Final oral Flashcards
- Place and significance of virology in biosphere; evolution of viruses
-History:
•Correlation bw diseases and bacteria – e.g. Tobacco Mosaic Disease – 1892
•Foot&Mouth 1898–no bacteria in microscope=>Toxins (Viruses)
•Electron microscopes – virus independent of bacteria
-Morphology – 2 forms existence:
a) Virion: Infectious particle outside body. 20-300nm
o Contains: NA; DNA or RNA, proteins, lipids
b) Vegetative virus: The infected cell – cell serves the Virus’ purpose
-Reproduction:
•Multiplication - 10³-10⁶ per virion/host cell – very efficient, but needs to be simple structure
•Few organelles: no Mito, SER etc.
•No E prod, no protein synt./no (or incomplete) NA enzymes
-Occurrence: Widespread. Infect bacteria, fungi, plants, animals, humans
-Significance:
•Pathogens of animals/humans
•Emerging new viruses: Influenza, Ebola, HIV, BSE, SARS – all from animals
•Simple living creatures, but a threat to man – Only protection is immunization
•Many viruses: non-pathogenic – do not cause disease - “Orphan viruses”
-Origin: simplest but not the most ancient of life forms - Very diverse origin. Do not fit phylogenetic tree.
-Theories:
•Cell degeneration – (Egg Chlamydia, Rickettsia) Parasite cell lost its independence; this explains sophisticated virus existence – pox, herpes etc.
•Runaway cell components (with genetic info) – e.g.:
o mRNA -> +ssRNA virus
o Chromosome fragment – dsDNA virus
o ssDNA, dsRNA, -ssRNA
- Propagation of viruses I– noculation of embryonated eggs, experimental infection of lab animal.
-Aims:
*Detect viruses – virology diagnostics
*Large scale prod of viruses – virus analytical studies; vaccine prod
*Cell parasites – therefore we need living cells for propagation
-2 Methods:
A) Inoculation of embryonated eggs
•The egg: embryonated, SPF, white shelled
•Before inoculation: transluminated, disinfected, drill through shell
•Place of inoculation:
1.Yolk sac (Rickettsia, Chlamydia - 5-7 d. old embryo)
2+3.Allantoic or amniotic cavity (Paramyxo - 9-12 d.)
4.Chorionic-allontoic membrane (Pox, Herpes - 10-13 d.)
5. Intravenous Inoculation (Bluetongue virus - 16-17 d)
•Sealing the egg: by paraffin/glue: incubated and controlled by translumination
*Egg necroscopy (after 4-5 days): Dwarfism, distortion, death. CAM inoculation; cause nodules-“Pocks”.
*Hemagglutination test with allantoic fluid
A) Experimental infection of living animals
•Decr. importance: animal welfare, costs
•Diagnostic approach: e.g. Rabies, African Horse Sickness, Arboviruses
•Intracerebral inoculation of suckling mice
*Classical Swine Fever: African Swine Fever differentiation
*Vaccine production: Rabbit Hemorrhagic Disease – experimentally infected. Classical Swine Fever vaccine strain.
*Vaccine control: Harmless and efficient
- Propagation & purification of Virus II – production of cell cultures
-Process where cells grow under controlled conditions
•In vitro maintenance and propagation of living cells
•Monolayer cell cultures used from organs – Esp. Kidneys, Testicles, Thymus, and Embryo
•In sterile conditions – steps:
a.Removal of outer membranes, cut tissue to small pieces
b.Sep of cells via digestion w. Trypsin and EDTA
c.Cell suspension removed regularly and replaced by renewed Trypsin solution
d.Trypsin blocked by ice bed and removed by centrifuge
e.Cells resuspended in a culture medium–Minimal Essential Medium (MEM) (optimal environment needed)
f.Cell counting (burker chamber)
g.Cell culture transferred to culture flask
h.Incubation: w/in 3-5 days, cover bottom of flask.
