Viruses and what they do -

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Viruses and what they do -
An overview
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Viruses (Encyclopedia Britannica)
..infectious agents of small size and simple
composition that can multiply only in living
cells of animals, plants and bacteria. Viruses
are obligate parasites that are metabolically
inert when they are outside their hosts. They all
rely, to varying extents, on the metabolic
processes of their hosts to reproduce themselves.
The viral diseases we see are due to the effects
of this interaction between the virus and its
host cell (and/or the hosts response to this
interaction).
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Viral Genomes
Single Stranded
DNA
Double Stranded
Double Stranded
Nucleic Acid
Positive
Single Stranded
RNA
Negative
RNA
DNA
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SS RNA genomes

• ve (sense) and -ve (anti-sense) RNA genomes

AUG GCA CGA
met ala arg
UAC CGU GCU
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Virion
nucleocapsid
envelope
capsomeres
enveloped virus
a Virion
Capsid
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Proteins produced by viruses

• Structural proteins
• Non-structural proteins

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Some viral shapes
papillomavirus
adenovirus
100 nm
parvovirus
1 nm 1 millionth of a mm 100 nm 1 ten
thousandth of a mm
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Some viral shapes
1 nm 1 millionth of a mm 100 nm 1 ten
thousandth of a mm
100 nm
influenzavirus
parainfluenza virus
poxvirus
herpesvirus
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Taxonomy

• What is it?
• On what is it based?
• Is it important?
• Do I need to remember all the details?

International Committee on Taxonomy of Viruses
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Viruses with ss DNA genomes
porcine circovirus
Circoviridae
canine parvovirus-2
Parvoviridae
feline panleukopenia virus
porcine parvovirus (SMEDI)
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Viruses with ds DNA genomes
papillomaviruses
Papovaviridae
adenoviruses

Adenoviridae
equine herpesviruses -1,4
bovine herpesvirus-1,2

Herpesviridae
porcine cytomegalovirus
malignant catarrhal fever virus
Poxviridae

poxviruses
African swine fever virus
african swine fever virus
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Viruses with ds RNA genomes
rotaviruses
Reoviridae
bluetongue virus
african horse sickness
Birnaviridae
infectious bursal disease (chickens)
infectious pancreatic necrosis (salmonid fish)
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Viruses with ve RNA genomes
foot and mouth disease virus
Picornaviridae

porcine enteroviruses
Caliciviridae
feline calicivirus
Coronaviridae
coronaviruses
Arteriviridae
equine arterivirus
Flaviviridae
flaviviruses (WNV)
pestiviruses (BVD)
Togaviridae
equine encephalitis viruses
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Viruses with -ve RNA genomes
influenzaviruses
Orthomyxoviridae
parainfluenza virus
canine distemper virus
Paramyxoviridae
Hendra, Nipah viruses
respiratory syncytial virus
Rhabdoviridae
rabies virus
vesicular stomatitis virus
Filoviridae
Ebola virus
Bunyaviridae
Haantan virus
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Viruses with reverse transcriptase
feline leukemia virus
Retroviridae

feline, bovine immunodeficiency
viruses
bovine, avian leukosis viruses
caprine arthritis-encephalitis virus
Hepadnaviridae
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Groups, types (sero-types), isolates and strains
Type -A specific antigen
Type -A
Type - B
Group specific antigen
isolate
Type - C
Group
Type - C specific antigen
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Group and type specific antigens
type specific antigen
naked virus (eg FMDV)
enveloped virus (eg influenza, FeLV)
group specific antigen
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Serotypes and neutralizing antibody (eg. FMDV)
A
O
C
SAT1
SAT2
SAT3
Asia
serotypes of FMD virus
receptor
receptor binding protein on viral surface
antibodies against receptor binding protein of
serotype A will neutralize viruses of serotype A
but not of serotype C
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example - influenza
type specific antigen
serotype H1
serotype H5
serotype H7
group specific antigen
test based on group specific antigen will detect
all three
vaccination against one serotype will not protect
against others
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Infection of a cell
Stage Biological implications Host defenses Drug intervention
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Distribution of the CCR5?32 mutation in human
populations
from PLoS Biology, Nov 2005
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Errors in replication lead to quasispecies
persistent infection
mixture of variant viruses (quasispecies)
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inclusion bodies
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Release of virus
or by budding (without death of cell,
non-cytopathic)
Release by lysis of cell (cytopathic)
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Infection of the animal

• Entry - the beginning of infection
• Local replication vs systemic spread
• Consequences of infection
• none to illness (signs, symptoms)
• Signs and symptoms
• Why some animals get sick while others do not
• Patterns of disease

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Why do some infected animals get sick and others
dont?

• Viral factors - virulence
• Host Factors

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Viral FactorsWhy are some isolates of a virus
more likely to cause severe disease than
others?determinants of viral virulence
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Host Factors

• Genetic Resistance
• loss of receptors
• CCR5-D32 mutation and resistance to HIV
• variation in immune response genes
• genetic defects in defenses

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Host factors

• age related susceptibility
• greater susceptibility of new born animals
• greater susceptibility of adults
• prior exposure, acquired resistance
• maternal protection
• concurrent infections, immuno-suppression,
  increase in susceptible cells

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Patterns of disease
clinical signs
virus shedding
acute
virus difficult to detect
recurrent
chronic or persistent
slow
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Incubation period
infection
incubation period - time between infection and
the appearance of clinical signs
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Resistance and Recovery
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Resistance and recovery

