Week 5

• Competitive ELISA


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

  Weaker signals indicates presence of antigens in samples
• Fluorescence antibody test

  Direct FAT: Labeled antibodies are added onto sample, Visible fluorescence appears at the binding sites of the specific antibodies
• Fluorescence antibody test

  Indirect FAT: Employs secondary antibody labeled with a fluorescent marker that recognizes the primary antibody bound to antigen
• Immunohistochemistry

  Antibody target with an enzyme (generally horseradish peroxidase)

  Enzyme reacts with a substrate to produce colored product that can be visualized in the infected cells with a standard light microscope
• Immunochromatography (lateral flow devices)

  Form of point-of-care test that is simple to perform, easy to carry, and does not require specialized equipment
• Agglutination

  Method using the property of specific antibodies to bind many antigens into single clumps thereby forming large complexes, Easily precipitated, Can be macroscopically or microscopically visible
• Hemagglutination & hemagglutination inhibition test

  Relies on the property of some pathogens to nonspecifically agglutinate erythrocyte
• Agar gel immunodiffusion


  Antigen and antibody placed in separate wells of an agar gel, Antigen and antibody diffuse toward each other, A thin white line is formed due to precipitation of antigen/antibody complex
• Complement fixation test

  Serum from patient has antibodies against virus A; Intact sheep RBCs settle at bottom = Positive reaction

  Serum from patient is negative to virus A (will not have antibodies); Hemolysis of sheep RBCs, destruction/hemolysis of sheep RBC = negative reaction
• Neutralization assay

  Loss of infectivity through reaction of the virus with specific antibody, Presence of unneutralized virus may be detected by reactions, Virus and serum are mixed under appropriate conditions and then inoculated into cell culture, eggs, or animals, Antibody-bound virus becomes noninfectious and cannot produce desired effects in eggs, cell-lines, or animals
• Polymerase chain reaction

  Amplification of viral genome/DNA, Denaturation, Annealing, extension/elongation
• Real-time PCR or quantitative PCR

  Advanced form of PCR, Allows monitoring and quantification of increasing accumulation of PCR products/nucleic acid load as the reaction progresses, Useful to study virus load in patient, Target specific probe, Intercalating dyes (SYBR Green), Fluorescence emitted by excited fluorophore is visualized and analyzed
• Genome sequencing

  DNA sequencing, Sequence of bases in a DNA molecule is elucidated/can be obtained and read
• Next generation sequencing

  Surveillance studies are more relying on, Gradually replacing conventional sequencing methods, Platforms are significantly cheaper, quicker, needs significantly less DNA, has high throughput, more accurate and reliable
• Metagenomics

  Study of the collective set of microbial populations in a sample

  Analyzing sample's entire nucleotide sequence content

  method for random detection of existing and new pathogens

  amplification and sequencing of whole genome content of a sample

  compare the genome databases and using different softwares

  Next generation sequencing platforms are being extensively used in this type of studies
• Importance of genome sequencing: Pathogen detection, Studies on genetic variation, Genotyping, evolution and interspecies transmission of pathogens, Identification of novel and undiscovered strains, Development of diagnostics, Genotyping primers or probes, Identifications of genes associated with drug resistance, Development of therapeutics, Judging the efficacy of current vaccines and formulating new vaccine strategies, Phylogenetic analysis
• Microarrays

  Several thousands of known DNAs are spotted onto glass or silicon chip, Amplified by PCRs/rtPCRs, target/sample DNA are fluorescently labeled and hybridized/added to the chip containing DNA probes, Positive reactions between probe-DNA and sample DNA generate a fluorescent signal from the sport where probe DNA is spotted in the chip
• Advantage of microarrays: 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 for replication of the virus
• Antiviral drugs

  Class of medication used specifically for treating viral infections
• Immune system stimulation

  • Interferons
  • Class of proteins that has antiviral effects and modulate functions of the immune system
• Acyclovir


  Antiviral activity primarily restricted to herpesviruses
• Acyclovir


  Administered as prodrug (inactive form)

  Interferes with virus replication
• Acyclovir - treatment of

  • Herpesvirus infection in humans
  • Feline herpesvirus-1 induced corneal ulcers
  • Equine herpesvirus-1 induced encephalomyelitis
• Acyclovir

  Synthetic nucleoside analog od deoxyguanosine (dGTP)
• Acyclovir mechanism of antiviral effect

  1. Herpes simplex DNA polymerase enzyme incorporates acyclovir monophosphate into growing DNA strand

  2. As if it were 2-deoxyguanosine monophosphate

  3. Further elongation of the chain is not possible
• Acyclovir

  Competitive inhibition of viral DNA polymerase
• Acyclovir

  Acyclovir triphosphate compete with dGTP for viral DNA polymerase
• Acyclovir

  Non-toxic to uninfected host cell
• Acyclovir

  • Enzymes herpesvirus thymidine kinase and herpesvirus DNA polymerase are viral enzymes
  • Not found in uninfected host cells
  • 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
• Amantadine mechanism of antiviral effect

  1. M2 ion channel is target

  2. Compounds clog the channel and prevent it from pumping protons into the virion

  3. Viral MRNs remain bound to M1 and cannot enter the nucleus

  4. Virus replication is inhibited
• Neuraminidase inhibitors

  • Oseltamivir (tamiflu)
  • Blocking the function of neuraminidase with NA inhibitors
  • Effective way to treat influenza
  • Prevents release of virus and spread of infection
• HA of virus is still bound to the sialic acid containing receptors on surface of already infected host cell


  Inhibition of neuraminidase slows virus spread
• Inhibition of neuraminidase

  Gives immune system opportunity to "catch up" and mediate virus clearance
• Nucleoside analog reverse transcriptase inhibitors

  • Zidovudine (AZT/ZDV)
  • Nucleoside analog of thymine
  • Competitive inhibition of reverse transcriptase activity
• Zidovudine mechanism of antiviral effect

  1. AZT-triphosphate competes with thymine deoxyribonucleotide triphosphate for reverse transcriptase

  2. Insertion of AZT-monophosphate 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 FIV-positive cats when administered at dose of 10 mg/kg twice a day subcutaneously for a period of 3 weeks
• Protease inhibitors

  • Inhibit protease
  • HIV polyproteins cannot be cleaved into functional proteins
• Protease inhibitor mechanism of antiviral effect

  1. Bind to the active site of the HIV protease and prevent the enzyme from cleaving HIV polyproteins into functional proteins

  2. HIV cannot mature

  3. Noninfectious viruses are produced