Week 7 methods to study viruses & cht 26 nervuos system(1)

 Diagnostics/detection
• Is there any virus in the sample
• What type of virus is it
• How many viruses are there in the sample / culture
• Are there and antibodies with specific binding to the virus?
 Virus replication
• Tropism - What type of cells can the virus infect
 Virus structure
• Protein structure
• Virus superstructure (how the different parts of the virus fits together)
 Virus – Host interactions
• Which proteins the virus interacts with in the host cell
• What effects these interaction has on the cell and the whole organism
• How the virus and the immune system interacts
Examples of research questions?

Techniques Detect
ion
Identifi
cation
Quantifi
cation
Struct
ure
Culture X X
Neutralization X
Sandwich ELISA X X +/-
Lateral Flow X X
Complement fixation test X X
Haemagglutination inhibition test X X
Immunofluorescence Assay (IFA) X X
PCR X X X
NASBA X X X
Transmission Electron microscopy
Transmission Electron microscopy (TEM) X X X X
Scanning Electron microscopy (SEM)
X-ray crystallography X
Cryo-Electron Microscopy X X
Nuclear Magnetic Resonance (NMR) X
Culture based
Serology based
Genetic
“Physics”

 Diagnostics:
• Detection of the virus by direct or
indirect means in a sample from a sick:
• Human;
• Animal; or
• Plant
 Detection:
• Same as for diagnostics (but usually “direct” detection), but the
sample is from the environment (in-door, out-door, air, water, soil,
food etc.)
• Usually environmental samples are more
complicated to work with:
• Complex matrix
• Background of related organisms
to the one you are looking for
Virus diagnostics and detection

 Is there any virus in the sample
• Virus culture
• Genetic methods
• Serological methods
• Electron microscope
 What type of virus is it
• Genetic methods
 How many viruses are there in the sample / culture
• Plaque assay (culture)
• Real-Time PCR
 Is there and antibodies with specific binding to the virus?
• Serology

 Culture of the virus is usually classified as the “Gold Standard”
• Pro’s:
• Concrete evidence of the virus
• The virus can be further studied
• Con’s:
• Usually not very sensitive
• Very slow (From days to months)
• Expensive (Manpower intensive)
Virus culture

Host
Zoonotic
viruses
“Human”
viruses Reason
 Most viruses are very “picky” regarding culture
conditions and might be difficult to culture
How to get a virus to grow?
Natural host
Suckling mouse
Embryonated egg
Primary cells
from host animal
Immortalized cell
line
++++
+++
++
++
+
+++
++
++
+
The virus is fully adapted to its
host. Unethical to infect humans
The suckling mouse immune
system is not fully developed
Relatively easy to purify viruses
from egg. Good for avian viruses.
Most similar to the host tissue. But
as it is made up of one cell type it
cannot compare to a whole organ or
complete host.
Depending on cell line the virus
might be able to infect the cells
-

Animals / Eggs
• Use of animals for culture of virus
– First method used for virus cultivation
– Inconvenient
– Safety issues in handling animals
• Mainly replaced by cell culture except
– Virus has no known host in vitro
• Hepatitis C virus in chimpanzees
• Influenza virus in chick embryo
– Study of viral pathogenesis in a whole host
• Polio studies in chimpanzees

 Isolation and cultivation of
many avian and some
mammalian viruses
 Only embryonated eggs
are suitable for virus
culture
 Eggs are candled to check
for embryo and for
monitoring the egg after
inoculation with the virus
Embryonated eggs

Embryonated eggs
Inject the virus in
an appropriate part
(different for
different viruses) of
the egg.
Culture in eggs has
been (and probably
still is) the most
common method to
produce Influenza
vaccine

Currently the great majority of Influenza virus needed
vaccine production is cultured in chicken eggs.
Embryonated eggs and vaccine

 Sensitive to a wide range of
viruses
 Has been used for vaccine
production (e.g. Japanese
Encephalitis virus)
Suckling mouse

 Cell tissue culture
• Cells grown in vitro
•Primary cell culture
•Continuous cell lines
 Some viruses do not grow in vitro (e.g. Hepatitis C)
Tissue Culture

Trypsination
Culture
Media
Release the cells
from the tissue with:
- gentle force and
- enzyme digestion
(e.g. trypsin,
collagenase etc.)

