(Final) Diagnosis of viral infections Flashcards
What are the risks levels associated with broad risk categories of viral infections?
Risk Group 1 = No or low individual risk
Risk Group 2= Moderate individual risk, low community risk
Risk Group 3 = High individual risk, low community risk
Risk Group 4 = High individual risk, High community risk
What risk category is Foot and mouth disease classified as?
Risk group 4
T/F: BSL4 is the maximum containment laboratory. BSL4 labs handle dangerous and exotic pathogens belonging to the highest risk group - 4
TRUE
ex: ebola virus
What is a biohazard?
Biological substances that pose a threat to the health of living organisms, primarily that of humans
What is biosafety?
Laboratory biosafety describes the containment principles, technologies, and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release
Very small droplets of fluid that can spread via air are called?
Aerosol
Viruses can spread in the lab through aerosol route
What does biosecurity refer to?
Lab biosecurity described the protection, control, and accountability for valuable biological materials within labs - in order to prevent their unauthorized access, loss, theft, and misuse
When should samples be taken for virus isolation?
Specimens should be collected as soon after onset of symptoms as possible. *maximum amounts (titers) of virus are usually present at the onset of signs
**chance of viral recovery is best during the first three days after onset - up to five
When should samples be taken for viral serological tests?
Two blood samples are best: One during the acute phase of illness and the second sample during the convalescence period
When should samples be collected for PCR or molecular diagnostics?
During the early part of illness
Viral swab samples should be sent to the lab in what medium?
Viral transport medium (VTM)
To prevent spillage, it is recommended to follow the _______________ system while transporting infectious materials
Basic triple packing system
What would be a good time frame to collect samples for virus isolation?
- within 3 days of onset of clincial signs
- within 7 days of onset of clinical signs
- within 14 days of onset of clinical signs
- within one month of onset of clincal signs
1 - within 3 days of onset of clinical signs
What process must be done to tissues prior to virus testing?
Tissue homogenization
Via tissue grinder or mortar and pestle
What are three simple ways to diagnose the presence of a specific virus?
Gross evaluation and histopathology:
Clinical signs
Necropsy
Histopath - inclusion bodies
What are two ways to detect the presence of a virus via cultivation?
Cultivation/isolation in cell culture or by egg inoculation
What kind of microscope is necessary to detect viruses that can not be grown in-vitro?
Electron microscope
Can use negative staining
What are the differences between the transmission and scanning microscope?
Transmission: Method based on transmitted elections: 2d image, seeks to see what is inside or beyond the surface, has higher magnification and greater resolution
Scanning: Method is based on scattered electrons. Focuses on the sample’s surface and its composition. **3D image
T/F: A transmission electron microscope yields a 3-D image
FALSE
Scanning electron microscope = 3D
Transmission = 2D
What is a gold standard test?
A dx test that is considered to be the most accurate and best available under a particular condition or set of conditions
Sensitivity is based on..?
The probability that cases WITH the infection will have a POSITIVE result using the test under evaluation
What is specificity based on..?
The probability that cases WITHOUT the infection will have a NEGATIVE result under test evaluation
How do you collect/prepare a serum sample?
Use a red top tube/vacutainer, allow the sample to clot, centrifuge the sample, supernatant = serum
How do you collect/prepare a plasma sample?
Use a lavender top EDTA tube/vacutainer, centrifuge, supernatant = plasma
Vacutainer tubes used to collect blood samples to obtain serum for diagnostic purposes usually have ___ colord caps?
Red
Blue
Lavender
Green
RED
What are the 6 steps of the basic Enzyme-linked immunosorbent assay (ELISA) test?
- antigen coated well
- Add antibody tagged with an enzyme
- Antigen binds to enzyme-tagged antibody
- Wash the excess unbound antiodies
- Add substrate
- Enzyme tagged to antibody which is bound to antigen will chnage color of substrate. Intensity of color indicates more positive reaction
How does the direct ELISA work?
Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antigen is very important
How does the indirect ELISA work?
Primary antibodies are not labeled, but detected instead with enzyme-conjugate secondary antibodies that recognize the primary antibodies.
How does the sandwich ELISA work?
The antigen to be measures is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-ractivity or competition of binding sites
What ELISA has a more positive result, when you see a weaker color change?
Competitive ELISA
A decrease signal when compared to assay wells with purified antigen alone, indicates the presence of antigens in the sample
T/F: When using a competitive ELISA, a weaker signal indicates the presence of antigens in a sample
TRUE
How does direct Fluorescence antibody test (FAT) work?