• innate resistance
• genetic
• serum, mucous factors (complement, defensins)
• induced resistance
• Toll-like receptors (dendritic cells)
• Pathogen Associated Molecular Patterns
• interferons and inflammatory cytokines
• acquired immunity
• humoral
• cell mediated

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After the Toll Rush, LAJ ONeill, Science
3031481 2004
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Interferons
I
I
I
I
I
activated
E
factor

antiviral
effects
infected

Immune Modulation
cell
Induced interferon
genes
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Acquired anti-viral immunity (antibody)
Virus neutralized
B
Virus infected cell
Antibody targets Fc receptor bearing cell To kill
virus infected cell
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Acquired immunity (CMI)
perforins
necrosis
CD8
Apoptosis trigger
apoptosis
CD4
cytokines
Virus purged
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Prevention of infection and/or disease
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Protection of the new born animal

• antibodies (CMI) in colostrum
• maternal immunization
• colostrum replacers
• implications for immunization of young animals
• caution when using modified-live vaccines
• interference by maternal antibodies

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Interference by maternal antibodies

window of susceptibility
minimum amount needed for protection

interferes with vaccination

passive antibody



can vaccinate in this range
2
4
6
8
10
12
weeks after birth
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Prevention of virus infections/disease

• vaccination
• inactivated vaccines
• attenuated vaccines
• subunit
• vectored
• DNA vaccines
• management

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Why vaccination sometimes fails to protect

• improper use
• genetic differences between animals
• antigenic differences
• blocking by maternal antibodies
• administration following infection (exceptions -
  rabies)

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Diagnosis of viral diseases
Why??
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Sensitivity and Specificity
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Diagnosis of viral diseases

• clinical signs
• virus detection
• detection of exposure

Laboratory
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Detection of virus
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Detection of virus

• isolation (isolation immunological detection)
• quantitation (plaque assay, TCID50)
• PCR
• haemagglutination (or HAI)
• ELISA (in clinic or lab)
• immunological detection (IH or IF)
• electron microscopy

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Virus isolation (tissue culture, experimental
animals)
cultured cells (two dimensional animals)
cytopathic effect
immunofluorescence (anti-herpesvirus antibody)
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Virus quantitation (plaques)
plaques
count plaques (plaque forming unit/ml)
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Virus quantitation (TCID50, LD50)
10-2
10-3
10-4
10-5
10-6
TCID50 104
50 Tissue culture infectious dose
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PCR
primers
Viral DNA
First cycle
BVDV-1a
BVDV-2
Second cycle
BVDV-1b
Twenty cycles
million
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PCR tests offered by PDS

• bov. resp. coronavirus 30
• rotavirus genotyping 65
• bov. RSV 20
• BVD genotyping 62
• porc. circovirus 27
• influenza 30
• swine influenza geotyping 50
• equine papillomavirus 20
• malignant catarrhal fever virus 27
• pan-herpesvirus
• West Nile virus 30

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Haemagglutination (HA)
No virus
virus
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Haemagglutination
Dilution
No virus
2
4
8
16
32
64
128
256
512
1024
titre
prozone
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Enzyme linked immunabsorbant assay (ELISA)
Enzyme -gt colour
Sample to be tested
Detecting antibody
virus
capturing antibody
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In-clinic ELISAs
Feline leukemia And Immunodeficiency viruses
Others canine parvovirus bovine herpesvirus -
1 bovine viral diarrhoea virus equine
infectious anemia virus influenza-A porcine
respiratory reproductive syndrome virus
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Immunological detection
Immunohistochemistry
Immunofluorescence
bovine herpesvirus antigens in endothelial cells
BHV-1 antigens in neuron In trigeminal ganglion
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Detection of exposure
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Detection of exposure (serology)

• virus neutralization
• haemagglutination inhibition
• ELISA

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Virus neutralization
Serially dilute serum
1/2 1/4 1/8 1/16.1/512
Add equal amount of virus (100 plaque forming
units) to each tube Infect cultured cells Last
dilution that can prevent plaque formation is
titer
--
No serum
1/2
1/4
1/256
1/512
1/128
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HAI
Serum dilution
Virus HA
1 wk
8 wks
1/20
1/40
1/80
1/160
1/320
1/640
1/1280
-ve control
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Limitations of serology

• detects exposure and not when exposure occurred
• for correlation with disease
• Paired sera
• IgM
• CSF

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Virology Diagnostic Laboratories

• PDS, Saskatoon
• Veterinary Services Branch,
• Manitoba Agriculture, Winnipeg
• Animal Health Monitoring Lab, Abbotsford
• Central Laboratories for Veterinarians Ltd.
  Calgary

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Sample collection and submission

• Choosing samples - considerations
• alive or dead
• suspected pathogen(s), tropism, pathogenesis
• phase of disease
• virus detection or exposure
• Live animals
• nasal swabs, transtracheal aspirates, respiratory
  secretions, scrapings
• vesicular fluid, covering epithelium, biopsy from
  margin of lesion
• feces or fecal swabs
• clotted and unclotted blood
• samples from unaffected animals

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• dead animals
• collect samples as soon as possible after death
• affected organs
• gut loops

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Shipping the samples

• transport medium (from lab or buffered saline
  with 50 µg/ml gentamycin)
• 10 buffered formalin - lt 1cm thick
• plastic, sealed containers, labeled with water
  proof ink
• ice packs vs frozen samples

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Samples should be accompanied by

• case history and suspected pathogen(s)
• treatment, vaccinations, numbers involved
• list of specimens

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Viruses can be useful too

• biological control of pests
• cancer therapy
• gene therapy
• nanotechnology
• symbiotic virus-host relationships