Primary cell lines
• Pro’s
– Have many of properties of the in vivo cells
– Can often more easily be infected with viruses
specific to the animal species
• Con’s
– Will only divide a few times
– Might be difficult to prepare (depending on the
cell type and animal species)
– Might contain other viruses

CONTINUOUS CELL LINE
HeLa cells

 Derived from primary cell lines
 Transformed / cancerous cells
 Often polyploid or multiploid
 Theoretically can be sub-cultured indefinitely
 Method of choice to cultivate virus, where possible
Continuous Cell Lines

Different viruses infect different types of cells = they have
different tropism
Cultivation of viruses
Cell line Origin
Vero African Green Monkey kidney cells
Hep-2 Human, probably a contamination of HeLa (Henrietta
Lack) cells cervix tumour
HEL Human embryonic lung
HEK Human embryonic kidney
MK Monkey kidney
BGM African Green Monkey kidney cells
LLC-MK2 Rhesus monkey kidney cells
MDCK Madin-Darby canine kidney (MDCK) epithelial
RK13 Rabbit kidney cells
BHK Baby hamster kidney fibroblast

Cytopathic Effect
Some viruses will kill some (not all) cells types. This is called
cytopathic effect effect.
If the cells are over laid with agar or cellulose after infection it is
possible to observe separate areas (plaques) of dead cells. This is
called plaque assay

CPE
A549
MRC-5
RhMK
HEp-2
HSV-2 Adenovirus
CMV Rhinovirus
Enterovirus Inﬂuenza A
virus
RSV Monkey B virus
Different effect
of
different viruses
in
different cell lines
Non-infected

• Virus culture
• Genetic methods
• Genetic methods
• Plaque assay
• Real-Time PCR
 Is there and antibodies with specific binding to the
virus?
• Serology

Sandwich ELISA
Lateral flow
Complement fixation test
Haemagglutination inhibition test
Direct and Indirect Immunofluorescence Assay
Serology

 Serological methods
• Neutralization test
• The virus is incubated with antibodies that blocks the
virus from infecting the cells (neutralization).
– Pro’s:
» Can be very specific
– Con’s
» Very slow (Days)
» Based on assumptions
What type of virus is it?

• Immunofluorescence assay
• Pro’s
– Direct visualization of
bound fluorescent
antibodies
– Sensitive
• Con’s
– Need an experienced
analyst
– Based on assumptions

• Sandwich ELISA
• Pro’s:
– Can be very specific
– Can be performed in a day (or faster)
• Con’s
– Based on assumptions

4) Excess buffer along
with any reagents not
captured at the test of
control line will then move
into the absorbent wicking
pad.
2)
- With the addition of the
sample, the detector
molecules are solubilized.
- When solubilized the
detector molecules mix with
and bind to the analyte in the
sample (if analyte is
present).
3)
- Capillary action draws the
fluid mixture up the
sample pad and into the
membrane.
- The sample/detector
molecule mix continues
to move up the
membrane until it
reaches the analyte
capture molecule.
- In these lines a second
(and third) antibody or
antigen, immobilized as a
thin stripe in the
nitrocellulose will then
capture the complex if it
is positive for the target
analyte.
 Lateral flow immunoassay
1) A sample is placed on the
sample pad at one end of the
strip

• Complement
fixation test
pospos
pos Neg
Neg
NegNeg
pos
Haemagglutination
inhibition test

• Virus culture
• Genetic methods
• Genetic methods
• Plaque assay
• Real-Time PCR
 Is there and antibodies with specific binding to the virus?
• Serology

 Polymerase Chain Reaction - PCR
 Nucleic Acid Sequence Based Amplification -
NASBA
Genetic methods

 PCR (Polymerase
Chain Reaction)
 Uses specific
primers
 One doubling per
cycle
PCR
1
2
4
cyc
le
Copies
1
2
3
4
5
6
7
8
9
10
11
12
cyc
le
Copies
13
14
15
16
17
18
19
20
21
22
23
24
1
2
4
8
16
32
64
128
256
512
1024
2048
4096
8192
1.6*10^4
3.2*10^4
6.5*10^4
1.3*10^5
2.6*10^5
5.2*10^5
1.0*10^6
2.1*10^6
4.2*10^6
8.4*10^6
8

 PCR (normal) animation
• Products have to be separated (usually
agarose gel, but DNA micro array is an
other possibility)
 Many different versions of PCR
• Nested PCR
• Reverse transcriptase (RT-PCR)
• PCR (Real-time)
• W. intercalating dye (e.g. Cybr-green)
• Probe based (e.g. Taqman, molecular
beacon, hybridization probes etc.)
PCR
Hydrolysis/TaqMan
Probe
Hybridization probe
Molecular beacon