Labelled antibodies are added onto the sample (antigen). Visible fluorescence appears at the binding site of the specific antibodies
How does indirect FAT work?
IFAT employs a secondary antibody labeled with a fluorescent marker that recognizes the primary antibody bound to antigen
How does immunohistochemistry work?
The antibody is tagged with an enzyme, generally horseradish peroxidase. The enzyme reacts with a substrate to produce a colored product that can be visualized in the infected cells with a standard light microscope
T/F: Immunochromatograhy is a point of care test
TRUE
A POC (point of care) test is one that is simple to perform, easy to carry, and does not require specialized equipment
What are agglutination tests?
Agglutination is a method using the property of specific antibodies to bind many antigens into single clumps - forming large complexes
What tests rely on the property of some pathogens to non specifically agglutinate erythrocytes?
Hemagglutiation and hemagglutination inhibition test
T/F: Agar gel immunodiffusion tests can be used to detect antibodies
TRUE
used for avian influenza
In compliment fixation test, what does no hemolysis mean?
POSITIVE reaction
When you perform a complement fixation test using a serum sample from a patient with no viral antibodies what is the expected result?
Hemolysis of sheep RBC = negative reaction (no virus antibodies)
What occurs in the hemadsorption inhibition assay test?
In cell culture: Antibodies bind to viral glycoprotein spikes in the cell membrane, they remain attached after washing techniques. Then when the infected cells are treated with RBCs - there will be NO hemadsorption noted bc the viral glycoproteins are bound to antibodies.
What is neutralization of a virus? What test uses this concept?
The loss of infectivity through reaction of the virus with specific antibody
Neutralization assays
What is the function of PCR?
Polymerase chain reaction –> amplification of viral genome/DNA
What are the three steps of PCR?
Denaturation
Annealing
Extension/Elongation
T/F: Both real time PCR and Quantitative PCR allow monitoring and quantification of increasing accumulation of PCR products/nucleic acid load as the reaction processes.
TRUE
What is genome sequencing?
DNA sequencing refers to the process by which the sequence of bases in DNA molecule is elucidated/can be obtained and read
What is next generation sequencing?
These are sequencing technologies that are significantly cheaper, quicker, needs less DNA, Has high throughput, and is MORE accurate and reliable than Sanger sequencing
T/F: Sanger sequencing is better than next generation sequencing
FALSE
Next generation is more accurate and reliable
What is metagenomics?
the study of collective set of microbial populations in a sample by analyzing the sample’s entire nucleotide sequence content, and is a powerful method for random detection of existing and new pathogens
What is a phylogenic analysis?
The use of virus genome sequence data to study evolution of viruses and genetic relationships among viruses
What is an advantage of microarrays?
Hundreds of pathogens can be screened for simultaneously using a single microarray chip
T/F: Microarrays will generate a fluorescent signal when a sample DNA sample is positive for a known DNA probe
TRUE
The study of virus evolution using genome sequence data is known as _______
phylogenetics
What are three ways to treat viral diseases?
Antiviral drugs
Immune system stimulation
Synthesize antibodies or administration of natural antiserum
T/F: there are many antiviral drugs to choose from when treating a viral disease
FALSE
antiviral drugs are a class of medication used specifically for treating viral infections and they are very limited
What is the antiviral that has activity restrcited to herpesviruses? How does it work?
Acylovir
This is administed in the prodrug, inactive form. Requires host cell to convert into active form and then it interferes with virus replication
What herpes viruses can Acyclovir be used to treat? (3)
Herpesvirus infections in humans
Feline herpesvirus 1 induced corneal ulcers
Equine herpesvirus 1 induced encephalomyelitits
__________ is a synthetic nucleoside analog of deoxyguanosine.
Acyclovir
MOA: The herpes simplex’s DNA polymerase enzyme incorporates the acyclovir monophosphate into the grown DNA stands as if it were 2-deoxyguanosine monophosphate – stops viral DNA growth
What are the two functions of acyclovir as an antiviral drug?
- Stop growing viral DNA chain: Further elongation of the viral DNA chain is impossible bc acyclovir monophosphate lacks the attachment point necessary for the insertion of any additional nucleotides
- Competitive inhibition of viral DNA polymerase: The acyclovir triphosphates compete with dGTPs for viral DNA polymerase
T/F: Acyclovir can be toxic to uninfected host cells
FALSE
The acyclovir cannot be phosphorylated and incorporated into the host DNA of uninfected cells
What antiviral drug inhibits the replication of most stains of influenza A viruses by blocking the uncoating of the virus
Amantadine
What is the mechanism of antiviral effect of amantadine?
