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Flashcards in Exam 2 Terminology Deck (65):
1

Polymerase Chain Reaction (PCR)

Technique used to make multiple copies of a segment of DNA. It is precise, fast and inexpensive. Result: Two new DNA molecules are obtained from the original one.

2

3 steps of PCR

1. Denaturation
2. Annealing
3. Extension/Elongation

3

Denaturation

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA.

4

Annealing

Two short stretches of nucleotides (20-30 nucleotides long), known as primers, attach to ends of each of the two separated DNA segments. Forward primer (+ sense) binds to reverse DNA strand (- sense strand) and vice versa

5

Extension/Elongation

An enzyme builds two new strands of DNA using the original strands as a template. Thus, a new DNA segment extending from the primer, and complementary to original DNA segment is created.

6

Taq polymerase

The enzyme used in extension/elongation. It keeps on adding free nucleotides (complementary to the corresponding nucleotides of original DNA segment) to the attached primer.

7

Real Time PCR

Also known as quantitative PCR. Allows monitoring and quantification of increasing accumulation of PCR product/nucleic acid load as the reaction progresses.

8

Genome Sequencing

Process by which the sequence of bases in a DNA molecule is elucidated/can be obtained and read.

9

Next Generation Sequencing (NGS)

Cheaper, quicker, less DNA, has high throughput, and is more accurate and reliable than Sanger sequencing.

10

Metagenomics

The study of the collective set of microbial population 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.

11

Why does genome sequencing play a crucial role in surveillance studies?

It allows:
1. Pathogen detection
2. Studies on genetic variation
3. Identification of unidentified strains
4. Development of diagnostics
5. Identifies genes associated with drug resistance
6. Judges the efficacy of current vaccine and formualting new vaccine strategies

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Phylogenetic Analysis

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

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Microarrays

Shows positive reactions between probe DNA and sample DNA (hybridization). Generates a fluorescent signal from the spot where probe DNA is spotted in the chip.

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Advantage of microarrays

Hundreds of pathogens can be screen for simultaneously using a single micoarray chip.

15

Antiviral Drugs

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

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Antimicrobial Chemotherapy

The treatment of infectious diseases by drugs (chemical compounds) that are inhibitory or lethal to the pathogenic microbe.

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Antibiotics

AKA antibacterials, are types of medications that destroy or slow down the growth of bacteria.

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Antimycotics

Medications used to treat fungal diseases.

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Antiparasitics

Are a class of medications which are indicated for the treatment of parasitic diseases.

20

What is the best logical approach of a new antiviral drug?

It will interfere with virus replication without harming or causing minimal harm to the infected host cell.

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Antiviral Difficulties

Difficulties encountered in development of broad spectrum antivirals with low cytotoxicity.

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How does an antiviral act?

Antivirals can stop the receptor binding, stop uncoating, stop protein nucleic and protein synthesis, stop assembly, and stop release.

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Acyclovir

-Activity primarily restricted to herpesviruses
-Is a synthetic nucleoside analog of deoxyguanosine

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Acyclovir Treatment of?

Herpesviruses infections in humans, Feline herpesvirus-1 induced corneal ulcers, and Equine herpesvirus-1 induced encaphalomyelitis.

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Acyclovir Mechanisms of Antiviral Effect

1. Acyclovir molecules entering the cell are converted to acyclovir monophosphate by virus induced thymidine kinase enzyme
2. Host cell enzymes add two more phosphates to form acyclovir triphosphate, which is transported to the nucleus
3. Cleavage of 2 phosphates from the acyclovir triphosphate by the herpes simplex's own enzymes to form acyclovir monophosphate
4. 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)

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Stop the growing viral DNA chain

Further elongation of the growing viral DNA chain is impossible because acyclovir monophosphate lacks the attachment point necessary for the insertion of any additional nucleotides.

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Competitive Inhibition of Viral DNA Polymerase

The acyclovir triphosphates compete the dGTPs for viral DNA polymerase.

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Acyclovir Mechanism for Resistance

- Absent production of viral thymidine kinase
- Decrease in the production of viral thymidine kinase
- Altered viral thymidine kinase substrate
- Decrease binding of acyclovir triphosphate

29

Amantadine

- Synthetic tricyclic amine of the adamantane family
- Acts as both antiviral and anti-parkinson disease
- Inhibits replication of most strains of Influenza A viruses by blocking uncoating of the virus

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Amantadine Mechanisms

M2 ion channel is the target of the antiviral Amantadine. Viral RNAs remain bound to M1 and cannot enter the nucleus. Virus replication is inhibited.

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Amantadine Mechanism for Resistance

Changes in amino acids that line the M2 channel. It prevents the drug from plugging the channel.

32

Neuraminidase Inhibitors

Inhibitor of neuraminidase [NA] enzymes synthesized by Influenza A and B viruses. Prevents the release of virus and spread of infection.

Examples: Oseltamivir (Tamiflu), Laninamivir, Zanamivir, and Peramivir

33

Oseltamivir phosphate (Tamiflu)

A prodrug that, after its metabolism in the liver, releases an active metabolite that inhibits neuraminidase.

