Final oral

Question 1

1. Place and significance of virology in biosphere; evolution of viruses

Answer 1

-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

Question 2

2. Propagation of viruses I– noculation of embryonated eggs, experimental infection of lab animal.

Answer 2

-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

Question 3

3. Propagation & purification of Virus II – production of cell cultures

Answer 3

-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

Question 4

4. Concentration and purification of viruses

Answer 4

•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

Question 5

5. Morphology of viruses

Answer 5

•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

Question 6

6. General characteristics and purification of viral nucleic acid – Methods of Nucleic acid investigation I

Answer 6

-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

Question 7

7. Nucleic acid investigation methods II

Answer 7

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.

Question 8

8. Viral proteins, their role + methods of investigation – viral proteins, lipids, carbohydrates

Answer 8

-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

Question 9

9. General features of virus multiplication

Answer 9

• 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)

Question 10

10. Transcription, translation and NA replication of DNA viruses

Answer 10

-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

Question 11

11. Transcription, translation and NA replication of RNA viruses

Answer 11

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

Question 12

12. Mutation of viruses, and its role in virus evolution – stabilization and storage viruses

Answer 12

• 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”

Question 13

13. Interactions between viruses and their environment

Answer 13

• 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)

Question 14

14. Viral Oncogenicity – (Virus: cell interactions)

Answer 14

• 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

Question 15

15 – Cytopathic effects, plaque formation

Answer 15

• 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

Question 16

17 - Types of viral infections

Answer 16

• 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

Question 17

18. Laboratory diagnosis of viral infections

Answer 17

• 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

Question 18

19. Direct demonstrations of viruses 1 – virus isolation, identification

Answer 18

-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)

Question 19

20. Direct demonstrations of Viruses II (EM, virus antigen, virus nucleic-acid demonstration)

Answer 19

-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

Question 20

21. Titration of viruses

Answer 20

-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

Question 21

22. Detection and titration of antiviral antibodies - Indirect virus demonstration

Answer 21

• 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

Question 22

23. Antiviral medicines, interferons, interferon inducers and immune sera in therapy

Answer 22

-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”

Question 23

24. Immunization against viral diseases, types of viral vaccines

Answer 23

-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

Question 24

25. Polyomaviridae, Papillomaviridae, Adenoviridae

Answer 24

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.

Question 25

26. Herpesviridae

Answer 25

• Genera:
  o Alphaherpesvirinae: Simplex-, Mardi-.
  o Betaherpesvirinae: Cytomegalo-, muromegalo-.
  o Gammaherpesvirinae: Lymphocrypto-, rhadino-.
  They all differ in replication time and latency site.
• Morphology: Linear dsDNA.
• Resistance: weak.
• Biological properties: Strong CPE. Oncogenic (Mainly gammaherpesvirinae)
• Antigenic properties:
  o Weak immunogenes.
  o Cross reaction within genera.
• Diagnosis:
  Aplha:
  o Simplex virus: Bovine Herpes Mamillaris, Simian Herpes, Human Herpes Simplex (HHV1-2)
  o Varicello virus: Infectious Bovine Rhinotracheitis, Equine Abortion, Aujeszkys disease (pseudorabies) in swine
  o Mardivirus genus: Marek´s disease (CNS, paralysis, lymph tumours), Turkey Herpes Virus
  o Litovirus genus: Infectious Laryngotracheitis
  Beta: Cytomegalovirus (Human+monkey)
  Gamma:
  o Lymphocryptovirus: Epstein-Barr virus
  o Meca-, Peca-, Rhadino virus: Malignant catharral fever of cattle, Bovine Herpes Virus 4, Equine Herpes Virus 2+5
  o Unassigned herpes virus: pigeon herpes, Pacheco´s disease
  o Fish Herpes Virus

Question 26

27. Pox Viridae

Answer 26

• Genera:
  o Chordopoxvirinae: Ortho-, Para-, Avi-, Capri-, Lepori-, Suipox virus
  o Entomopoxvirinae: Insect poxvirus
• Morphology: Linear dsDNA, complex capsid.
• Resistance: usually good.
• Biological properties: Baltimore I (multiplication in cytoplasm), CAM inoculation. Mainly stenoxen.
• Antigenic properties: Strong Ag. Cross neutralization w/in genera
• Diagnosis:
  Chordopoxvirinae:
  o Orthopox virus: Cowpox (teats, skin)
  o Parapox virus: Psuedo Cowpox, Bovine Papular Stomachitis
  o Avipox virus (birds)
  o Capripox virus (goat)
  o Leporipox virus (rabbit)
  o Suipox virus (swine)