=> Prim. monolayer cell culture produced
-Use of monolayers:
•Propagation of viruses
•Maintenance of cell cultures
•Sub culturing (passaging): tranferring cells to new vessel -> secondary culture – fresh, incr. amount (can only be reused 2-3x)
•Cloning: cultivation of a single cell
*types: diploidic or aneuploidic
*advantages : Genetically homogenous, standardized, unlimited nr of passages
*long-term storage: -80⁰C liquid N
*disadvantages: Sensitivity to infection varies, contamination (virus, leptospira), presence of active oncogen (esp. tumour Cells)
-Special virus propagation - Cell Culturing methods
a) Co-cultivation: isolate cell-associated and latent viruses i.e. mix healthy + infected cells
b) Suspension cultures: continuous stirring stops settling => fewer, more conc. cells
- Concentration and purification of viruses
•Aim: virus analysis needs clean cultures
•Steps: Isolate virus – plaque purification – viral strain – propagation of identical viruses on large scale
•Release: Freeze-thaw, Sonication (heat), detergents (for NA)
•Purification:
o Centrifugation
o Filtration – removes larger particles than virus – 450nm filter
•Concentration – method depends on viral resistance
o Precipitates - in ammonium sulphate, ethanol etc.
o Adsorption – aluminium hydroxide
o Ultrafiltration – hydrostatic pressure
o Dialysis
•Methods of concentration and purification:
o Affinity chromatography
o Density gradient ultra-centrifuge
*Stokes laws 1 + 2: sediment ~ to size particle
*Zonal or isopycnic technique
o Virion buoyant density ~ NA/protein/lipid ratio
- Morphology of viruses
•Virion
o Core = NA + proteins
o Capsid = proteins
o Envelope (+/-) = lipid membrane + proteins – may/may not be enveloped
•Examination:
o Electron microscopy – ultra thin sections
o Negative contrast staining – uranyl acetate
o Shadow casting = 3D structure
•Morphology – determined by the capsid
o Composed of “capsomers” – protein units
•Helical: Capsomers + NA = nucleocapsids
•Quasihelical (Orthomyxo, Paramyxo): always enveloped
•Cubic (Icosahedral) (Adenovirus, Parvovirus): 20 faces
*20 equilateral triangles-proteins arranged in capsomers
*May have envelopes (Herpes, Flavivirus)
•Binal: Icosahedral head/tail – tailed bacteriophages
•Complex: (Poxvirus) – no capsomers
•Pleomorphic: (Arenavirus, G type phages): no capsid, always enveloped
- General characteristics and purification of viral nucleic acid – Methods of Nucleic acid investigation I
-Viral NA (Virion–core): carries genetic info and determines viral properties
*dsDNA, ssDNA, dsRNA, ssRNA
*Linear or circular; continuous or segmented
*Usually haploid, but may contain alien NA
-NA purification: from purified virus suspension - 2 methods:
1.Proteinase K enzyme digestion
2.Protein lysis + chromatography (faster, simpler)
-Investigation of viral nucleic acid:
*Morphology + biochemical structure studied
-Digestion w. nucleases: DNA or RNA virus exposed
-Restriction endonuclease analysis: dsDNA
*Endonucleases: Enzymes that defend bacteria
- Investigation of RNA infectivity (RNA polarity):
* If neg: needs viral enzymes for transcription
– EM observation of NA: Linear or circular.
– Electrophoresis: Agarose gel electrophoresis (sometimes polyacrylamide-gel)
– Enzyme cleaving: Smaller fragments -> easier to handle.
* ID and taxonomy.
– Physical mapping: Localization of cleavage sites.
-Molecular cloning of viral DNA: propagation of virus DNA fragments in bacterial plasmids
*Makes DNA prod – quicker, cheaper
*Safe bacteria needed
-Blotting – Southern blot: easier to handle – allows NA hybridization
- Nucleic acid investigation methods II
Heteroduplex technique
* dsDNA heating -> the double helix.
* Cooling down -> the complimentary threads rejoin.
* D-Loop: Mix 2 diff virus DNAs: heating->cooling->EM investigation. Loops form and can indicate level of homology.
* R-Loop: Virus DNA+mRNA: Heating->Cooling->EM investigation. Where mRNA anneals, a loop is formed.
Nucleic acid hybridization:
* Step 1 - Probe: Labeled oligonucleotides (DNA or mRNA).
* Step 2 – Sample: HeatingCoolingWashing
* Step 3 – Audioradiography.
DNA Microarray technique:
* Step 1 – DNA samples are bound to glass slides (DNA chip)
* Step 2 – Hybridization.
* Step 3 – Laser scanning and computer analysis.
