MMG 565 – Microbiology – Flashcards
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| - Direct Fluorescent Antibody (FA) Test |
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| 1. Process a) Sample frozen & sliced in cryostat b) Conjugate w/fluorescent-labeled antiviral Abs added c) Sample washed to remove excess Ab not attached to virus d) Ab attaches to virus if present and fluoresces, examined w/UV light source 2. Immunohistochemistry – same principle as above, but the “flag” is an enzyme that creates an enzyme-substrate rxn that is colored |
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| - Virus Isolation (VI) |
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| 1. Process a) Take sample, homogenize & centrifuge b) Filter supernatant to eliminate bacteria c) Make serial dilution d) Add virus to culture of susceptible cells e) Examine cells to see if they undergo cytopathic effect a. Could also see a giant cell/synctium (fuses adjacent cells together) f) Double-check results w/IFA (some viruses are not cytopathic) |
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| - Virus Isolation (VI) |
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| 2. Virus Quantitation (plaques) – maybe to see how much virus shed after animal vaccinated a) Process – count plaques that form on plate a. TCID50 – highest serial dilution with detectable virus 3. Virus Growth in Embryonated Eggs – can inject virus into chorioallantoic membrane, yolk, etc |
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| - Antigen Detection ELISA |
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| a) Add sample to well containing Capture Ab b) Ab attaches to/captures vius c) Add Detection Ab that has a substrate/chromagen attached to it a. Chromagen – chemical that changes color as fxn of product formation d) Color indicates positive & color strength = amount of Abs bound |
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| - Negative Staining Electron Microscopy |
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| 1. Process – specimen bombarded by e- beam, get a negative image 2. Viral Shapes – look at PPT |
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| - Polymerase Chain Reaction (PCR) |
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| 1. Process a) Must know DNA sequence, then get primers based upon sequence b) Use heat-stable DNA Polymerase, add nucleic bases (ATCG) c) Denaturation > Annealing > Extension d) Exponential doubling of template DNA strands e) PCR products in agarose gel 2. Multiplex RT-PCR – looking for multiple viruses in single assay |
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| 3. Probe-based Real-time PCR – |
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| a probe w/2 labels added (Q label controls R label), as strand is being made it attaches to R end, the R end is released and no longer controlled by Q so shows signal |
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| - Hemagglutination (HA) |
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| 1. Ex – influenza, parvovirus 2. Process a) Make serial dilution in wells that contain RBCs b) If virus present, see a carpet of RBCs in well c) Measure dilution that last produces reaction, this is the tire (if 1:128 dilution, titer = 128) |
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| - Restriction Endonuclease Analysis |
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| tests vaccines for strength, now PCR used instead |
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| Restriction Endonuclease Analysis |
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| 1. Process a) Take swab from sick cow & from vaccine to see if DNA is the same b) Digestion using enzymes breaks up DNA c) Agarose gel electorphoresis of pieces d) Used for viruses w/segmented genomes |
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| - Automated DNA Sequencing |
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| Didnt talk about |
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| Tests for Detection of Viruses/Viral Components |
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| Direct Fluorescent Antibody (FA) Test Virus Isolation (VI) Antigen Detection ELISA Negative Staining Electron Microscopy Polymerase Chain Reaction (PCR) Hemagglutination (HA) Restriction Endonuclease Analysis Automated DNA Sequencing |
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| Laboratory Diagnosis: Viral Serology |
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| - Indirect Fluorescent Antibody (IFA) Test - Antibody Detection ELISA Test - - Hemagglutination Inhibition (HI) Test - Agar Gel Imunodiffusion (AGID) Test Virus Neutralization (VN) Test - Immunoblotting (IB) or Western Blotting (WB) Test |
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| - Indirect Fluorescent Antibody (IFA) Test |
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| 1. Process a) Add patient serum sample to tissue that contains virus, incubate & wash b) Patient Ab should attach to virus c) Add fluorescence-labeled Ab that attach to the patient antibodies *detects viral Ab, NOT Ag |
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| - Antibody Detection ELISA Test |
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| 1. Process a) Add patient serum to virus on plate, patient Ab binds b) Add Ab w/ligand that attached to patient Ab c) Add chromagen that attaches to ligand-Ab |
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| - Hemagglutination Inhibition (HI) Test |
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| – use for viruses that attach to RBCs, can test a vaccine titer 1. Process a) Add patient sample w/patient Ab to hemaggluinating virus b) If positive, patient Ab will bind to virus & inhibit agglutination c) If negative, Ab will not bind and virus can hemagglutinate, see red field |
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| - Agar Gel Imunodiffusion (AGID) Test |
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| used to be used for Coggins, but now use ELISA more 1. Process a) 5 wells punched into agar, 4 surrounding central hole b) Known viral Ag added to center well, positive reference controls added to 2 wells c) Patient sample added to other 2 wells d) Ag & patient Ab diffuse into agar, form line of precipitation if Ab present |
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| - Virus Neutralization (VN) Test |
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| 1. Process a) Prepare serial dilution of patient serum, add equal amounts of virus b) Add susceptible cells c) Measure lowest amount of patient Ab dilution that prevents cytopathic effect (CPE) |
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| - Immunoblotting (IB) or Western Blotting (WB) Test |