 Specific primers are used, one of them carrying the T7 promoter.
 RNA and DNA targets are amplified up to one billion fold in within
60 to 90 minutes reaction time.
 The amplification reaction is isothermal, proceeds at 41°C, and
 results in single stranded RNA molecule synthesis
 Uses 3 enzymes:
• Reverse Transcriptase:
for cDNA synthesis
• RNase H:
for degradation of the
RNA in the heteroduplex
RNA-DNA
• T7 RNA polymerase:
for synthesis of RNA from
the T7 promoter
Nucleic Acid Sequence Based Amplification (NASBA)

 What is an electron microscope?
• A microscope that uses an electron
beam instead of a light beam to
investigate the sample
 Why electrons instead of light?
• Due to that the electrons have much shorter wave
length is can resolve structures down to 0.1 nm
(compared to ~ 200 nm for light)
 2 main types
• Transmission electron microscopy (TEM)
• Scanning electron microscopy (SEM)
Electron microscope
This is a pretty good lecture on electron microscopy:
http://www.youtube.com/watch?v=nkGRhYv01ag&list=PL0D6F7A92E4E47489
TEM of Influenza virus

Electron microscopy (EM)
http://science.howstuffworks.com/scanning-electron-microscope2.htm
Transmission Electron Microscope
Scanning Electron Microscope
Sample

Avian poxvirus
 Transmission Electron Microscope (TEM) is most
common
• TEM pictures are produced by electrons that are
scattered as they pass through the sample.
• To increase the contrast the sample is often stained with
electron-dense heavy metals. This called Negative
Staining
• The harsh environment and dehydration of the samples
affects the results. To minimize theses deleterious effects
the sample can be
embedded in vitreous ice and analysed
in a cold environment (using liquid
nitrogen (-196 ºC)). This is called:
• Cryo Electron Microscopy
(Cryo-EM)
Different types of Electron microscopy

 Scanning electron microscopy (SEM)
 SEM pictures are produced by:
• Backscatter - Electrons that bounce back (back
scatter) from the sample
• Secondary electron - Electrons that are “ripped off”
from the sample atoms
• X-rays generated by absorbed electrons
Different types of Electron microscopy

Brief summary
 Culture (gold standard, based on assumptions (what cell
line to use), slow, laborious)
 Serology (based on assumptions (what antibodies to use)
• Sandwich ELISA (Moderate sensitivity, relatively high throughput)
• Lateral Flow devices (very fast, variable sensitivity)
 Genetic methods (based on assumptions (need primers))
• PCR for DNA (several methods, fast. High throughput)
• Reverse Transcriptase (RT) PCR for RNA (similar sensitivity to
NASNA, but more reliable)
• NASBA for RNA
 Electron microscopy (NOT based on assumptions, need
highly experienced operator, slow, laborious)
Diagnostics and detection

 Serology (assumption based)
• Uses antibodies that are specific to the virus
• Sandwich ELISA
• Immunofluorescence assay (IFA)
• Serological neutralization assays
 Genetic (assumption based)
• PCR
• RT-PCR
• NASBA
 Electron microscopy
(NOT assumption based,
but not very specific)
Typing of viruses
Can be sequenced or
hybridized for higher
specificity

 Is there any virus in the sample
◦ Virus culture
◦ Serological methods
◦ Genetic methods
◦ Electron microscope
 What type of virus is it
◦ Serological methods
◦ Genetic methods
 How many viruses are there in the sample / culture
◦ Plaque assay
◦ Real-Time PCR
 Is there and antibodies with specific binding to the virus?
◦ Serology

 Culture
• Plaque assay (counts functional virus particles)
 Genetic (counts number virus genomes)
• Real Time PCR
• Real Time NASBA
 Direct counting
• Electron microscopy (counts number of virus particles)
Quantification of viruses

 Quantitative Real Time PCR & NASBA
• The amount of genetic material is amplified and
• Compared to the amounts amplified from known
amounts of genetic template
• In Real Time
Genetic methods

 Electron microscope
• Pro’s
• Direct counting
• Insensitive to mutations
• No assumptions needed
• Can give other information about the virus
• Con’s
• Expensive
• Skilled analyst needed
• Laborious
• Low throughput
How many viruses are there in the sample / culture?

