Block 5

Question 1

PCR

Answer 1

Amplification of viral genome/DNA

Question 2

3 steps of PCR

Answer 2

1) Denaturing
2) Annealing
3) Extension/Elongation

Question 3

Real-Time PCR assay

Answer 3

1) A target specific probe (labelled with a fluorescent dye), such as the 5’-nuclease TaqMan probes, molecular beacons, or FRET hybridization probes
2) or Intercalculating dyes, such as SYBR Green
are used in an otherwise conventional PCR reaction. The fluorescence emitted by the excited fluorophone (Fluorescent probes or dye) is visualized and analyzed.

Question 4

Genome Sequencing

Answer 4

DNA sequencing refers to the process by which the sequence of bases in a DNA molecule is elucidated/can be obtained and read

Question 5

Real-Time PCR or Quantitative PCR

Answer 5

Advanced form of PCR which allows monitoring and quantification of increasing accumulation of PCR products/nucleic acid loads as the reaction progresses. Useful to study virus load in patient.

Question 6

Next Generation Sequencing

Answer 6

Are cheaper, quicker, needs significantly less DNA, has high throughput, and is more accurate and reliable than Sanger sequencing

Question 7

Metagenomics

Answer 7

The study of the 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.

Question 8

How does Metagenomics work?

Answer 8

Amplification and sequencing of whole genome (DNA and/or RNA) content of a given sample followed by filtering and analysis of obtained data by comparing with genome databases and using different softwares.

Question 9

Genome sequencing plays a crucial role in surveillance studies, as it allows (7):

Answer 9

1) Pathogen detection
2) Studies on genetic variation, such as genotyping, evolution and interspecies transmission of pathogens
3) Identification of novel and undiscovered strains
4) Development of diagnostics, such as genotyping primers, or probes
5) Identification of genes associated with drug resistance
6) Development of therapeutics
7) Judging the efficacy of current vaccines and formulating new vaccine strategies

Question 10

Phylogenetic Analysis

Answer 10

The use of Virus genome sequence data to study evolution of viruses and genetic relationships among viruses

Question 11

Microarrays

Answer 11

In microarray, several thousands of known DNAs (probes), amplified by PCRs/RT-PCRs, are spotted onto a glass or a silicon chip.
The target/sample DNA are fluorescently labeled and then hybridized/added to the chip containing DNA probes.
Positive reactions between probe-DNA and Sample DNA (hybridization) generate a fluorescent signal from the spot where probe DNA is spotted in the chip.

Question 12

Advantage of microarray in detection of pathogens in surveillance studies

Answer 12

Hundreds of pathogens can be screened for simultaneously using a single microarray chip

Question 13

Antiviral Drugs

Answer 13

Interfere with the ability of a virus to infiltrate a target cell or target different stages of replication/synthesis of components required of the virus

Question 14

Immune system stimulation

Answer 14

Interferons, class of proteins that has antiviral effects and modulate functions of the immune system

Question 15

Acyclovir

Answer 15

Antiviral activity primarily restricted to herpesvirus.
Administered as a prodrug, inactive form.
Requires virus enzymes in infected host cell to convert itself into active form, which then interferes with virus replication.

Question 16

What does Acyclovir Treat? (3)

Answer 16

1) Herpesvirus infections in humans
2) Feline herpesvirus 1 induced corneal ulcers
3) Equine herpesvirus 1 induced encephalomyelitis

Question 17

Acyclovir is a synthetic nucleoside analog of ______.

Answer 17

Deoxyguanosine

Question 18

Mechanism of antiviral effect of Acyclovir

Answer 18

The herpes simplex’s DNA polymerase enzyme incorporates the acyclovir monophosphate into the growing DNA strand as if it were 2-deoxyguanosine monophosphate (a “G” base). Further elongation of the chain is now impossible because acyclovir monophosphate lacks the attachment point necessary for the insertion of any additional nucleotides. Viral DNA chain synthesis stops.
Also, competitive inhibition of viral DNA polymerase, as acyclovir-triphosphate compete with dGTP for viral DNA polymerase.

Question 19

Is Acyclovir toxic or non-toxic to the uninfected host cell?

Answer 19

Non-toxic because acyclovir cannot be phosphorylated and incorporated into the host DNA.

Question 20

Amantadine

Answer 20

Inhibits replication of most strains of Influenza A viruses by blocking uncoating of the virus.

Question 21

Mechanism of antiviral effect of Amantadine

Answer 21

The M2 ion channel i the target of the antiviral Amantadine.
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. Virus replication is inhibited.

Question 22

Neuraminidase Inhibitors

Answer 22

Inhibitor of Neuraminidase enzyme synthesized by Influenza A and B viruses.

Question 23

Example of Neuraminidase Inhibitors

Answer 23

Oseltamivir (Tamiflu)

Question 24

Mechanism of antiviral effect of Neuraminidase Inhibitors

Answer 24

Blocking the function of neuraminidase with NA inhibitors is an effective way to treat influenza.
This prevents the release of virus and spread of infection, as the HA of virus is still bound/attached to the sialic acid containing receptors on surface of already infected host cell.
Inhibition of neuraminidase, therefore, slows virus spread, giving the immune system the opportunity to “catch up” and mediate virus clearance.

