viral infection are they found?

Inclusion bodies that help identify rabies Indicative of when rabies are affecting the BRAIN cell (brain cell slide in powerpoint) STILL can't confirm but can help you come to a more narrow conclusion of what your patient has

Detected the presence of Antibodies Direct Elisa OR Indirect Elisa ?

Indirect ELISA AGAIN this one is occur LATER on in the stage of infections. Includes a SECONDARY antibody conjugate

Viral nucleic acids, either the viral genome or viral mRNA can be detected within the patients blood or tissues with complementary DNA or RNA (cDNA/cRNA) used as a PROBE Strand of complementary nucleic acid will hybridize with the viral nucleic acid. The complementary probes are linked to radioactive label and samples are exposed to film to determine if hybridization has occurred.

Nucleic Acid Hybridization

Which antibody (primary or secondary) causes the color change in ELISA testing?

Secondary antibody which is linked to an enzyme causes a COLOR change when the appropriate substrate is added and is digested by the enzyme!

This method utilizes the specificity and sensitivity of antigen antibody interactions It also detects the presence of virus or of ant viral antibodies in the patient Ex - ELISA - Immuno - fluoresence assay

Serological Methods

T/F Cytopathic effects (CPE) help in diagnosis of viral infections

TRUE

T/F Electron microscopy can detect individual viral particles.

TRUE

T/F Examination of tissues for cytopathic effects is important in diagnosis of viral infections.

TRUE

T/F PCR - once large enough quantities of nucleic acids are obtained, it can be sequence to identify the exact agent.

TRUE

T/F Serologic techniques take advantage of the SPECIFICITY and sensitivity of Ag-Ab interactions.

TRUE

T/F Serological testing is extremely specific and sensitive and will provide definitive diagnosis.

TRUE

T/F The amount of color present in the ELISA testing is directly related to how much antigen is present

TRUE

T/F Virus culturing although providing a definitive diagnosis, takes longer to accomplish

TRUE

T/F: Cytopathic effects include changes in shape or size, presence of inclusion bodies, etc.

TRUE

T/F: Hybridization quantity can give you a good value of how much viral load there is in the sample

TRUE

T/F: some cytopathic effects may cause death of the cell

TRUE

T/F Rabies uses dFA testing

TRUE dFA - direct immunofluorescence assays

T/F Using the Light microscopy, one can examine tissues or cells that can reveal virus induced changed (CPE's) to the cell. (Syncytial & inclusion bodies)

TRUE You can see if the cells have single large or multiple small inclusion bodies as well as if there is an altered shape BUT light microscopy will NOT give you a view of the actual pathogen - you NEED Electron Microscope

T/F: Many viruses cause similar CPEs

TRUE, that is why a more concrete/specific diagnosis is required

Why is a bird embryo a good place to cultivate viruses?

The embryonic development within the protected confines of the egg which is a HIGHLY NUTRITIOUS ENVIRONMENT

Animal cell cultures are grown in sterile chambers using special media These cells grow a MONOLAYER

Tissue/Cell Culture technique

What type of light is used to excite the flour?

UV light

Proteins (naturally negatively charged) are extracted from a sample and separated in an acrylamide gel through a process called ELECTROPHORESIS. Electrophoresed proteins are transferred from the gel onto a solid membrane and washed with enzyme linked antibodies When a substrate is added, color develops on the membrane where antibodies have bound. THIS method identifies the PRESENCE and SIZE of specific viral proteins

Western Blot

Sample subjected to electrophoresis - IDs presence and size of specific viral proteins

Western blot (simplified)