Polymerase chain reaction (Used often):
* Step 1 – Amplification of specific DNA fragments.
* Step 2 – Template + primer + free nucleotides + Thermo resistant polymerase HeatingCooling in cycles.
* Step 3 – Agarose-gel electrophoresis.
Real-time PCR:
* Step 1 – Fluorescent labeling.
* Step 2 – Laser detection.
* Step 3 – Computer analysis, quantification.
Reverse transcriptase conversion:
* Step 1 – Investigation of RNA viruses.
* Step 2 – RNA dependent DNA polymerase (transcriptiondsDNA.
*Step 3 – DNA investigation methods.
- Viral proteins, their role + methods of investigation – viral proteins, lipids, carbohydrates
-Viral Proteins: both a component+product of viruses
*Roles: Define + target genome
*Shape virion
*Act as multiplication enzymes
-Surface receptors: allow virus to recognize host cells
Groups:
1) Structural proteins
a. Surface proteins (Capsid/Envelope): Build virus shape, antigenicity, influenced by Ag as exposed to immune system. Can have agglutinating qualities – attach to RBCs
b. Core proteins: Protect NA. May be enzymes of replication
2) Non-structural proteins: coded in the viral genome
*Only found in vegetative viruses
a. Early or immediate early proteins: regulate cells, inhibit cell defence, enzymes of transcription/translation
b. Late proteins: enzymes that organize structural proteins + viral assembly
-Methods of protein investigation:
a) PAGE: Creates a Polypeptide map of virus.
*Needs conc. viral suspension that contains only virions.
*Shows nr and size of proteins in virus
b) Immunoblotting – “Western Blot”: transfer of viral proteins from PAGE to a nitro cellular filter
c) Immunoperoxidase staining: colour reaction. Steps:
1.Detect antigens – cover with antibodies
2.Cover with conjugate – labeled with peroxide AB
3.Cover with substrate + H2O2 -> O2
d) Monoclonal AB prod: IGs prod by clones of B-lym. Mouse immunized with viral Ags, then lym. isolated in spleen
-Viral lipids: w/in enveloped viruses - form a lipid bilayer
-Viral Carbohydrates: ribose/deoxyribose surface glycoproteins
- General features of virus multiplication
- Multiplication cycle:
a) ADSORPTION: virus attaches to host - Triggered by host cell – genetically coded
- Cell receptors can also be infected. (Egg CD4 receptors – HIV, Acetylcholine receptor – rabies)
- Virus Surface Anti-receptors: adapted by virus during evolution. Some target same receptor => competition (Egg-Coxackie-adeno receptor)
b) PENETRATION–into host. Requires x4 E of attachment. Must be into living cells. Only over 4⁰C - General forms:
i) Translocation: allows virus into cell (Picornavirus)
ii) Endocytosis: esp. non-enveloped viruses (herpes, pox)
iii) Membrane fusion – only for enveloped viruses - Alternative forms:
i) Injection: “Tailed bacteriophages”
ii) Sexfimbria: Lack of a cellular wall => “bacterial sexual disease”
iii) Passive – plant viruses
c) DECAPSIDATION – releases NA from capsid. Dangerous for virus but needed for transcription. - Strategies:
i) Use of cellular proteases
ii) Viral uncoating proteins
iii) Partial decapsidation – NA hidden until early virus protein prod.
iv) Sometimes simultaneous penetration + decapsidation
d) ECLIPSE – Expression and copy of viral genetic info - Transcription (mRNA) ->Translation (viral protein prod) -> Replication (copying of NA)
e) MATURATION: assemble virions - Polypeptides => proteins - Glycolysation, dimer formation, Ag development
- Sites: RNA+Pox; in cytoplasm, DNA viruses; in nucleus
f) VIRUS RELEASE: explosive incr in nr of cells - Passive or active
- Non-enveloped virus: cytolysis
- Enveloped virus: budding
- Cell associated viruses – Released at cell death
- Cell fusion => membrane tunnels (Herpes)
- Transcription, translation and NA replication of DNA viruses
-Eclipse – three events that run simultaneously: transcription, translation, NA-replication
-Transcription: DNA copied to mRNA
-Translation: Synt. of proteins by ribosomes.
-Replication: NA multiplication
-Diff. NA require a diff. strategy -> Baltimore system
-Baltimore system: Virus classification system. Gives the multiplication strategy.