Virus Dilution
Sample
Transfer 0.5 ml Transfer 0.5 ml Transfer 0.5 ml
10-fold
dilution
10-fold
dilution
10-fold
dilution
Original Sample 4.5 ml diluent
1:10 dilution
4.5 ml diluent
1:100 dilution
4.5 ml diluent
1:1,000 dilution
Dilution of sample
0.5 ml

Plaque Assay
diluted virus cell culture dish
cell monolayer
infected cells
plaques

 𝑉𝑖𝑟𝑢𝑠 𝑡𝑖𝑡𝑟𝑒 =
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑣𝑖𝑟𝑢𝑠 𝑝𝑙𝑎𝑞𝑢𝑒𝑠 ∗(𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟)
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑝𝑙𝑙𝑖𝑒𝑑 𝑣𝑖𝑟𝑢𝑠 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝑚𝐿)
 E.g. After applying 200 µL of a virus 104 dilution,
40 plaques in well A, 45 plaques in well B. What is
the pfu/ml?
Formula for calculating virus concentration (Titre)
𝑉𝑖𝑟𝑢𝑠 𝑡𝑖𝑡𝑟𝑒 𝑖𝑛 𝑤𝑒𝑙𝑙 𝐴 =
40 ∗ 104
0.2 (𝑚𝐿)
= 2 ∗ 106
𝑝𝑓𝑢
𝑚𝐿
𝑉𝑖𝑟𝑢𝑠 𝑡𝑖𝑡𝑟𝑒 𝑖𝑛 𝑤𝑒𝑙𝑙 𝐵 =
45 ∗ 104
0.2 (𝑚𝐿)
= 2.25 ∗ 106
𝑝𝑓𝑢
𝑚𝐿
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑖𝑟𝑢𝑠 𝑡𝑖𝑡𝑟𝑒 =
(2 + 2.25) ∗ 106
2 (𝑚𝐿)
= 2.125 ∗ 106
𝑝𝑓𝑢
𝑚𝐿
≈ 2 ∗ 106

 Very time-consuming
 Very simple method
 Only works for viruses that:
 infect monolayer cells
 cause cell lysis
 Uses principle of one virus on the monolayer
produces one plaque
Plaque Assay - Features

 Centrifuge the (liquid) sample at high gravitational
forces (up to 2.000.000 g)
 Sample is mixed with a solution that will form a
gradient under a high gravitational field (e.g. sucrose
or cesium chloride)
 The sample will be separated
based on weight density, with
the lightest components on top
and the heaviest below.
 The viruses will form a “band”
in the gradient
Ultra centrifugation

Ultracentrifugation
Sucrose
concentration(%)
virus
concentration
sucrose density
purified virus
centrifugal force

 Ion exchange or Immunoaffinity
 Viruses can be bound onto an ion-exchange or
antibody coated resin
 They can then be eluted out by applying different
concentration salt (ion exchange) or antibody ligand
solutions
Chromatography

 Same as for purification
 But also:
• filtration; and
• precipitation
Virus concentration

 Tangential flow filtration can filter large volumes of
liquids without getting “clogged”
Filtration

 2 strategies
1. Add flocculent that is easily precipitated. The flocculent
will “trap” the viruses that then co-precipitates.
2. Add Polyethylene Glycol (PEG) and salt to the sample.
The PEG will absorb water while excluding the viruses,
thus the viruses get concentrated up to a point where
they precipitate
• The sample is then centrifuged to collect the precipitate
at the
bottom of the tube
Precipitation

METHODS FOR STUDYING VIRUS
STRUCTURE

Bacteriophage epsilon15
Rabies nucleoprotein
Why do we want to know the structures of a virus?
 To understand:
• The virus replication process
• Virus-host interactions
• Viral and host receptors
• Viral hijack of the cell
• Evasion of the immune system
• How “weaknesses” in the virus that can be used to develop
medical interventions
 2 levels of structure
• Protein structure (how an individual protein is folded)
• Virus superstructure (how the different parts of the
virus fits together)
Virus structure

 Methods
• Reverse genetics
• Crosslinking
• Cryo-Electron Microscopy
• Nuclear Magnetic Resonance (NMR)
• X-ray crystallography
• Computer modelling
Virus structure

 Reverse genetics
• The complete viral genome has been cloned expression
plasmids
• A complete virus can be created by expressing the these
plasmids
 Why “reverse genetics” and not “normal” cloning?
• It is very difficult to make specific mutations in a “live”
virus.
• Creating mutations in a plasmid is much easier
 Mutate parts of the virus and look for changes in
how the virus can infect, replicate and affect the
host cell or host organism
Virus structure