Question 25

2 examples of Nucleoside Analog Reverse Transcriptase Inhibitors (NRTIs)

Answer 25

1) Zidovudine (ZDV) or AZT [Azidothymidine]
2) ddI [Didanosine]

Question 26

ZDV/AZT

Answer 26

Nucleoside analog reverse transcriptase inhibitors (NRTIs).
Nucleoside analog of thymine, ie resembles the deoxyribonucleotide containing the base thymine.

Question 27

Mechanism of antiviral effect of ZDV/AZT

Answer 27

Competitive inhibition of Reverse Transcriptase activity: AZT-triphosphate competes with thymine deoxyribonucleotide triphosphate for reverse trasncriptase.
Insert of AZT-monophosphate into cDNA blocks the growth of the cDNA being transcribed from the viral RNA by reverse transcriptase.

Question 28

AZT has been shown to reduce clinical signs in _________ when administered at a dose of 10mg/kg twice a day, subcutaneously, for a period of 3 weeks

Answer 28

FIV-positive cats

Question 29

_______ are required to cleave the HIV polyproteins into functional proteins.

Answer 29

Proteases

Question 30

Live-Attenuated Virus Vaccine (4)

Answer 30

1) Vaccines produced from naturally occurring attenuated viruses
2) Vaccines produced by attenuation of viruses by serial passage in cultured cells
3) Vaccines produced by attenuation of viruses by serial passages in heterologous hosts
4) Vaccines produced by attenuation of viruses by selection of cold-adapted mutants and reassortants

Question 31

Non-replicating Virus Vaccines

Answer 31

1) Vaccines produced from inactivated whole virions
2) Vaccines produced from purified native viral proteins

Question 32

3 types of vaccines

Answer 32

1) Live-attenuated vaccines
2) Non-replicating vaccines
3) Vaccines produced by recombinant DNA and related techniques

Question 33

Differentiating infected from vaccinated animals (DIVA)

Answer 33

Subunit ‘marker vaccines’ DIVA vaccines have only a portion (subunit) of the pathogen in the vaccine, ie has less antigens than natural strains.
If antibodies to other parts/antigens of the pathogen not included in the vaccine are detected the animal has been infected with the pathogen.
If only antibodies to the vaccine subunit/antigens are detected, the animal has not been infected.
An accompanying diagnostic test allows us to actually make that differentiation.

Question 34

Isolation

Answer 34

Applies to animals/persons who are known to be ill with a contagious disease.
Separate animal if shows clinical sings and/or tests positive by diagnostic test.

Question 35

Quarantine

Answer 35

Applies to those who have been exposed to a contagious disease.
Not effective with diseases involving chronically infected healthy shedders.
Separate animal if exposed to a contagious disease, even if does not show clinical signs, and/or animal test negative by diagnostic test.

Question 36

Quarantine and Culling

Answer 36

• To separate and restrict the movement of animals
• Culling (killing) of animals
• Proper disposition of culled animals

Question 37

Decontamination

Answer 37

A process or treatment that renders a medicinal device, instrument, or environmental surface safe to handle

Question 38

Sterilization

Answer 38

Process that destroys or eliminates all forms of microbial life/pathogens, including highly resistant pathogens, such as bacteria with spores.

Question 39

Disinfection

Answer 39

A process that eliminates many or all pathogenic microorganisms, except bacterial spores, or inanimate objects. Less effective than sterilization, does not necessarily kill all microorganisms.

Question 40

Antisepsis

Answer 40

The application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microoganisms.

Question 41

Moist heat

Answer 41

A sterilization method that uses an autoclave

Question 42

Dry Heat

Answer 42

A sterilization method that uses a hot air oven

Question 43

Chemical methods

Answer 43

A sterilization method that uses gases like Ethylene oxide, Ozone

Question 44

Radiation

Answer 44

A sterilization method that uses UV Radiation (non-ionizing) and Gamma rays, X-rays (Ionizing)

Question 45

Sterile Filtration

Answer 45

A sterilization method that uses microfiltration using membrane filters.

Question 47

Hemagglutination & Hemagglutination inhibition test

Answer 47

Relies on the property of some pathogens (mainly viruses) to nonspecifically agglutinate erythrocytes

Question 48

Agglutination

Answer 48

A method using the property of specific antibodies to bind many antigens (antigens on pathogen, or antigen coated particles-latex beads) into single clumps thereby forming large complexes, which are easily precipitated. The precipitation can be macroscopically or microscopically visible.

Question 49

Immunochromatography (lateral flow devices)

Answer 49

A form of POC (point-of-care) test that is simple to perform, easy to carry, and does not require specialized equipment

Question 50

Immunohistochemistry

Answer 50

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.

Question 51

Indirect FAT (IFAT)

Answer 51

Employs a secondary antibody labeled with a fluorescent marker that recognizes the primary antibody bound to antigen.

Question 52

Direct FAT (Fluorescence Antibody Test)

Answer 52

Labeled antibodies are added onto the sample (antigen). Visible fluorescence appears at the binding sites of the specific antibodies (antigen-antibody binding).

Question 53

T/F: Weaker signal in a Competitive ELISA indicates presence of antigens in sample

Answer 53

TRUE

Question 54

Competitive ELISA

Answer 54

A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample

Question 55

Sandwich ELISA

Answer 55

The antigen to be measured 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-reactivity or competition of binding sites.

Question 56

Indirect ELISA

Answer 56

Primary antibodies are not labeled, but detected with enzyme-conjugated secondary antibodies that recognize the primary antibodies

Question 57

Direct ELISA

Answer 57

Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important.