I. dsDNA, II. ssDNA, III. dsRNA, IV. +ssRNA, V. -ssRNA, VI. Viruses using reverse transcriptase
-Properties of virus multiplication:
a) Use cellular enzymes, ribosomes, aa. and E
b) For DNA – NA is similar to genetic material in cell
c) For RNA – no info stored in cell, so needs an enzyme of replication: “RNA dependent RNA polymerase” – coded by the virus
-Group I. dsDNA – e.g. Papilloma, Polyoma, Adeno, Herpes, Pox
*Early transcription: Viral DNA enters nucleus - Cellular transcriptase -> mRNA (not Pox virus)
*Early translation: On surface of ribosomes - Cellular translation -> nonstructural proteins formed
*Replication: By viral replicase – in abundant amounts
*Late transcription/translation: progeny act as a template -> many structural proteins created
-Group II. ssDNA – e.g. Parvo-, Circo-viridae
*DNA thread forms “hairpins” – cellular polymerase synthesizes complimentary threads
*Transcription: mRNA synthesis from coding thread
*Translation: Via polycistronic mRNA
*Replication: Cellular polymerase copies dsDNA and then one thread removed
*Parvo-circo-viridae: Multiply only in “S” phase when cells divide – esp. bone marrow cells
- Transcription, translation and NA replication of RNA viruses
GROUP III. DSRNA VIRUSES: (Reo-, Birnaviridae)
* Segmented genome.
* Alien nucleic acid from cell.
* Transcription: viral RdRp -> mRNA synthesis
* Translation: monocistronic
* Replication: mRNA enters inner capsid. Replicated by RdRp -> mRNA & a(-)thread
* Late transcription and translation.
GROUP IV. +SSRNA VIRUSES:
(Picorna, Calici, Toga, Flavi, Coronaviridae)
* Transcription: genomic RNA -> mRNA
* Translation: polycistronic (Picorna, Flaviviridae) or monocistronic (Calici, Togaviridae)
* Replication: intermediate forms: dsRNA, –ssRNA
* The –ssRNA is used as a tamplate for +ssRNA genome.
* Finally the + thread incorporates into the progeny virions.
GROUP V. –SSRNA VIRUSES: (Orthomyxo, Paramyxo, Borna, Filo).
* Transcription: neg sense RNA (3’->5’). Not readable for Ribosomes so the Viral RdRp = structural!. Complementary + thread synthetized => mRNA.
* Translation: monocistronic mRNA (Orthomyxo, Arena)
* Replication: dsRNA in capsid -> +ssRNA synthesis -> ssRNA synthesis.
* -ssRNA inorparates into the capsid of progeny virus.
GROUP VI. VIRUSES USING REVERSE TRANSCRIPTION: (Retro, Hepadnaviridae).
* Transcription: mRNA -> dsDNA
* The DNA integrates into the cellular genome of host cell.
* Translation: Polycistronic mRNA
* Replication = Transcription. Full length transcripts produced -> circularization of dsDNA
- Mutation of viruses, and its role in virus evolution – stabilization and storage viruses
- Mutation: changes in genetic material.
- Key source of evolution = allows adaption
- Risk of harmful effects - occurs accidentally and rarely beneficially
- Cause - Failure in NA replication
- Types of mutations: spontaneous or induced: Irradiation and mutagenic drugs
- Forms of mutations:
a) Point mutations
b) Sequence mutations (frame shift) - Results:
a) “Silent mutations”: no change in phenotype. Can occur in non-translated regions, redundant code or non-important aa.
b) “Lethal mutations”: mutation causes a stop codon (No-sense), or an AA change (Mis-sense)
c) “Conditionally lethal mutations”: esp. on Polymerases – i.e. thermo-sensitive mutations
d) “Beneficial mutations”: viral protein benefits survival – occur very rarely - Influences on viral phenotype:
a) Antigenic structure: allow virus to evade AB of host
b) Changes in host species specificity: finds new hosts
c) Changes in organ specificity
d) Different tissue tropism (w/i cell cultures) - Evolution: Selective advantage. Viral evolution = a million times quicker than the host.
- Opportunity for protection: Vaccine strains developed from virulent strains. Biotechnology – genetic engineering.
