Transcript Isolation of Emerging Viruses

Emerging Viral Pathogens
The Nipah Virus Experience
9/98
First cluster of patients
with acute febrile encephalitis.
Outbreak preceded by occurrence
of respiratory illness and
encephalitis in pigs
Initial detection of JE-specific IgM
led to suspicion of Japanese
encephalitisvirus as causal agent
2/99
Disease spread south to Nipah.
3/99
Cluster of 11 human cases of
respiratory and encephalitic illness
in abbatoir workers in Singapore,
but only in those who handled pigs from outbreak
regions in Malaysia
Final Toll: 265 human cases of acute encephalitis, 105 deaths
(~40% mortality rate)
Culling of > 1 million pigs
Nipah Virus
• Novel paramyxovirus
– Negative sense, nonsegmented RNA virus
• Natural host are fruit bats
– Bats urinate on pigs, pigs
urinate on humans
– Virus can be isolated from
urine of wild-free-roaming
fruit bats,
– serological evidence of
Nipah virus infection in
many animal species
• Isolation of Emerging
Viruses
– Vero Cells
• African Green Monkey
Kidney Fibroblast Cells
• Especially susceptible to
the cytopathic effect of
many viruses
• Spontaneous gene
deletions leading to lack of
interferon response; make
cells more permissive for
virus growth
Nipah Virus:
Epidemiological features
• Mortality in pigs is only 5% but transmission is 100%
• Mortality in humans is 40%, but no reported case of
nosocomial transmission (human to human)
transmission in healthcare workers) in 1st outbreak
• Strong evidence of human-to-human transmission in
Bangladesh outbreaks (2004); mortality rate is up to
70%
• Transmission is attributed to direct contact with
excretions and secretions (urine, saliva, pharyngeal and
lung secretions)
• Mechanical transmission to dogs and cats(?)
Order
Mononegavirales
Family
Filoviridae
Paramyxoviridae Bornaviridae
Rhabdoviridae
Sub-family
Paramyxovirinae
Pneumovirinae
Genus
Rublavirus Respirovius Morbillivirus Henipahvirus
(Mumps)
(Newcastle
(Measles)
disease virus)
Pneumovirus
(Nipah virus)
(Hendra virus)
Metapneumovirus
Species
Nipah Virus Genome
3’ and 5’
UTR**
3’ Leader
N
P
1.6 kb
2.2 kb
Intergenic
region
M
5’ Trailer
F
~18 kb
Multiple open reading frames
G
L
6.8 kb
Bioterrorism Concerns
•
•
•
•
•
•
•
Extreme pathogenicity (40%); latest outbreak in Bangladesh (April,
2004) has mortality rates up to 74% (similar to smallpox-30% and
Ebola-40-90%)
3-7% experience late or relapsed encephalitis; increased
community exposure
No effective anti-virals, limited diagnostic capability
Paramyxoviruses can be grown to high titers in vitro (1011 IU/ml)
without concentration
Aerosolization of other paramyxoviruses has been demonstrated
Symptoms take a week or two to develop during which time,
asymptomatic carriers can be infectious
Prodromes of fever, headaches, myalgia (muscle ache), dizziness,
areflexia, hypotonia etc. are relatively non-specific and not as
dramatic as those caused by viral hemorraghic fevers (e.g. Ebola)
Economic Bioterrorism
•
NiV outbreak in Malaysia (1999)
– 265 affected individuals
– > 1 million pigs were culled (military operation)
– economic losses totaled far more than their export value of US$100 million
•
In U.S.A.
–
–
–
•
Production value of hogs/pigs in 2002, ~US$8.6 billion
Farms in just 3 states (Iowa, Minnesota, North Carolina) accounts for 50% of value (>$4 billion)
If Nipah-like agents released in any one of those States, loss of production alone could cost
more than $1 billion
Needed:
–
–
–
Effective vaccine compatible with goals of efficient animal husbandry
Vaccine that can protect animals and handlers
Better understanding of pathogenesis of disease
Classsical
“herringbone”
morphology of
paramyxoviral
nucleocapsid
Virus Emergence
(El Nino)
Nipah Virus (BSL-4 ):
Category C Priority Pathogen
•
•
40%-74% mortality from fatal
encephalitis
Pathognomonic features: Endothelia
syncytia formation
– Mediated by envelope glycoproteins
(F and G)
anti-F2
NH2
F2
F1 F3
F4 F5
** *
**
COOH
G6
* *
COOH
F
F1
F2
G7
TM
anti-AU1
F0
G5 G4
G3
G2
* *
*
*
G1
*
NH2
TM
anti-AU1
Extracellular
anti-G
Intracellular
G
NiV also infects
Smooth Muscle Cells
(surrounding small arteries)
Neurons
Fusion of ectodomain of NiV-Gopt allows for
immunoadhesin that binds to NiV receptor
Fusion
70
293T
60
A
50
40
B
30
20
Permissive
10
P = 0.45
0
0
10
1
10
2
10
3
10
4
10
70
50
40
Permissive
30
Cell number
20
10
0
0
10
70
1
10
2
10
3
10
4
10
HeLa
60
50
100
80
60
40
20
0
40
30
No
Treatment
20
10
NonPermissive
Nuclei in syncytia/
syncytia /
100X field
P = 0.01
Vero
60
Permissive
Fc-NiV-G blocks
fusion mediated
by NiV F&G
0
0
10
80
70
60
50
40
30
1
10
2
10
3
10
Fc
FcFc-NiV-G
NiV-G
4
10
PK13
NiV-Gecto
20
10
0
0
10
1
10
2
10
3
10
ectodomain
4
G7
10
Fluorescence Intensity
COOH
G6
* *
G5 G4
G3
G2
* *
*
*
G1
*
NH 2
TM
huIgG1-Fc
Extracellular
Intrace llular
G
CHAPS
TX-100
CHAPS
TX-100
CHAPS
• Biotinylate cell surface
proteins
• Pre-clear with Fc-only coated
Protein-G Dynal beads
• IP pre-cleared supernatant
with Fc-only construct or FcNiV-G
• Blot IPed lysate with
Streptavidin-HRP
TX-100
Fc-NiV-G can IP cognate
NiV receptor
kDa
64
48
Fc-only
Pre-cle ar
Fc-only
IP
Fc-NiV-G
IP
Receptor identity must explain
NiV tropism
• Receptor is expressed on endothelial cells
and neurons and smooth muscle cells
surrounding small arteries
• (contrast and compare with CCR5 on
macrophages and CXCR4 on T-cell lines)
Other “exotic” emerging viruses (hemorrhagic fevers)
All RNA viruses