The M2 iron channel is the target of this drug. These compounds clog the channel and prevent it from pumping protons into the virion. In the presence of amantadine, viral RNAs remain bound to M1 and cannot enter the nucleus –> inhibits virus replication
T/F: Acyclovir is a pro-drug
TRUE
What class of antiviral is Oseltamivir (Tamiflu)? What viruses is it used to treat?
Neurominidase inhibitors
Influenza A and B
T/F: Oseltamivir (Tamiflu) is a pro-drug
TRUE
If neuraminidase is blocked by Oseltamivir, what occurs in the infected cell?
Sialic acid receptors on the infected cell surface can not be cleaved, so they will hold onto the new developed virion –> preventing further infection/spread
What are areas of target for retroviruses?
Inhibit fusion
Inhibit integrase
inhibit reverse transcriptase
Inhibit protease
How does Zidovudine (ZDV)/AZT work?
it is a nucleoside analog reverse transcriptase inhibitor
It is an analog an thymine - so it competes for reverse transcriptase. Insertion of AZT into cDNA blocks growth of the cDNA being transcribed from the viral RNA by reverse transcriptase
AZT has been shown to reduce clinical signs in _______ postive cats when administered at a dose of 10mg/kg BID, SQ for 3 weeks
FIV positive cats
T/F: ZDV triphosphate competes with the dGTP for reverse transcriptase
FALSE
Acyclovir competes with dGTP
ZDV competes with dTTP
___________ are required to cleave HIV polyproteins into functional proteins
Proteases
What is the mode of action of protease inhibitors?
They inhibit proteases - proteases cleave polypeptides into functional proteins - wtihout the cleavage, the virus can not mature, so non infectious viruses are produced
What are the four Ws of immunization?
Where? endemic areas
when? ifs the dz has a distinct season or an outbreak occurs
who? population at risk
why? The loss caused by dz must e greater than the cost of immunization
What are live attenuated vaccines?
Vaccines produced from naturally occurring attenuated viruses
what are different methods of making live attenuated vaccines?
- produced by serial passage in cultured cells
- Produced by serial passage in heterologous hosts
- produced by selection of cold-adapted mutants and reassortants
What are non replicating virus vaccines?
Vaccines produced from inactivated (killed) WHOLE virions
or
Vaccines produced from purified native viral proteins
What is another type of vaccine besides live attenuated and non replicating vaccines?
Recombinant DNA vaccines (and related technologies)
Vaccines produced by serial passage of viruses in heterologous hosts is a ______ vaccine
live-attenuated
What is DIVA?
Differentiating infected from Vaccinated Animals
**Vaccination with live attenuated vax will produce antibodies that do not differ from the antibody response to a natural infection, so subunit ‘marker vaccines’ are used instead - it makes it possible to differentiate vax antibodies vs natural antibodies
T/F: DIVA vaccines do not include entire virions, therefore they have less antigens than the pathogen it is protecting against
TRUE
So, if antibodies to other antigens of the dz are present in addition to the vaccine induced antibodies - the animals has been exposed to the natural virus
What are methods of vector control?
Source reduction (where vectors multiple)
Biological control
Chemical control
Are isolation and quarantine the same?
NO
Isolation: separate animals that show clinical signs or have a positive diagnostic test
Quarantine: segregation of animals based on exposure to a contagious dz
What is the standard time of quarantine for a contagious dz?
Typically enforced for the longest incubation period of the dz - gold standard testing should be run on the animal(s) during this time
What is decontamination?
A process or treatment that renders a medical device, instrument, or environment surface safe to handle
What is sterilization?
A process that destroys or eliminates all forms of microbial life/pathogens, including highly resistant pathogens like bacterial with spores
What is disinfection?
Process that eliminates many or all microorganisms, except bacterial spores, on inanimate objects
**less effective than sterilization
What is antisepsis?
Application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms
What are 5 methods of sterilization?
Moist heat (autoclave)
Dry heat (hot air oven)
Chemical methods (gases like ethylene oxide/Ozone)
Radiation (ultra violet - non ionizing / Gamma rays - ionizing)
Sterile filtration
Autoclaving is primarily considered as a method of ________
Sterilization