- Stabilisation of virus strains: creates virus multiplication -> virus mutations. Hence vaccines kept from mutations - “low passage viruses”
- Interactions between viruses and their environment
- Types of environment: other viruses, host cells, host organism
- Interactions bw viruses: only during multiplication – vegetative virus
- Types of interaction:
a) Advantageous: “recombination” or “complementation”
b) Disadvantageous – “Interference”
c) Neutral – “Virus exultation”
1) “Recombination”: exchange of genetic material bw viruses
a) Intramolecular: occurs during NA replication - Occurs frequently – esp. Herpes, Aujeszky’s disease
b) Genetic reassortment: need a segmented genome (Orthomyxoviridae) Genetic info not mixed.
c) Reactivation: - Cross-reactivation: vaccine strain+related virus (Herpes)
- Multiple-reactivation: bw 2 sep virus strains
2) Complementation: bw defective+“helper”viruses => exchange of polymerase enzymes
3) Phenotype mixing
a) Exchange of structural proteins
b) Transcapsidation: similar capsid (Polio+ Coxsackievirus)
4) Interference: one virus inhibits multiplication of another
a) “Absorption Interference”->competition for surface receptors
b) Autointerference: Complete & incomplete form of same virus
c) Heterologous interference: bw unrelated viruses (Herpes inhibits pox)
5) Virus exaltation: Virus can multiply independently, but change the viral influence on host cell - Incr pathogenicity (Egg Polio + Coxsackie in Monkeys)
- Cytopathic effect appears (Classic swine fever, Newcastle Disease, BVD)
- Viral Oncogenicity – (Virus: cell interactions)
- Oncogenicity: cells divide in irregular fashion
- Oncogenic viruses:
a) DNA viruses: often more oncogenic(Adeno,Herpes,Pox)
b) RNA viruses: (mainly Retroviridae)
c) Hepatitis C: damages cells => tumours (indirect method) - Types of tumours:
a) Benign – less invasive, less active
b) Malignant – invasive, destructive => metastatic tumours in other body areas - Mechanism of viral oncogenesis: Retro-virus – via their NA
- Activate cellular oncogenes (e.g. Avian/Feline Leukosis Virus)
- Enter Host’s cellular genome – promote oncogenesis
- C-Oncogene activated => cell proliferation => lymphatic tumours develop
- In stem cells of BM: leukemia/myeloma/lymphatic tumours
- Expression of viral oncogenes: typical for retrovirus – esp. Sarcoma virus (Avian/Feline)
- Recombine from Host and Viral genome – enters virus genome – V-Onc
- Travels from cell to virus: oncogen carried by virus -> rapid development of tumours
- Viral tumours – usually malignant
- Viral proteins with oncogenetic effect:
- Oncogenetic DNA viruses => cause less cell division as viral proteins control the cell
- Inactivate cellular anti-oncogentic proteins
- Inhibits apoptosis proteins: esp. Adenovirus
- Usually benign tumours (e.g. warts)
- In Vitro cell cultures: Malignant transform seen => form microtumours
15 – Cytopathic effects, plaque formation
- CPE: Structural changes in host cell due to viral invasion
- Causes cytolysis: alterations in cell -> cell death
- If not, it causes inability to reproduce
- Adenoviruses develop “Cytopathogenic effects” (CPE)
- Viral proteins:
- Inhibit translation of some cell components (herpes, pox)
- inhibit cellular synt of DNA & RNA (herpes, adeno)
- squeeze out cellular components
- Cytoskeleton depolimerisation->cell rounding (herpes, CDV)
- Fusion proteins -> Syncytia form. (herpes, paramyxo)
- Inclusion bodies: nuclear or cytoplasmic aggregates of stable substances, usually proteins
- Cell organelles squeezed out
a) Nucleur inclusion bodies: in nucleus replicating DNA virus (e.g. parvo, adeno, herpes)