Question 27

28. Asfarviridae and Hepadnaviridae

Answer 27

ASFARVIRIDAE:
* Genus: Asfivirus
* Morphology: Linear dsDNA, enveloped
* Resistance: Moderate
* Biological properties: Baltimore I. Hemadsorption. Stenoxen.
* Antigenic properties: No neutralization AB! No vaccine available.
* Diagnosis: African swine fever (most deadly swine disease)
HEPADNAVIRIDAE:
* Genera: Ortho-, Avihepadna virus
* Morphology: Circular ss/dsDNA, enveloped capsid.
* Resistance: Weak (low pH), detergents
* Biological properties: Baltimore VI or VII, stenoxen virus, chronic. Transmission; blood, sexual, close contact
* Antigenic properties: HBsAg - protective
* Diagnosis:
o Orthohepadnavirus genus: Human hepatitis B
o Avihepadnavirus genus: Duck hepatitis B

Question 28

29. Parvoviridae and Circoviridae

Answer 28

PARVOVIRIDAE:
* Genera:
o Parvoviridae: Parvo-, Amdo-, Boca-
o Densoviridae: Denso-, brevidenso
* Morphology: Linear ssDNA, self-priming, non-enveloped capsid.
* Resistance: VERY STRONG!
* Biological properties: Baltimore II – multiplication in dividing cells, Hemagglutination, Stenoxen.
* Antigenic properties: Very good Ag – long immunity
* Diagnosis: Isolation in dividing cells.
Parvo:
o Parvovirus: Porcine, Canine (enteritis, myocarditis)
o Amdovirus: Aleutian mink disease
o Bocavirus: Bovine+Canine
o Erthyrovirus
o Dependovirus: Derzsy´s disease (goose)
CIRCOVIRIDAE:
* Genera: Circo-, Gyrovirus genus.
* Morphology: Circular ssDNA, non-enveloped capsid.
* Resistance: VERY STRONG!
* Biological properties: Baltimore II, Stenoxen virus.
* Antigenic properties: Good Ag, immunosuppression
* Diagnosis:
o Circovirus genus: PCV1 And PCV2, beak and feathers disorder.
o Gyrovirus genus: Chicken anemia

Question 29

30. Reoviridae, Birnaviridae

Answer 29

REOVIRIDAE:
* Genera: Ortho-, orbi-, Colti-, Rota-virus
* Morphology: Segmented dsDNA, non enveloped capsid
* Resistance: Ortho-, Rota- : STRONG, Orbi-, colti-: weaker
* Biological properties: Baltimore III – viral transcriptase,
Orbi-, Rota-: hard to propagate.
* Antigenic properties: good Ag – cross reaction.
* Diagnosis:
o Orthoreovirus genus: Mammalian reovirus (pneumoenteritis), Euryxen, Avian reovirus
o Orbivirus: African Horse Sickness (edema, hemorrhage, pneumonia), Blue Tongue, Ibaraki disease (cattle, abortion), Equine encephalosis
BIRNAVIRIDAE:
* Genera: Avi-, Aquabirna
* Morphology: segmented dsRNA, non-enveloped
* Resistance: STRONG
* Bio. prop.: Baltimore III, propagation in specific tissues
* Antigenic properties: Good Ag, no croos reaction
* Diagnosis:
o Avibirnavirus: Gumboro disease, Infectious bursa Fabricii
o Aquabirnavirus: Infected pancreas-Salmonidae

Question 30

31. Picornaviridae, Caliciviridae, Astroviridae

Answer 30

PICORNAVIRIDAE:
* Genera: Entero-, Hepato, Cardio-, Erbo-, Tescho-, Rhino-
* Morphology: +ssRNA genome, non eveloped
* Resistance: Generally good
* Bio. prop: Baltimore IV, generally stenoxen
* Antigenic properties: Good Ag, no cross reaction
* Diagnosis:
o Enterovirus genus: Swine vesicular disease (SVD)
o Hepatovirus genus: Hepatitis A human
o Cardio: Encephalomyocarditis
CALICIVIRIDAE:
* Genera: Vesi-, Noro-, Sapo-, Lago…
* Morphology: +ssRNA, non enveloped capsid.
* Resistance: good.
* Bio. prop.: Baltimore IV, Stenoxen, CPE: Lysis.
* Antigenic properties: Good antigens.
* Diagnosis:
o Vesivirus genus: Feline calicivirus - respiratory disease
o Norovirus genus: Food-borne gastroenteritis
ASTROVIRIDAE:
* Genera: Astro-, Mamastro-…
* Morphology: Linear, +ssRNA, non enveloped capsid.
* Resistance: good - pH resistant
* Biological properties: Stenoxen – host specific.
* Antigenic properties: Good antigens.
* Diagnosis:
o Astrovirus: Chicken nephritis
o Mamastrovirus: Gastroenteritis