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| used for PPM in horses 1. Process – prepare sample of viral proteins, do gel electrophoresis, add patient Ab |
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| Feline Calicivirus |
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| - Diagnosis – clinical signs + test 1. RT-PCR – method of choice, on fresh or fixed 2. Other Tests – virus isolation (easy), direct FA (not used anymore), VN testing on paired sera 3. Cannot specifically test for virulent systemic form |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Diagnosis – clinical signs + test 1. PCR – definitively dxs 2. Virus Isolation – works well, but PCR best (nasal/conjunctival/oral swabs) 3. Ab Titers – if owner brings in cat post-acute inf,take 2 swabs 3w apart, and if titer has 4x difference you can say cat had it |
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| Felid Herpesvirus-1 (FeHV-1) |
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| a) Linear dsDNA |
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| Feline Calicivirus |
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| 1. Non-enveloped 2. +strand, ssRNA |
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| Feline Calicivirus |
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| 1. Open Reading Frame 1 (ORF 1) – encodes a polyprotein & RNA polymerase a) ORF 2 – encodes capsid precursor protein b) ORF 3 – encodes small basic protein 2. Antigenic Variation – all FECV are from ONE serotype, but there is Ag-variation a) Hypervariable Regions C & E of ORF2 – created Ag-variation (has parts A-F) |
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| Feline Calicivirus |
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| - Clinical Presentations 1. Acute Oral/Resp Dz – most common, assoc w/temporary lingual ulceration 2. Chronic Stomatitis a) If chronic stomatitis & gingivitis, think of calic and FIV 3. Limping Syndrome – some Ag-variations have affinity for synovial joints 4. Virulent Systemic Form – systemic inflmm response, DIC, multi-organ failure, mortality 67% a) Signs – lesions on foot bads & tongue are erosive, facial edema 5. Feline Urinary Syndrome? – some calicivirus found in bladder stones 6. Subclinical |
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| Feline Calicivirus |
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| - Pathogenesis 1. Exposure to virus from other cats/fomites, enters through resp/oral route (virus also in urine) a) Must clean w/bleach 2. Primary virus replication in oral/nasal/conjunctival epithelium 3. Viremia leads to secondary replication at secondary sites (pulmonary, synovial, UT epithelia), strain-dependent 4. Virus shed from urine & RT |
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| Feline Calicivirus |
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| - Clinical Signs 1. Distinguishing Signs – oral ulceration, lameness 2. Other Signs – lethargy, sneezing, conjunctivitis, ocular/nasal discharge, pneumonia |
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| Feline Calicivirus |
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| - Epidemiology 1. Most cats still shedding infective forms 30 days post-infection a) 50% cats still shedding 75 days-post 2. Asymptomatic Carriers – some cats carry for life in tonsillar/pharyngeal tissues (20% of all cats admitted to clinics for non-resp dz) 3. Virus Shedding – high in some, low in others |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Etiology – enveloped dsDNA, Herpesviridae-Alphaherpesvirinae-Varicellovirus 1. Narrow Host Range – felidae ONLY (large & small) 2. 45% of Feline URD – causes feline rhinotracheitis |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Attachment, Fusion, Penetration 1. Viral glycoproteins interact w/cell receptors (attachment) a) First, viral *gC forms loose contact w/host receptor b) Second, *gD forms tighter bond w/different host receptor 2. Viral envelope fuses w/cell membrane (fusion) 3. Tegument + nucleocapsid enter cell (penetration) 4. Capsid fuses w/nuclear membrane at nuclear pore 5. Viral DNA + *Vp16 (viral protein 16) enter nucleus, Vp16 activates virus replication 6. 3 stages of viral DNA processing a) Immediate Early – immediate early genes transcribed/translated into imm-early (?) proteins, they regulate upshift/transcription of early proteins (?) b) Early – early proteins (?) made, they are enzymes that stimulate viral DNA replication c) Late – after several DNA replications the late genes make late proteins (?), structural components of virus particle 7. Newly made capsid moves to ER, acquires envelope & surface glycoproteins, now infective 8. May see inclusion bodies inside nucleus, *made of nucleocapsid |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Transmission 1. Aerosol – oral/nasal/ocular discharges infections 7-21d 2. Fomites – don’t have to use bleach to clean 3. Asymptomatic Carriers – reactivation of latent infection (boarding, parturition, steroids, etc.) |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Pathogenesis 1. Susceptible animal exposed via aerosol/fomite 2. Virus replicates in upper RT or conjunctival epithelium ONLY, no lung involvement a) Virus is temp-sensitive and can only replicate at temps below normal body temp 3. Inflmm response w/cytokine production (high fever) |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Clinical Signs (Feline Viral Rhinotracheitis/Classical Acute Dz) 1. HIGH fever (105F) 2. Conjunctivitis & conjunctival edema, dendritic ulcers in cornea (more common in reactivation) a) Corneal ulcers also point to FHV & Chlamydophila 3. Dyspnea (occlusion of trachea by necrotic plaques), sneezing 4. Limited viremia & transplacental infections b/c virus can’t replicate at body temp 5. Generalized infection causes high mortality/encephalopathy 6. High morbidity (age-independent), low mortality (age-dependent) 7. Distinguishing Signs – keratitis & hypersalivation, lethargy, sneezing somewhat 8. Uncommon Signs – abortion, encephalitis, death |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Other Disease Types of FeHV-1 1. Atypical Acute Dz – skin dz, viraemia, pneumonia, sever systemic signs, kitten deah 2. Chronic Dz – stromal keratitis, chronic rhinosinusitis, blindness 3. FHV-related – corneal sequestra, eosinophilic keratitis, neuro dz, uveitis |
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| Felid Herpesvirus-1 (FeHV-1) |
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| - Viral Latency – feature of all herpesviruses 1. Lifelong infection of tissues w/FeHV-1 DNA 2. Reactivation – produces infectious virus, mild/no clinical signs 3. Process a) Induction – virus enters nerve endings at small necrotic areas in nasal epithelium during acute dz, moves upstream to CNV ganglion, targets NEURONS b) Maintenance/Latent Phase – viral DNA sits dormant c) Reactivation – replication restarted, must express LAT |