Crosslinking
 Use of crosslinking chemicals (see lecture on protein
interactions)
 Chemicals with two reactive groups (one at each end of a
linker structure) will crosslink neighbouring epitopes in a
protein.
 After crosslinking, the protein is digested with protease(s).
 Parts of the protein that is found to be cross-linked can be
assumed to be near each other…
Virus structure

 Cryo-Electron Microscopy
• Samples are studied using EM at cryogenic temperatures
(usually at liquid nitrogen temperatures)
• Pro’s:
• Protein and virus
structures are in more
natural form (than if
crystallised)
• Con’s:
• The resolution is not as
good as for X-ray, but the
resolution is steadily
increasing
Virus structure

 Virus / Protein structure:
• X-ray
• Based on the refraction pattern of x-rays
passing through a crystal of protein or
even a whole virus
• Pro’s
– Can give a very clear picture of the protein structure
– Can handle large proteins and even whole viruses
• Con’s
– The protein need to be in the form
of a crystal. Protein crystallization is
notoriously difficult
– The structure is unnatural as the
protein is in the form of a crystal
and not in solution or in a membrane
Virus structure

 Nuclear Magnetic Resonance (NMR)
spectroscopy
• Theory: magnetic resonance results from the interaction of an
atomic nucleus (of certain atoms) with an external
magnetic field. Different functional groups give
different NMR “shifts” which can be correlated
to other functional groups
• Pro’s:
• No need to crystalize the protein
• Protein studied in solution
• Con’s
• Only works for smaller proteins
(generally up to 150 kDa)
Virus structure

 Computer modelling
• Using energy-minimization modelling it is currently
possible to predict the structure of proteins that have
close “relatives” with a known structure
• Pro’s:
• Fast
• Cheap (unless there is a need
for super computers)
• Con’s
• Need the structure of a closely
related protein
• Is based on assumptions (how the
computer model is made)
Virus structure

74Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chapter 26
-
Nervous system

Nervous System Infections
 Frightening Infections
• Threaten ability to move, feel, even think
• Poliomyelitis can result in a paralyzed limb or
inability to breath without mechanical assistance
• Hansen’s disease (leprosy) can result in loss of fingers
or toes or deformity in the face
– Historically, lepers were feared and loathed,
shunned from society; in the Middle Ages, attended
their own symbolic burial before being sent away
• Infections of brain or membranes can
render a child deaf or disabled
• Before antibiotics, bacterial infections
of nervous system often fatal
• Fortunately, CNS infections
are uncommon

26.1. Anatomy, Physiology, and Ecology
 Nerve cells transmit electrical impulses
• Each neuron has different regions
• Dendrites are branching projections, receive information
• Convey information to cell body, the command center
– Contains cell nucleus
• Long, thin extension called axon transmits to another cell
• Neurons communicate with neurotransmitters
– Chemicals produced in cell body, stored in vesicles at
end of axon
– When released, diffuse to neighboring cell
– Cell has receptors, receives signal, responds
– Region between cells is a synapse
– Some viruses can move through axon toward cell
body in process called retrograde transport

 Central Nervous System (CNS)
• Brain and spinal cord
• Brain is complex; distinct parts have different functions
– Generalized inflammation or infection is encephalitis
 Peripheral Nervous System (PNS)
• Nerves composed of bundles of axons
• Motor neurons carry messages from CNS to parts of body
– Cause them to respond
• Sensory neurons transmit sensations to the CNS
• Most nerves mixed, carry sensory and motor information
• Cell bodies of sensory neurons from skin located in ganglia
near vertebral column; cell bodies of motor neurons located
within the CNS

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Nerves
Leprosy
Botulism
Spinal cord
Meningitis
Poliomyelitis
Brain
Encephalitis
Meningitis
Rabies
African sleeping sickness
Spongiform encephalopathies
Central nervous system (CNS)
is surrounded by meninges.
Peripheral nervous system (PNS)
carries information to and from the CNS.
(b)
(a)
Dendrites
Axon
Synapse
Cell body
Ganglia

 CNS much better protected than PNS
• Encased by bone: skull, vertebral column
• Only rarely do infections spread
• Three layers of membranes cover surface of brain and
spinal cord; called meninges
• Outer dura is tough and fibrous, adheres closely
– Provides barrier to spread of infection from bones
• Two inner membranes are arachnoid and pia
– Separated by subarachnoid space within which flows
cerebrospinal fluid
– Inflammation of these membranes is meningitis
– When both infected, meningoencephalitis