b) Cytoplasmic inclusion bodies: in cytoplasm replicating RNA viruses and ASFV + pox DNA viruses
c) Cell rounding: frequent CPE (adeno, herpes, entero)
d) Syncytia form: only by enveloped v. (alpha-herpes) - Spread IC - have an advantage vs immune system
e) Other CPE: - Hemadsorption – Virus proteins on RBC’s
- Lumpy cell nucleus (ASP in lymph tissue)
- Cell vacuolization (nucleus-adeno, cytoplasm-flavi)
- Non-specific CPE:
a) Alteration of pH range of medium, or incubation temp
b) Bacterial contamination
17 - Types of viral infections
- Virus multiplication: effect on cell and host organism dictates course of disease
- Parameters:
a) Presence of infective virion in host: 90% IC, 10% EC
b) Shedding of infective virions: to incr spread of diseases
c) Clinical symptoms of the disease
d) Immune response – AB prod. - Acute infections: (Parvoenteritis, influenza): virus multiplies in a normal distribution
- Delayed immune response – either recovers or death
- Chronic infections:
a) Latent infection (Aujesky’s disease, herpes simplex) - A secondary infection after suppressing the immune response – “reactivation”
b) Tolerated infection (BVD, rubella) - Infection occurs 2nd trimester of pregnancy
- Self-recognition phase – virus Ag regarded as “self” -> no immune response -> continuous shedding
- Persisting infections (African Swine Fever)
a) Hidden neutralization Ag: IG is not effective -> Immunocomplex deposition
b) Hidden virus: - Equine arteritis: within stallion venereal tract
- Foot and mouth – within lymph tissue
- Canine distemper – within CNS
- Slow infection
a) Retroviruses (equine anaemia, Aids): integrated into genome -> Antigenic changes and immune system destroyed
b) Prions: non immunogenic – no shedding occurs in living animals
- Laboratory diagnosis of viral infections
- Cell parasites -> cell damage -> pathogen viruses -> viral diseases -> orphan viruses
- Diagnosis: must be a) fast and b) specific
- Viral diagnosis:
i) Restricted possibilities to practicing vets: (e.g. ELISA kits, urine testing) - shows many viruses but not all
ii) Need specific labs in some cases: - If regulations or rules govern disease (Foot and Mouth, African Swine Flu, BSE)
- Suspected zoonosis (human infecting) (Rabies)
- Eradication and control of free status – (Leucosis (cattle), IBR (cattle herpes), Ay)
- Certification – Free status, SPF herd, exhibitions, shipping
- Clinical signs => pathology needed for diagnosis
- Herd diagnosis
- Methods of virus diagnosis:
i) Direct virus demonstration: whole virus/its components
ii) Indirect processes (virus serology): - 2-3 days – virus develops strongly (acute phase)
- If we find AB, we know virus present
- After 14 days, this levels off (chronic phase)
- Aim of examination – sample and its collections
i) Where and where from: - Direct method - from dead animals, living animals (anticoagulated blood, secretions) - Usually in early phase
- Indirect method - coagulated blood, AB also in milk, serum etc.
- Herd diagnosis - Statistical samples taken
- Individual diagnosis - “Paired sera” test – take blood in both acute and then chronic phase. If no AB initially, but high antibodies (>x4) = disease present
ii) Unambiguous mark – roman numerals
iii) Period of shipping. As fast as possible but within 24 hrs. + 4C – do not freeze
iv) Letter of basic information, date of start disease,o bservations of clinical signs, vaccination, treatmenti, instructions
- Direct demonstrations of viruses 1 – virus isolation, identification
-Virus isolation:
* Period of diagnosis: 2-3 weeks.
* In vitro propagation of infective virions is required.
