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Viruses and what they do -
An overview
Wednesday, August 25, 2010
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 host’s
response to this interaction).
Viral Genomes
Single Stranded
DNA
Double Stranded
Nucleic Acid
RNA
Double Stranded
Positive
Single Stranded
Negative
RNA
DNA
SS RNA genomes
• +ve (sense) and -ve (anti-sense) RNA genomes
Positive
(sense)
AUG GCA CGA
Negative
(anti-sense) UAC CGU GCU
met ala arg
herpes
capsid
adeno
Virion
“naked” virus
particle or
Virion
envelope
enveloped
Virus or Virion
capsomeres
Capsid
Proteins produced by viruses
• Structural proteins
• Non-structural proteins
Some viral shapes
papillomavirus
adenovirus
“naked” viruses
100 nm
1 nm = 1 millionth of a mm
100 nm = 1 ten thousandth of a mm
parvovirus
Some viral shapes
1 nm = 1 millionth of a mm
100 nm = 1 ten thousandth of a mm
100 nm
influenzavirus
parainfluenza
virus
Enveloped viruses
herpesvirus
poxvirus
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
Viruses with ss DNA genomes
porcine circovirus
Circoviridae
canine parvovirus-2
Parvoviridae
feline panleukopenia virus
porcine parvovirus (SMEDI)
Viruses with ds DNA genomes
papillomaviruses
Papovaviridae
Adenoviridae
Herpesviridae
adenoviruses
equine
bovine
herpesvirus-1,2 herpesviruses -1,4
porcine cytomegalovirus
malignant catarrhal fever virus
Poxviridae
poxviruses
African swine fever virus
african swine fever virus
Viruses with ds RNA genomes
rotaviruses
Reoviridae
bluetongue virus
african horse sickness
Birnaviridae
infectious bursal disease (chickens)
infectious pancreatic necrosis (salmonid fish)
Viruses with +ve RNA genomes
Picornaviridae
Caliciviridae
Coronaviridae
Arteriviridae
Flaviviridae
Togaviridae
foot and mouth disease virus
porcine enteroviruses
feline calicivirus
coronaviruses
equine arterivirus, PRRS
flaviviruses (WNV)
pestiviruses (BVD)
equine encephalitis viruses
Viruses with -ve RNA genomes
Orthomyxoviridae
Paramyxoviridae
influenzaviruses
parainfluenza virus
canine distemper virus
Hendra, Nipah viruses
respiratory syncytial virus
Rhabdoviridae
rabies virus
Filoviridae
vesicular stomatitis virus
Ebola virus
Bunyaviridae
Haantan virus
Viruses with reverse transcriptase
feline leukemia virus
Retroviridae
feline, bovine immunodeficiency
viruses
bovine, avian leukosis viruses
caprine arthritis-encephalitis virus
Hepadnaviridae
Isolates, strains, serotypes and
groups
no selective pressure on
internal proteins
selective pressure
on
external viral proteins
antibodies to all
viral proteins
antibodies to
external proteins
neutralize virus
selective pressure
on
external viral proteins
antibodies to all
viral proteins
antibodies to
external proteins
neutralize virus
selective pressure
forces slight
change in external
proteins
virus, including
changed virus,
passed on to new
host
selective pressure
on
external viral proteins
antibodies to all
viral proteins
antibodies to
external proteins
neutralize virus
selective pressure
forces slight
change in external
proteins
process repeated, over time…..
neutralizes
neutralizes
neutralizes
neutralizes
does not neutralize
serum from original cat
NOTE: Only external
proteins change. Internal
proteins do not change
process repeated, over time
same serotype
neutralizes
neutralizes
neutralizes
new serotype
neutralizes
does not neutralize
serum from original cat
NOTE: Only external
proteins change. Internal
proteins do not change
Serotype - all isolates of a virus
that can be neutralized by a
common antiserum are said to
belong to the same serotype.
…..because of changes in external protein (internal proteins do not change)
external proteins are called TYPE SPECIFIC antigens
internal proteins are called GROUP SPECIFIC antigens
process repeated, over time
same serotype
new serotype
different serotypes
same group
Group and type specific antigens
type
specific
antigen
“naked”
virus
(eg FMDV)
enveloped virus
(eg influenza, FeLV)
group specific
antigen
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
Serotypes and neutralizing antibody (eg. FMDV)
A
C
O
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
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
Infection of a cell
•
•
•
•
attachment
entry and uncoating
viral gene expression
genome replication
– DNA viruses
– RNA viruses
• assembly and release
– naked viruses
– enveloped viruses
Distribution of the CCR532 mutation in
human populations
from PLoS
Biology, Nov
2005
Errors in replication lead to “quasispecies”
persistent
infection
mixture of variant viruses
(quasispecies)
inclusion bodies
A
B
C
D
Release of virus
Release by lysis of cell
(cytopathic)
or by budding (without
death of cell, non-cytopathic)
Infection of a cell
Stage
Biological Host
Drug
implications defenses intervention
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
Why do some infected animals
get sick and others don’t?