26.3. Viral Diseases of the Nervous System
 Viral Meningitis
• Signs and Symptoms
• Abrupt onset;
– fever, – severe headache above or behind eyes,
– sensitivity to light, – stiff neck
• Nausea, vomiting common
• May be sore throat, chest pain, swollen salivary glands,
skin rash
• Causative Agents
• Non-enveloped RNA viruses of enterovirus subgroup of
picornaviruses responsible for at least half of cases
– Most common are coxsackie viruses (cause throat,
chest pain) and echoviruses (cause rash)

 Viral Meningitis (continued…)
• Pathogenesis
• Infect throat, intestinal epithelium, lymphoid tissue
• Spread to bloodstream, cause viremia
– Can result in meningeal infection
• Inflammatory response differs from bacterial meningitis;
fewer cells enter cerebrospinal fluid
– Typically less severe; causes little lasting damage
• Epidemiology
• Enteroviruses relatively stable in environment
– Can sometimes persist in chlorinated swimming pools
• Transmitted via fecal-oral route
• Feces of infected individuals contain viruses for weeks

 Viral Meningitis (continued…)
• Treatment and Prevention
• No specific treatment available
• Handwashing
• Avoiding crowded
swimming pools
• No vaccines against
coxsackie, echoviruses

 Viral Encephalitis
• Onset abrupt; – fever, – headache,
– vomiting
• Possible disorientation, localized paralysis, seizures, coma
• Causative Agents
• Usually arboviruses (arthropod-borne viruses), group of
enveloped, single-stranded RNA viruses
• Transmitted by insects, mites, or ticks
• Leading causes in
U.S. transmitted
by mosquitoes

 Viral Encephalitis (continued…)
• Pathogenesis
• Viruses multiply at site of bite and in local lymph nodes
• Produce mild, brief viremia
• If viruses infect cells of blood-brain barrier, can enter brain,
replicate in neurons, destroy brain tissue
• Neutralizing antibodies stop disease progression
• Case-fatality rates range from ~2% with LaCrosse
encephalitis to 35–50% with eastern equine encephalitis
– Emotional instability, epilepsy, blindness, or
paralysis may occur in 5–50% of those who recover
– Depends on kind of virus and age of patient
– Very young, elderly most affected
Sequela

Japanese encephalitis virus (JEV)
 Previously known as “Japanese B encephalitis”
 Causes encephalitis (acute inflammation of the brain) in
humans
 Vast majority of infections are asymptomatic: only 1 in 250-
500 infections develop into clinical disease
 >50,000 cases annually
• 10,000 deaths
• 15,000 long term nerve disease
 Rural areas are at highest risk
 Case-fatality rates range from
0.3% to 60% and depends on
population and on age
 Serious neurologic sequelae: 30%
 Incubation period of 5 to 15 days
Brain fever image:
'Somebody save my son'

JEV - Prevalence
Age-standardised disability-adjusted life year per 100.000 inhabitants

JEV - Recent outbreaks
 India – Utar Pradech
 400 deaths
 2300 patients
 30% neurological sequel
• ~700 cases

JEV - Transmission
 Zoonotic
 Domestic Pigs and birds (herons) are hosts
 Transmitted to humans by culex mosquitoes

JEV - Symptoms
 Severe rigors (chills) mark the onset of this disease in
humans.
 Fever, headache and discomfort are other non-specific
symptoms last for a period of between 1 and 6 days.
 Signs which develop during the acute encephalitic stage
include:
• neck rigidity,
• fatigue,
• hemiparesis, (weakness of one side of the body)
• convulsions and
• fever between 38 and 41 degrees Celsius.
 Mental retardation developed from this disease usually leads
to coma.
 Mortality varies but is much higher in children.
 Life-long neurological defects such as deafness, emotional
lability and hemiparesis (partial paralysis) may occur in those
who have had central nervous system involvement.

JEV - Diagnosis
 Serological
• Detection of viral antigen
• IFA
• Detection of specific IgM
antibodies from serum
cerebrospinal fluid
 Genetic:
• RT-PCR

JE treatment
 There is no specific treatment,
 Only supportive such as control of:
• Breathing,
• Seizures and
• Intracranial pressure.

