Block 5 Flashcards
PCR
Amplification of viral genome/DNA
3 steps of PCR
1) Denaturing
2) Annealing
3) Extension/Elongation
Real-Time PCR assay
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.
Genome Sequencing
DNA sequencing refers to the process by which the sequence of bases in a DNA molecule is elucidated/can be obtained and read
Real-Time PCR or Quantitative PCR
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.
Next Generation Sequencing
Are cheaper, quicker, needs significantly less DNA, has high throughput, and is more accurate and reliable than Sanger sequencing
Metagenomics
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.
How does Metagenomics work?
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.
Genome sequencing plays a crucial role in surveillance studies, as it allows (7):
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
Phylogenetic Analysis
The use of Virus genome sequence data to study evolution of viruses and genetic relationships among viruses
Microarrays
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.
Advantage of microarray in detection of pathogens in surveillance studies
Hundreds of pathogens can be screened for simultaneously using a single microarray chip
Antiviral Drugs
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
Immune system stimulation
Interferons, class of proteins that has antiviral effects and modulate functions of the immune system
Acyclovir
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.
What does Acyclovir Treat? (3)
1) Herpesvirus infections in humans
2) Feline herpesvirus 1 induced corneal ulcers
3) Equine herpesvirus 1 induced encephalomyelitis
Acyclovir is a synthetic nucleoside analog of ______.
Deoxyguanosine
Mechanism of antiviral effect of Acyclovir
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.
Is Acyclovir toxic or non-toxic to the uninfected host cell?
Non-toxic because acyclovir cannot be phosphorylated and incorporated into the host DNA.
Amantadine
Inhibits replication of most strains of Influenza A viruses by blocking uncoating of the virus.
Mechanism of antiviral effect of Amantadine
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.
Neuraminidase Inhibitors
Inhibitor of Neuraminidase enzyme synthesized by Influenza A and B viruses.
Example of Neuraminidase Inhibitors
Oseltamivir (Tamiflu)
Mechanism of antiviral effect of Neuraminidase Inhibitors
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.
2 examples of Nucleoside Analog Reverse Transcriptase Inhibitors (NRTIs)
1) Zidovudine (ZDV) or AZT [Azidothymidine]
2) ddI [Didanosine]
ZDV/AZT
Nucleoside analog reverse transcriptase inhibitors (NRTIs).
Nucleoside analog of thymine, ie resembles the deoxyribonucleotide containing the base thymine.
Mechanism of antiviral effect of ZDV/AZT
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.
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
FIV-positive cats
_______ are required to cleave the HIV polyproteins into functional proteins.
Proteases
Live-Attenuated Virus Vaccine (4)
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
Non-replicating Virus Vaccines
1) Vaccines produced from inactivated whole virions
2) Vaccines produced from purified native viral proteins
3 types of vaccines
1) Live-attenuated vaccines
2) Non-replicating vaccines
3) Vaccines produced by recombinant DNA and related techniques
Differentiating infected from vaccinated animals (DIVA)
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.
Isolation
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.
Quarantine
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.
Quarantine and Culling
- To separate and restrict the movement of animals
- Culling (killing) of animals
- Proper disposition of culled animals
Decontamination
A process or treatment that renders a medicinal device, instrument, or environmental surface safe to handle
Sterilization
Process that destroys or eliminates all forms of microbial life/pathogens, including highly resistant pathogens, such as bacteria with spores.
Disinfection
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.
Antisepsis
The application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microoganisms.
Moist heat
A sterilization method that uses an autoclave
Dry Heat
A sterilization method that uses a hot air oven
Chemical methods
A sterilization method that uses gases like Ethylene oxide, Ozone
Radiation
A sterilization method that uses UV Radiation (non-ionizing) and Gamma rays, X-rays (Ionizing)
Sterile Filtration
A sterilization method that uses microfiltration using membrane filters.
Hemagglutination & Hemagglutination inhibition test
Relies on the property of some pathogens (mainly viruses) to nonspecifically agglutinate erythrocytes
Agglutination
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.
Immunochromatography (lateral flow devices)
A form of POC (point-of-care) test that is simple to perform, easy to carry, and does not require specialized equipment
Immunohistochemistry
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.
Indirect FAT (IFAT)
Employs a secondary antibody labeled with a fluorescent marker that recognizes the primary antibody bound to antigen.
Direct FAT (Fluorescence Antibody Test)
Labeled antibodies are added onto the sample (antigen). Visible fluorescence appears at the binding sites of the specific antibodies (antigen-antibody binding).
T/F: Weaker signal in a Competitive ELISA indicates presence of antigens in sample
TRUE
Competitive ELISA
A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample
Sandwich ELISA
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.
Indirect ELISA
Primary antibodies are not labeled, but detected with enzyme-conjugated secondary antibodies that recognize the primary antibodies
Direct ELISA
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.