• Viral factors - virulence
• Host Factors
Viral Factors:
Why are some isolates of a virus
more likely to cause severe disease
than others?
determinants of viral virulence
Host Factors:
• Genetic Resistance
– loss of receptors
• CCR5-32 mutation and resistance to HIV
– variation in immune response genes
– genetic defects in defenses
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
Incubation period
infection
incubation period - time between infection and the appearance of clinical signs
Patterns of disease
clinical signs
virus shedding
acute
recurrent
chronic
or persistent
slow
virus difficult to detect
Resistance and Recovery
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
After the Toll Rush, LAJ O’Neill, Science 303:1481 2004
Interferons
I
I
I
I
I
E
antiviral
effects
infected
cell
activated
factor
Induced interferon
genes
Immune
Modulation
Acquired anti-viral immunity
(antibody)
Virus neutralized
B
Virus infected
cell
Antibody targets
Fc receptor bearing cell
To kill virus infected cell
Acquired immunity (CMI)
perforins
CD8
CD4
Apoptosis
trigger
necrosis
apoptosis
cytokines
virus replication
suppressed
Prevention of infection and/or
disease
Protection of the new born animal
antibodies (possible CMI as well) in colostrum
– maternal immunization
– colostrum replacers
– implications for immunization of young animals
• caution when using modified-live vaccines
• interference by maternal antibodies
Interference by maternal
antibodies
*
window of
susceptibility
passive antibody
*
2
4
*
6
*
8
weeks after birth
*
10
minimum
amount
needed for
protection
*
12
interferes
with
vaccination
can
vaccinate
in this
range
Prevention of virus
infections/disease
• vaccination
– inactivated vaccines
– attenuated vaccines
– subunit
– vectored
– DNA vaccines
• management
Why vaccination sometimes fails to
protect
•
•
•
•
•
improper use
genetic differences between animals
antigenic differences
blocking by maternal antibodies
administration following infection (exceptions - rabies)
Diagnosis of viral diseases
Why??
Sensitivity and Specificity
Diagnosis of viral diseases
• clinical signs
• virus detection
• detection of exposure
}
Laboratory
Detection of virus
Detection of virus
•
•
•
•
•
•
•
isolation (isolation +immunological detection)
quantitation (plaque assay, TCID50)
PCR and real-time PCR
haemagglutination (or HAI)
ELISA (in clinic or lab)
immunological detection (IH or IF)
electron microscopy
Virus isolation (tissue culture,
experimental animals)
cultured cells
(two dimensional
animals)
cytopathic effect
immunofluorescence
(anti-herpesvirus antibody)
Virus quantitation (plaques)
plaques
count plaques
(plaque forming unit/ml)
Virus quantitation (TCID50, LD50)
10-2 10-3 10-4 10-5 10-6
TCID50 = 104
50% Tissue culture infectious
dose
primers
PCR
Viral DNA
First cycle
BVDV-1a
BVDV-2
Second cycle
BVDV-1b
Twenty cycles
million
PCR tests offered by PDS, 2009
($35)
•
•
•
•
•
•
•
•
•
•
•
•
Avian influenza
Avian paramyxovirus
Bovine viral diarrhoea virus
Equine influenza
Equine herpesvirus (1 vs 4, neurotropic)
Influenza A – pan species
Malignant catarrhal fever
Porcine circovirus
PRRS
Rotavirus (genotyping)
Swine influenza
West Nile virus
Haemagglutination (HA)
virus
No virus
Haemagglutination
Dilution
2
4
prozone
8
16 32 64 128 256 512 1024
titre
No
virus
Enzyme linked immunabsorbant assay (ELISA)
Sample to be tested
virus
capturing antibody
Enzyme -> colour
Detecting antibody
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
Immunological detection
Immunohistochemistry
Immunofluorescence
bovine herpesvirus antigens
in endothelial cells
BHV-1 antigens in neuron
In trigeminal ganglion
Detection of exposure
Detection of exposure or measure or
humoral immunity (serology)
• virus neutralization
• haemagglutination inhibition
• ELISA
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
-1/2
1/4
1/128
1/256
1/512
No serum
HAI
Virus
HA
8 wks
1 wk
Serum
dilution
1/20
1/40
1/80
1/160
1/320
1/640
1/1280
-ve control
Limitations of serology
• detects exposure and not when exposure
occurred
• for correlation with disease
– Paired sera
– IgM
– CSF
Virology Diagnostic Laboratories
• PDS, Saskatoon
• Veterinary Services Branch,
Manitoba Agriculture, Winnipeg
• Animal Health Monitoring Lab, Abbotsford
• Central Laboratories for Veterinarians Ltd.
Calgary
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
• dead animals:
– collect samples as soon as possible after death
– affected organs
– gut loops
Shipping the samples
• transport medium (from lab or buffered saline with 50 µg/ml
gentamycin)
• 10% buffered formalin - < 1cm thick
• plastic, sealed containers, labeled with water proof ink
• ice packs vs frozen samples
• For PCR
– Regular (ice, frozen)
– RNALater
Samples should be accompanied by
• case history and suspected pathogen(s)
• treatment, vaccinations, numbers involved
• list of specimens
Viruses can be useful too
•
•
•
•
•
biological control of pests
cancer therapy
gene therapy
nanotechnology
symbiotic virus-host relationships