Laboratory

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Detection of Viruses or Viral componenets: list-o-tests

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1) Fluorescent Anitbody Staining (FA)
2) Virus Isolation (VI)
3) Antigen Detection ELISA (ACE)
4) Negative Staining Electron Microscopy
5) Polymerase Chain Reaction (PCR)
6) Restriction Endonuclease Analysis
7) DNA sequencing

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Direct Fluorescent Antibody Staining

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1) Prepare sample
2) section tissue in a cryostat
3) Apply conjugate to acetone fixed tissue section
4) Wash tissue to remove excess conjugate
5) View under UV light microscope

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Virus Isolation

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1) Select sample
2) Homogenize sample
3) centrifuge homogenate
4) Filter centrifuged supernatant
5) Inoculate susceptible cells
6) Examine inoculated cells under microscope

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Negative Staining Electron Microscopy

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1) Prepare sample
2) Examine w/ EM

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Polymerase Chain Reaction

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1) operates on the Exponential Target Amplification Principle
2) PCR cycle segments: a) denaturation, b) annealing, c) extension (1 min. each)
3) Duplication of template DNA strands
4) Run on gel

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Restriction Endonuclease Analysis

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1) Restriction enzyme cuts are specific digestion site sequency
2) Run on gel

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Viral serology: list-o-tests

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1) Indirect FA
2) Antibody detection ELISA
3) Hemagglutination Inhibition
4) Agar Gel Immunodiffusion
5) Virus Neutralization
6) Immunoblotting

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Antibody detection ELISA

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1) sensitize plate
2) wash
3) add test antibody
4) wash
5) add ligand
6) wash
7) add chromogen
8) develop plate

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Agar Gel Immunodiffusion

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1) prepare plates
2) create patterns of 1 central and 6 peripheral wells
3) apply test and control sample

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Immunoblotting

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1) sample prep
2) gel electrophoresis
3) transfer
4) blocking
5) antibody addition
6) detection

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Virus-induced Immunosuppression

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1) Infections usually stimulate immune response, but some infx can cause global immune suppression or specifically dampen immune system

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Mechanisms of Immunosuppression (on exam)

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1) Virus infection and depletion of immune cells (monocytes, T cells, APCs...)
2) Tolerance induction
3) Perturbation of immune response through secreted viral proteins

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Influenza virus neuraminidase functions in viral replication

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1) NA cleaves new virus particles from host cell membrane to free it
2) NA prevents aggregation of said particles to optimize dissemination
3) NA helps virus penetrate respiratory epi (by binding sialic acid) through which the virus must spread and replicate

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Influenza virus neuraminidase functions in immunosuppression by T cell depopulation and reorientation

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1) NA binds sialic acid in hinge region of IgA--> IgA denuded of sialic acid--> recognized, bound, and cleared by hepatic asialoglycoprotein receptor (ASGPR)
2) NA removes sialic acid from mucosal gamma/delta T-cells or IgA-producing B cells. Causes these cells to move away from mucosa, reducing localized lymphocyte population

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Virus infection and depletion of immune cells (monocytes, T cells, APCs) by HIV

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1) HIV replicates primarily in CD4+ T cells: destroys T-cells (CD4+ preferentially infected); Monocytes/macrophages and dendritic cells can also be infected--> non-cytopathic infection (reservoir?)

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Real Time PCR (RT-PCR)

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1) primers and probe anneal to target
2) Taq begins to displace 5' end of probe as extension proceeds
3) 5' nuclease activity of Taq cleaves off 5' fluorophore on probe
4) Probe begins to fluoresce as it separates from Quencher, fluorescence builds up as PCR products accumulate

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DC-Sign

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1) a cell surface lectin molecule that captures HIV-1 onto T-cells
2) may provide a conduit for transfer of HIV to lymphoid organs

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Virus infection and depletion of immune cells (monocytes, T cells, APCs) by lymphocytic choriomeningitis virus

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1) LCMV and APCs: Clone 13 preferentially infects and destroys macrophages and DCs (APCs are destroyed first, before cognate antigen presentation can take place)
2) Armstrong strain is immunogenic and thus well-controlled by responding immune system
3) Clone 13 strain of LCMV is globally immunosuppressive (Tolerance induction when newborns are infected)

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Tolerance Induction: Virus as Self

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Tolerance
1) required for prevention of autoimmunity: a) thymic deletion of forbidden clones; T-cell clones are educated in thymus. b) peripheral exhaustion of forbidden clones
2) Characteristic of tolerogenic viral infections: a) most easily induced during fetal development, b) most readily induced if viral replication produces high levels of viral antigens, c) virus specific, d) leads to viral persistence

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LCMV and tolerogenic Infections

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1) Neonatal mice infected w/ LCMV (clone 13) are tolerant only to LCMV but respond well to other viruses
2) Thymic deletion: LCMV infects immature thymocytes; T cells being educated in the presence of LCMV expressing T cells in the thymus are thus tolerant (LCMV reactive clones are deleted)
3) peripheral exhaustion of reactive clones: a) CTL induction is faster with a higher viral inoculum but the majority of activated CTLs apoptose instead of self-renewing and differentiating into effector cells. b) virus-induced high-dose tolerance produces virus-specific immunosuppression

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Viral proteins that perturb immune responses

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Effect on complement:
1) accessory proteins encoded by HSV (gC and gI) can prevent complement activation by binding Fc protion of anti-HSV (gC) antibodies
2) Down-regulation of MHC Class 1: a) MHC Class 1 essential for Ag presentation of viral-derived peptides, b) MHC Class 1 down-regulation prevents CTL lysis of virus infected cells, c) Different mechanisms: HIVnef, Adeno E1A, E3
3) Interference w/ cytokine fx: IFN-gamma receptor homology encoded by poxviruses blunts effects of IFN-gamma secreted by Ag-specific T cells

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Animal Viruses Associated with Immunosuppression

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1) LCMV
2) Morbilliviruses: Canine Distemper Virus-- a) acute febrile and fatal dz in dogs, b) encephalitis, pneumonitis, gastroenteritis, c) immunosuppression results in inability to control bac/fungal superinfection, d) CDV infects lymphocytes, monocytes resulting in severe leukopenia (loss of lymphoproliferative response; loss of recall entigen response)
3) Retroviruses: Mouse leukemia virus/murine AIDS

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PCR components

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1) one or more molecules of target DNA or RNA
2) 2 oligonucleotide primers
3) thermostable DNA polymerase
4) reverse transcriptase enzyme, if RNA is the nucleic acid target
5) dNTPs

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PCR principle

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rx mix heated/cooled 25-35x to amplify target DNA

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PCR Amplification Cycle

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1) heat denaturation of DNA (Temp 1)
2) single stranded region allows primer annealing (Temp 2)
3) DNA extension via Taq polymerase
4) visualize on agarose gel

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Mosquito PCR: Procedures ala Ned Walker

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1) sort mosquitos
2) grind, mix, and spin mosquitoes to get viral particles suspended in diluent
3) extract RNA
4) prepare reaction mix (reverse transcription, then PCR)
5) PCR product visualized on gel using electrophoresis