* Sample from living or dead animal
* Helps diagnose new viruses or mutations
METHODS:
-Cleaning process: (diseased organ - dead animals)
a) Diluted 1:10 in physiological solution
b) Homogenisation w. sand–to release ECF
c) Rinsing 2 hours
d) Centrifugation
e) Filtration
-Inoculation of cell culture or embrionated egg:
a) “Adsorption technique”: eliminate CPE of toxins
b) Suspension cell culture technique (parvo)
-Cell viral assay (herpes viruses):
a) Inoculate b) Centrifuge c) Incubation d) Map staining
-Cell culture:
a) Daily examination: check for cell cultures
b) Accessory examinations: EM, Staining
c) Blind passage: specimen in a fresh cell – to see CPE
-Embryonated egg:
a) Sample on slide+fresh cells -> agglutination or not
b) Accessory examinations: Histopathology, EM, staining
-Virus identification test:
a) Virus neutralization: serotype of species
b) Physio-chemical tests
c) Electron microscopes
-Diagnostic experimental animal infections: (African horse sickness, blue tongue, rabies, FMD, Classical + African swine fever)
*In vivo method of viral propagation
*Period of diagnosis: 8-10 days (faster, more reliable than virus isolation)
- Direct demonstrations of Viruses II (EM, virus antigen, virus nucleic-acid demonstration)
-ELECTRON MICROSCOPE INCESTIGATION:
* Morphological recognizable virions
*Types: i) Ultra-thin sections, ii) Negative contrast methods, iii) Immune electron microscope method
-VIRUS ANTIGEN detection: w. specific ABs => virus ID
Reliable. Types:
a) Immune Fluorescence (IF) Test:
*Direct (rabies, classical swine fever) or indirect (sensitive)
b) Complement fixation (CF) test: Ag released off the cells (e.g. hemolysis of RBCs)
c) Agar Immune-diffusion test (Adeno-virus, Blue Tongue)
d) Counter-current immune electrophoresis (Parvo-, Adeno)
f) Enzyme Linked Immunosorbant Assay (ELISA):
uses AB and color change to id. a substance
g) RIA – Radio Immune Assay
-VIRUS NUCLEIC ACID Detection:
* Nucleic acid hybridization method:
o From histological section virus DNA
o From nasal swabs
* Polymerase chain reaction (CPR):
o NA can be detected from autolysed sample
o Small amounts of NA can be amplified.
o Second strand of DNA can arise.
* DNS Microarray test: Oligo DNA fixation on glass slide. Data saved to computer–pathogens detected by fluorescence
-Newer methods:
* Random PCR and PEER.
* Proximity ligation
- Titration of viruses
-Aim of titration: Standardization of the virus suspension, so infective viruses of known conc. are added to bio. test
-Biological test:
* Diagnosis: virus-neutralization test.
* Vaccine prod
* Experimental animal infection.
* Need infective titer’s determination
- Infective titer:
1) Endpoint dilution:
o Serial tenfold dilution -> Inoculation -> Incubation
o Method of calculation: Reed-Muench, Spearman-Karber
o CPE in 50% of the inoculated cell cultures=Infective titer
2) Plaque counting:
* Plaque: locally appearing CPE (spots on cell cultures)
* Evaluation: conc of the virus suspension is given in plaque forming units (PFU)
3. Hemagglutination titer’s determination:
o On micro hemagglutination plates
o Serial tenfold dilution
- Detection and titration of antiviral antibodies - Indirect virus demonstration
- Virus serology – serum neutralization and hemagglutination inhibition
- Serological methods – Indirect methods:
- Advantages: High chance of demonstration, cheap
- Disadvantages: can´t differentiate maternal/vaccine/seroconversion AB
- Aim:
a) Survey: viruses in stock -> random sampling
b) Individual diagnosis: Paired sera investigation - Types of serological tests: (for AB detection)
- AGIR-Agar Gel Immune Detection (blue tongue)
- CF-Complement Fixation (FMD)
- VN-Virus Neutralization (all cytopathogen viruses)
- ELISA (Discriminating) (Aujeszky)
- Virus Neutralisation (VN) – main AB test – Heat inactivates serum to eliminate non-specific ABs
- Blocking AB around virus -> virus can´t attach to cell receptors -> no CPE
- 2 methods:
1. Constant virus varying serum dilution method – for AB detection - Serial twofold dilution – Incubated, inoculated, incubated
- Results: Any CPE proves no blocking AB are present
a) Serum Neutralising Titer
b) Plaque reduction test
2) Constant serum varying virus dilution method: - Neutralisation index calculation -> for virus ID
- Hemagglutinating Inhibition (HAI) - AB test (Only for hemagglutinating viruses)