JE - Prevention
 Vaccines that protect are available
 Avoid being bitten by mosquitoes
 Control of mosquitoes
 However, the prevalence in Singapore is very low and
routine vaccination is not generally recommended

• Epidemiology
• All zoonoses; natural reservoir in birds, small animals
• Humans are accidental dead-end host, do not develop
sufficient viremia to
transmit to arthropod
vector
• LaCrosse virus infects
Aedes mosquitoes
directly by semen or
via feeding on blood
• West Nile virus introduced
in NY in summer of 1999,
spread by migrating birds
Per 100,000 population
0.00
0.01–0.24
0.25–0.49
0.50–0.99
1.00
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
www.cdc.gov/mmwr/preview/mmwrhtml/mm534a4.htm

 Poliomyelitis
• Similar to meningitis: headache, fever, stiff neck, nausea
• Muscle pain and spasm, followed by paralysis
• Muscles shrink, bones do not develop normally
• In severe cases, respiratory muscles paralyzed
– Artificial respirator must be used
– Survivors recover some function
• Survivors may develop post-polio syndrome
– Muscle pain, weakness, muscle degeneration 15–50
years post-recovery
– Thought to be secondary effect of initial damage
– During recovery, surviving nerve cells branch out, take
over functions; probably ultimately die from extra work

 Poliomyelitis (continued…)
• Causative Agent
• Three types of polioviruses named 1, 2, and 3
• Non-enveloped, Baltimore group IV
• Members of enterovirus subgroup of picornavirus family
• Pathogenesis
• Enter body orally, infect throat and intestinal tract,
invade bloodstream
• Symptoms usually mild; immune system conquers
• Rarely, virus binds to specific receptors on motor
neurons, replicates, destroys cells during release
• Mostly asymptomatic
– presence of a single case means the virus is rampant

• Epidemiology
• Endemic where sanitation is poor; fecal-oral transmission
• Newborns partially protected for 2–3 months; usually
exposed during this time, develop mild infections
– Develop lifelong immunity from maternal antibodies
• Most devastating in countries with good sanitation
– Disappears, cannot spread
– When reintroduced, high
incidence of paralysis results
due to lack of immunity
– Occurred in U.S. in 1950s
– Most now routinely vaccinated

• No treatment; supportive care, ventilator if required
• Polioviruses stable under natural conditions, can be found in
swimming pools
• Inactivated by pasteurization,
chlorination of drinking water
• Control using vaccines is
great success story
• Ironically, all cases of paralytic
polio in U.S. since 1980 from
Sabin’s oral attenuated
vaccine introduced in 1961
– In rare cases, virus
mutates, becomes virulent
0
1951 1955 1960 1965
Year
1970 1975 1980 1985 1990
0.001
Casesper100,000population
Inactivated
virus (Salk)
vaccine
Attenuated
oral (Sabin)
vaccine
0.01
0.1
1
10
100 20
15
10
5
2005
Year
200019951990
Casesreported
CDC, 1993, 1999. Summary of Notifiable Diseases, United States, 1992, 1998. MMWR 41 (55): 46, 47(53): 54

• Small risk (~1 in 2.4 million doses) of polio from Sabin vaccine
– led U.S. to return to use of inactivated Salk vaccine in 1999
• Sabin vaccine remains key
where wild virus occurs
– Better immunity
– Person to person
– No injection
– Herd immunity
– Less expensive
• Global eradication efforts:
today only found in Afghanistan,
Nigeria, Pakistan

 Rabies
• Fever, head and muscle aches, sore throat, fatigue,
nausea
• Tingling or twitching sensation at site of viral entry
• Symptoms appear 1–2 months after infection
• Progress rapidly to agitation, confusion, hallucinations,
seizures, increased sensitivity, encephalitis
• Later stages characterized by increased salivation,
difficulty swallowing, causing “frothing at the mouth”
– Swallowing, even site of fluids, leads to severe spasms
• Within a few days, coma, death from respiratory failure or
cardiac arrest

 Rabies (continued…)
• Causative Agent
• Rabies virus, member
of rhabdovirus family
• Has striking bullet
shape, is enveloped,
contains Baltimore
group V.
• On single strand of RNA
=> Mononegavirales
20 µm
75 nm
(a)
(b)
a: © Tektoff-BM/CNRI/SPL/Photo Researchers, Inc.; b: © Tobey Sanford