Question 31

32. Togaviridae, Flaviviridae

Answer 31

TOGAVIRIDAE:
* Genera: Alpha-, Rubvirus
* Morphology: Linear +ssRNA, enveloped
* Resistance: weak.
* Biological properties: Baltimore IV. NO CPE!
* Antigenic properties: Good antigens, cross reaction.
* Diagnosis:
o Alphavirus: Equine encephalitis, Semliki forest virus
o Rubvirus genus: Rubella (human red rash, røde hunder)
FLAVIVIRIDAE:
* Genera: Flavi-, Pesti-, Hepaci-virus
* Morphology: Linear +ssRNA, enveloped
* Resistance: weak.
* Biological properties: Baltimore IV, Flavivirus; arbovirus
* Antigenic properties: Good antigens, cross reaction.
* Diagnosis:
o Flavivirus: Yellow fever in Humans
o Pestivirus: Classical swine fever, BVD
o Hepacivirus: Hepatitis C in humans

Question 32

33. Nidovirales – (Coronaviridae, Arteriviridae)

Answer 32

CORONAVIRIDAE (Corona – solar crown)
* Genus: Alpha-, Beta-, Gammacorona-, Toro-virus
* Morphology: +ssRNA genome, long, enveloped
* Resistance: Good – pH resistance 3-9
* Biological properties: Unique gene expression, Baltimore IV (subgenomic mRNA synt), specific CPE
* Antigenic property: Good antigens, cross reactions
* Diagnosis:
o Alphacorona-virus: Porcine transmissible gastroenteritis virus (TGE), Porcine epidemic diarrhea virus (ED), Feline infectious peritonitis virus (FIP)
o Betacoronavirus: Bovine coronavirus, Severe acute respiratory syndrome-SARS
o Gammacoronavirus: Avian infectious bronchitis – (IB)
o Torovirus: Berne virus-Foal gastroenteritis, Breda virus-Calf gastroenteritis
ARTERIVIRIDAE
* Genus: Arterivirus
* Morphology: +ssRNA genome, enveloped
* Resistance: Weak
* Biological properties: Baltimore IV, Use sub-genomic mRNA, No typical CPE, sexual or aerogenus
* Antigenic properties: Good antigens, No cross reactions
* Diagnosis:
o Arterivirus genus: Equine arteritis virus – (EAV), Porcine respiratory & reproductive syndrome (PRRS)

Question 33

34. Orthomyxoviridae

Answer 33

ORTHOMYXOVIRIDAE (Myxa- mucous)
* Genus: Influenza A, B, C; Thogotovirus, Isavirus
* Morphology: -ssRNA genome, 8 segments, helical capsid, enveloped
* Resistance: Weak
* Bio. prop.: Baltimore V (viral transcriptase enzymes), Matrix M2 protein, 2 key proteins in envelope
* Virulence: Hemagluttinin cut by a protease, In cell mucous -> infects respiratory organs (Flu!)
* Propagation: Embryonated egg, Influenza A – Euryxen
* Antigenic properties: Core + matrix proteins are conservative – A,B, C Genus. Depends on HA & NA combination
* Diagnosis:
o Influenza A virus: Porcine Influenza, Equine Influenza, Avian Influenza, Human Influenza
o Influenza B and C: Human influenza
o Thogotovirus: Tick bourne
o Isavirus: Infectious Salmon Anaemia virus (Norway)

Question 34

35. Paramyxoviridae

Answer 34

PARAMYXOVIRIDAE (para – like, myxa – mucous)
* Similar to Orthomyxoviridae
* Subfamily: Paramyxovirinae – Respiro-, Morbilli-, Avula -
Pneumovirinae – Peneumo-, Metapneumo -
* Morphology: -ssRNA, helicalcapsid, enveloped
* Resistance: Weak
* Biological properties: Baltimore V, HN and NA, F protein, CPE – Inclusion bodies, Syncytia, Hemadsorption
- Respiro-, Rubula- - Euryxen – infect several hosts
- Morbillivirus, Pneumovirinae – infect one host
* Antigenic properties: Good antigens, some cross reactions
* Diagnosis:
o Respirovirus: Parainfluenza virus 1 + 3
o Morbillivirus – (Morbilli – measles): Canine Distemper virus, Rinderpest, Measles
o Rubula-virus: Parainfluenza Virus 2 (kennel cough), Parainfluenza Virus 4, Mumps
o Avula-virus: Avian-Newcastle disease, Avian paramyxovirus 2-9