- Non-specific hemagglutinating AB exhausted from serum
- Serial twofold dilution - 4-8 hemagglutinating units added
- Incubated then washed w. RBCs
- Hemagglutination shows virus not inhib by AB -> no AB
- Antiviral medicines, interferons, interferon inducers and immune sera in therapy
-ANTIVIRAL MEDICINES:
*Effects: a) Inhib viral replication b) Decr virus shedding
c) Localize infections
*Problems: a) drugs toxic for cells b) Need specific agents
c) High viral mutation ratio -> resistance
1. Nucleoside analogues:
a) 1st generation NA analogues: w/in NA – varied toxicity
*Generally unsafe
b) 2nd generation NA analogues: safer
*Activated by virus specific enzymes
2. Non-nucleoside RT inhibitors: Inactivates reverse transcriptase
3. Protease inhibitors – VS retroviral protease enzyme
4. Other: Methisazone – viral polymerase inhibitor Vs Pox
Tamiflu - neuraminidase inhibitors Vs influenza
-Problems with antiviral drugs: Use in vet. practice is very rare, expensive. Narrow spectrum resistance develops
-INTERFERONS: group of signaling proteins made and released by host cells when presence of pathogens
*Benefits: a) High efficacy b) Not Ag specific
*Problems: a) Short-term effects b) Parenteral administration – difficult to apply c) Expensive
-IMMUNE SERA and purified IGs: serum containing ABs specific for one/more Ags
*Benefits: Passive immunization
*Problems: a) Offer short-term protection
b) Can cause anaphylaxis or serum-sickness
c) Large animals need huge doses
d) High cost – “hyperimmunisation”
- Immunization against viral diseases, types of viral vaccines
-Active immunization: Preventative (Ø after infection)
*Most widespread, effective defence to limit economic loss
*General rule:
1) Inhibitory effect of maternal AB
2) Negative phase – if AB already in situ -> lower immunity short-term
3) Vaccination schedules
4) Avoid immunosuppression – not to lower already existing immunity
VACCINES
*Type: Monovalent–for one virus/Polyvalent–several
-Live virus vaccines:
1) Virulent (wild type) virus vaccines: Rare(sheep pox in HL)
2) Attenuated vaccines:
i) Naturally occurring mutants (Newcastle disease strains)
ii) Artificially attenuated viruses:
a) by cultivation, in vitro
b) selected thermo sensitive mutants (rabies)
c) genetic modification-marker vaccines (Aujeszky disease)
iii) Heterotypic virus (Marek’s disease – Turkey herpes)
iv) Virus vector vaccines: Introduce Ag into apathogen viruses (vaccination of foxes vs rabies)
-Inactivated (killed) vaccines: virus not able to multiply
*Inactivating agents: e.g. formaldehyde (carcinogenic)
*Adjuvants: a) Slow the Ag release b) Immunostimulant property c) Saponine, Oil
*Boostering: Often required for inactivated viruses
-Subunit vaccines: Contains only proteins needed for neutralization
*Production: Purified virus after normal prod
*Advantages: Safe – No NA, Marker vaccines – Elisa – no AB, Good efficacy
*Disadvantage: Expensive, Parenteral administration, in vivo – less effective
-Anti-idiotype vaccines: Epitope and paratope groups – “lock and key” theory
- Polyomaviridae, Papillomaviridae, Adenoviridae
2) POLYOMAVIRIDAE (Poly=many, oma=tumor):
* Genus: Polyomavirus.
* Morphology: Circular dsDNA, superhelical.
* Resistance: STRONG.
* Biological properties: Baltimore I. Oncogenic – SV40 model virus. Airborne. Microtumors in cell culture.
* Diagnosis: Bovine or goose polyomavirus->nephritis
2) PAPILLOMAVIRIDAE (Papilla=wart, oma=tumor):
* Genus: papillomavirus (16)
* Morphology: Circular dsDNA, superhelical.
* Resistance: STRONG.
* Biological properties: Baltimore I. Oncogenic - Benignant tumors.
* Antigenic properties: Good Ag -> auto vaccines. No cross-reaction.
* Diagnosis: Equine/Bovine papillamatosis, Horse Sarcoid, Canine Oral Papillamatosis
3) ADENOVIRIDAE (adenos – adenoid gland):
* Genera: Mastadeno-, Aviadeno-, Atadeno, and Siadenovirus.
* Morphology: Linear dsDNA, no envelope.
* Biological properties: Baltimore I. Oncogenic – apoptosis inhibition.
* Antigenic properties: Good Ags. Cross-reaction.
* Diagnosis: Isolation:
I) Mastadenovirus: Pneumoenteritis in calves and lamb, Canine Laryngotracheitis (kennel cough)
II) Aviadenovirus: Inclusion body hepatitis, gizzard eruption
III) Atadenovirus: Pneumoenteritis in calves and lamb, Egg Drop Syndrome (EDS)
IV) Siadenovirus: Frog adenovidus.