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Hemagglutinin (HA)

Glycoproteins found on the surface of influenza virus. It binds to receptors containing sialic acid on host cell membrane.

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Neuraminidase (NA)

Glycoproteins found on the surface of influenza virus. It will cleave the sialic acid containing cell surface receptors and release HA. It is critical in cell to cell spread.

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Replication of Retrovirus and Targets for Anti-retroviral therapy

Inhibit fusion, integrase, reverse transcriptase, and protease.

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Nucleoside Analog Reverse Transcriptase Inhibitors (NRTIs)

1. Zidovudine (ZDV) or Azidothymidine (AZT)
2. Didanosine (ddI)

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Azidothymidine (AZT)

Nucleoside analog of thymidine. Phosphorylated by kinases present in host cell to AZT-triphosphate. The reverse transcriptase cleaves two phosphates and inserts AZT monophosphate into the cDNA that is being synthesized from viral RNA. Blocks the growth of the cDNA being transcribed from the viral RNA by reverse transcriptase.

39

Zidovudine (ZDV)

Phosphate groups of free deoxyribonucleotide can only bond to OH groups, they are unable to bind to N3 of ZDV. This results in an incomplete cDNA/provirus.

40

Maintenance drugs

It slows down the progression of HIV infection, but does not eradicate or eliminate the virus. Such as ZVD and AZT.

41

HIV Protease Inhibitors

Saquinavir, Ritonavir, Indinavir, Nelfinavir

Bind to the active site of HIV protease and prevent the enzyme from cleaving HIV polyproteins into functional proteins. (Gagpol polyprotein: p120, p55 and Env polyprotein: gp160). As a result, HIV cannot mature and non-infectious viruses are produced.

42

Immunization: Vaccination

Protect susceptible individuals from infection or disease. Prevent transmission of infectious agents by creating an immune population.

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Live-Attenuated Virus Vaccines

Vaccines produced from naturally occurring attenuated viruses.

Examples: human smallpox vaccines from cowpox virus, Marek's disease vaccine from herpesvirus of turkeys, and Porcine rotavirus vaccine from bovine rotavirus.

44

Vaccines produced by attenuation of viruses by serial passage in HETEROLOGOUS hosts

Rinderpest (deadly disease of cattle) and classical swine fever (deadly disease of pigs) viruses were each adapted to grow in rabbits and, after serial passage, became sufficiently attenuated to be used as vaccines. Are live attenuated vaccines.

45

Vaccines produced by attenuation of viruses by selection of COLD ADAPTED MUTANTS AND REASSORTMENTS

Would be safer vaccines for intranasal administration, in that they would replicate well at the lower temperature of the nasal cavity (33oC in most mammalian species), but not at the higher temperature (37oC) of the more vulnerable lower respiratory tract and pulmonary airspaces.

46

Non-replicating virus vaccines

Vaccines produced form inactivated whole virions
and
Vaccines produced from purified native viral proteins

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Vaccines produced form inactivated whole virions

Inactivated (syn. Killed) virus vaccines. Have destroyed infectivity but maintain immunogenicity. Need to contain relatively large amount of antigen to elicit an antibody response commensurate with that induced by a much smaller dose of live-attenuated virus vaccine.

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Vaccines produced from purified native viral proteins

Virion is solubilized and its components released, including the glycoprotein spikes of the viral envelope.

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Vaccines produced by recombinant DNA and related Technologies

Vaccines produced by attenuation of viruses by gene deletion or site directed mutagenesis.

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Differentiating Infected from Vaccinated Animals (DIVA)

Contain subunit 'marker vaccine' DIVA vaccines have only a portion (subunit) of the pathogen in the vaccine. i.e. has less antigens than natural strains.

51

Source reduction

A vector control by stopping where breeding of vectors occurs such as stagnant water.

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Biological control

Vector control by use of natural enemies to manage mosquito population.

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Chemical control

Vector control such as insecticides, larvicides, adulticides.

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Isolation

Applies to animals/persons who are KNOWN to be ill with a contagious disease.

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Quarantine

Applies to those who have been EXPOSED to a contagious disease. Enforced for the longest incubation period of the disease.

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Population control program

Population reduction may be used to control spread of zoonosis within a reservoir population.

57

Quarantine and culling

To separate and restrict the movement of animals.

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Decontamination

A term used to describe a process or treatment that renders a medical device, instrument, or environmental surface safe to handle. Sterilization, disinfection, and antisepsis are all forms of decontamination.

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Sterilization

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

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Disinfection

Describes a process that destroys or eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects.

61

Antisepsis

Is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms.

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Sterilization methods

Moist heat, dry heat, chemical methods, radiation, sterile filtration.

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Farm Biosecurity

All measures taken to minimize the risk of the introduction and the spread of infectious agents.

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External Biosecurity

Measures taken to prevent an infectious disease from entering or leaving the farm.

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Internal Biosecurity

Measures taken to combat spread of an infectious disease within the farm.