Question 35

36. Rhabdoviridae, Filoviridae

Answer 35

RHABOVIRIDAE (Rhabdos – rod, stick – plant virus)
*Genus: Vesiculo-, Lyssa-, Ephermero -, Novirhabdo-virus
* Morphology: -ssRNA genome, helical capsid, enveloped
* Complete virions: bullet shaped (B) or Truncated (T)
* Resistance: Weak
* Biological properties: Baltimore V, cell bound infections, CPE – weak, Fish vesiculovirus, Zoonoses
* Antigenic properties: Good, cross reactions
* Diagnosis:
o Vesiculovirus: Vesicular stomatitis virus
o Lyssavirus (Lyssa–Rabies) Rabies, Urban rabies, Sylvatic rabies
o Ephemerovirus: Bovine ephemeral fever
o Novirhabdovirus: Viral haemorrhagic septicemia
FILOVIRIDAE – (Filum – thread)
* Genus: Ebola-, Marburg-virus
* Morphology: -ssRNA genome, helical capsid, envelope
* Resistance: Weak
* Biological properties: Baltimore V, CPE – inclusion bodies, Zoonoses
* Antigenic properties: Good antigens – but rapid course of lethal disease, no cross reactions
* Diagnosis:
o Ebola virus (Ebola – African river)
o Marburg virus – (German city)
o Reston virus

Question 36

37. Bunyaviridae, Arenviridae, Bornaviridae

Answer 36

BUNYAVIRIDAE (Bunyamwera – African region)
* Genus: Orthobunya-, Phlebo-, Nairo-, Hantavirus
* Morphology: Segmented, -ssRNA genome, enveloped, helical capsid – RNA polymerase enzyme – synthesizes mRNA
* Resistance: Weak
* Biological properties: Baltimore V, Zoonoses, mosquito or tick bourne
* Antigenic properties:Good antigens, cross reaction
* Diagnostic:
o Orthobunyavirus: Akabane disease, La Crosse virus
o Phlebovirus – Sandflies: Rift valley fever
o Nairovirus – Nairobi sheep fever, Crimean-congo haemorrhagic fever
o Hantavirus – (Korea): Haatan Haemorrhagic Fever
ARENOVIRIDAE – (arena – sand)
* Genus: Arenaviridae
* Morphology: Segmented, ssRNA genome, no capsid, enveloped, have ribosomes inside, 2 ends of NA hybridize together, 2 key proteins on surface
* Resistance: Weak
* Biological properties: Baltimore V, Zoonoses,
* Antigenic properties: Cross reactions
* Diagnosis:
o Arenavirus - Old world arena virus, Lymphocytic choriomeninigitis (LCM), Lassa fever – Africa Severe haemorrhages
BORNAVIRIDAE – (Borna- Horse disease - Germany)
* Genus: Bornavirus
* Morphology: -ss RNA genone, helical capsid, enveloped
* Resistance: Weak
* Biological properties: Baltimore V
* Antigenic properties: Good antigens – was a vaccine
* Diagnosis: histopathology lesions
o Bornavirus – Borna diseases (BDV), Avian Borna virus

Question 37

38. Retroviridae

Answer 37

RETROVIRIDAE – (Retro-reverse transcription)
* Genus: Orthoretroviridae - Alpha, Beta, Gamma, Delta, Epsilon-retrovirus, Lentivirus, Spumaretrovirus–Spumavirus
* Morphology: Diploid +ssRNA genome, enveloped, Helical + Icosahedral capsid
* Resistance: Very sensitive!
* Bio. prop: Baltimore VI (forms a pro-virus, if cell divides, viral genome is also transmitted=life-long infection), tumours (Oncogenic)
* Multiplication competent viruses: Strong CPE + tumours
* Transmission: sensitive->special methods of infection
* Antigenes: Good Ag in theory, destroys immune system!
* Clinical signs – tumours (Leukemia)
Ortheroretroviridae family:
o Alpha-retrovirus: Avian Leucosis, Avian sarcoma
o Beta-retrovirus: Ovine pulmonary adenocarcinoma virus
o Gamma-retrovirus: Feline Leukemia
o Delta-retrovirus: Bovine leukaemia virus, Human T-lymphotrophic virus 1-2 (HTLV)
o Epsilon-retrovirus: Fish virus – Walleye
o Lentivirus – (Lente – slow): rarely cause tumours, Immuno suppressive, Equine infectious anaemia virus (EIA), Human Immunedeficiency Virus – HIV 1-2, Feline immune deficiency virus (FIV)