• Pathogenesis
• Multiplies in cells at site of infection for several weeks
• Enters sensory neuron, travels by retrograde transport up
axon to spinal cord, eventually to brain
– Location of bite, infectious dose, host condition impact
length of time before symptoms appear
• Virus multiplies in brain tissue extensively, causes
symptoms of encephalitis
– Characteristic Negri bodies found in most cases
• Virus spreads outward via nerves to various body tissues,
notably salivary glands, eyes, fatty tissue under skin, heart,
other vital organs
• Can be diagnosed before death by identifying virus in smears
collected from surface of eyes

• Epidemiology
• Transmission via saliva from bite of rabid animal
– Inhalation of aerosols containing virus (e.g., from bat
feces) has been reported but not documented
• Main reservoir in U.S. is wild animals such as raccoons,
bats, skunks, and foxes
– >5,000 wild animal cases/year
– Raccoons top list
– Nearly all human cases from bats
– Domestic dogs most host in
Southeast Asia
– Only 1–3 cases/year in U.S.

• Epidemiology
• 40,000–70,000 deaths globally
• Most from dog bites in areas without regular vaccination
– 30% risk of developing rabies from bite if virus in
saliva
– Most rabid dogs excrete in saliva, sometimes even a few
days before onset of symptoms
• Person bitten by animal should wash wound thoroughly with
soap and water, then apply antiseptic
• If animal might be rabid, anti-rabies antibody is injected
– Then four injections of inactivated vaccine
– Provokes immune response that neutralizes free virus, kills
infected cells during long incubation period

• No effective treatment once symptoms appear
– Only two known to have recovered without vaccine
• In U.S., ~30,000/year receive
vaccine after bite from animal
suspected of having rabies
• Vaccination of dogs, cats in
U.S. highly successful
• DNA vaccine developed, may
help vaccinate dogs in
developing countries
• Programs to administer
oral vaccine to wild animals

26.6. Diseases Caused by Prions
 Spongiform encephalopathies
• Named after spongy appearance of affected brain tissue
• Chronic, degenerative brain diseases caused by prions
• Found in wild animals (mink, elk, deer), domestic animals
(sheep, goats, cattle) and humans
• Scrapie: sheep, goats have difficulty standing, “scrape” along
fences for support
• Cattle, presumably fed meat, bone meal from infected sheep,
developed bovine spongiform
encephalopathy, or
“mad cow disease”
• Outbreak of spongiform
encephalopathy in humans
in UK tied to earlier outbreak
of mad cow disease Normal Infected
© BSIP/Photo Researchers

 Transmissible Spongiform Encephalopathy in
Humans (TSE)
• Rare, occurs in only 0.5–1 case per million
• Most are Creutzfeldt-Jakob disease (CJD)
– Affects individuals over 45, sometimes runs in families
– Disease acquired by eating affected animals is different,
but has same result; is variant of CJD, vCJD
• Another TSE, kuru, is linked to cannibalism in New Guinea
• Vague behavioral changes, anxiety, insomnia, fatigue
• Progress to characteristic muscle jerks, lack of
coordination, memory loss, dementia
• Incubation period may last years; once symptoms appear,
death generally occurs within a year

 Transmissible Spongiform Ecephalopathy in
Humans (TSE) (continued…)
• Causative Agent
• Proteinaceous infectious particles, or prions (PrP)
• Misfolded form of normal cellular protein (PrPC)
– PrP encoded by normal gene, modified after
translation; misfolding results in protein that is
protease-resistant; normal is protease-sensitive
• Pathogenesis
• PrP acts as template, promotes misfolding of normal
PrPC on surface of neurons, which aggregate in plaques in
brain
• May be taken up by neurons or phagocytic cells
• Death of neurons produces spongy appearance
• Prions differ in range, incubation, areas of CNS attacked

 Transmissible Spongiform Ecephalopathy in Humans
(TSE) (continued…)
• Epidemiology
• CJD generally occurs in individuals over 45
• Has been transmitted human to human through corneal
transplants, contaminated surgical instruments, injections
of human hormone replacements
• Can be experimentally transmitted to chimpanzees
• Scrapie known for more than two centuries
– No evidence of transmission to humans
• Cattle prions can be transmitted to humans, cause vCJD
– vCJD differs from CJD in symptoms, brain pathology,
age of onset

 Transmissible Spongiform Ecephalopathy in
Humans (TSE) (cont…)
• No treatment; always fatal
• Important to avoid eating
any animals that show
neurological symptoms
• Prions highly resistant to
disinfectants including
formaldehyde
• Resistant to heat, UV,
ionizing radiation
• Can be inactivated by
extended autoclaving in
1M NaOH

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