Question 38

39. Bacteriaphages

Answer 38

BACTERIAPHAGES – (Phagos – eating) – eat Bacteria (no harm to humans, animals)
* Genus: Numerous!
* Morphology: Very diverse – grouped A-G, NA: dsDNA, ssDNA, dsRNA, ssDNA
* Biological properties: Bacteria has no immunesystem, so antigenic features less important
* Penetration: Needs special routes to penetrate
* Species specific: Phage typing – used to id a bacterial species
* Lysogen infection: After virus infection, instead of gene expression, the genomes merge to form pro-phages – “lytic infection”
* Lysogen conversion: Changed bacterial properties -> Causes Toxin production – β-phage
* Transduction: Viruses aid gene transfer bw bacteria
*Important phages:
o Tbilissi – Tb phage – Brucella
o T4 phage – E.Coli – very complicated
o β-phage – Diptheria
o λ phage (Lambda) – E.Coli – gene sequence known – used as a molecular marker

Question 39

40. Viroids, Prions

Answer 39

• Sub-viral agents: Viruses = NA + proteins in all cases
  VIROIDS – contain only NA (no proteins!)
• Morphology: Circular ssRNA – superhelical
• Resistance: Extremely strong
• Biological properties: Plant pathogens, host spectrum
• Antigenic properties: Not antigenic
  PRIONS - “Protein of infectious nature” - Agents of spongiform encephalopathies
• Morphology: Pure protein -> does not contain NA!
• Resistance: Very high, lipid solvents – lower their heat resistance!
• Biological properties: Cellular Prion Protein, normal part of neurons, Infectious Prion Protein
• Pathogenesis: Gut -> Spleen. Finally enter neurons very slowly and CNS after several months. In grey matter; induce proteins to produce. Slow process of cell death
• Leads to: Spongiform encephalopathy
• Antigenic property: None. Seen as “self” tissue, therefore no AB
  Diagnosis: Clinical signs – Histiopathology
  o Transmissible spongiform encephalopathies – (TSE’s): Scrapie (sheep), Bovine Spongiform Encephalopathy, Feline spongiform encephalopathy (FSE), Transmissible Mink Encephalopathy (TME), Chronic Wasting Disease
  o Humans: Progressive dementia, Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeld Jakob Disease – (vCJD), GSS – Gerstmann Straussier Scheiker Syndrome, Fatal Familial Insomnia

Question 40

16 – General features of viral infection – (Virus: host interaction)

Answer 40

• Survival of viruses: rely on successful infection
• The extent to which they propagate w/i the host – localized or generalized
• Infection routes:
  a) Transcutaneous: keratinized cells-no virus multiplication
• Injuries – papilloma, rabies (bite, lick)
• Arthropod/Vector – arbovirus – mosquito, tick
• Special – conjunctiva (herpes)
  b) Airbourne: via respiratory tract – droplet/aerosol particles or UV light
• Defence systems: mucosal immunity (IgA, lymph cells), temp - 33⁰C = polymerase activity slows
• Respiratory diseases – (rhinovirus)
  c) Peroral: via the enteric system
• Oral cavity – Lysozyme
• Stomach – acidic pH, proteolytic enzymes
• Small intestine – enzymes + bile
• Resistant viruses – mainly non-enveloped – adeno, reo, picorno, astro…
  d) Venereal: via urogenital tract - sexual intercourse
• Sensitive viruses also: often cell-associated transmission
• Herpes, papilloma, arteri, retro….
  e) Transplacental:
• Placenta structures: different barriers
• Viruses control age/development of embryo
• E.g. Herpes, parvo, flavi
  f) Germinative: via the egg – birds
• On surface of the egg – at hatching (e.g. herpes)
• Inside egg (circo, adeno, retro)
• Yolk = immunity!
  g) Latrogenic: by vets
• Non-sterile equipment (papilloma, flavi)
• Needle (flavi, retro)
  h) Mixed forms: oronasal – influenza
• Factors influencing virus:
• Susceptibility – abortive infection - non pathogenic
• Host spectrum – Stenoxen, Euryxen
• Infectibility – viral receptors, NA
• Amount of virus - ~ chances of successful infection/capacity of immune system
• Pathogenicity
• Virulence – degree of expression