Emerging Viral Pathogens The Nipah Virus Experience.
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Transcript of Emerging Viral Pathogens The Nipah Virus Experience.
Emerging Viral Pathogens
The Nipah Virus Experience
9/98First cluster of patients with acute febrile encephalitis.Outbreak preceded by occurrence of respiratory illness and encephalitis in pigsInitial detection of JE-specific IgM led to suspicion of Japanese encephalitisvirus as causal agent
2/99Disease spread south to Nipah.3/99Cluster 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, non-
segmented 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(?)
MononegaviralesMononegavirales
FiloviridaeFiloviridae
ParamyxoviridaeParamyxoviridae BornaviridaeBornaviridae RhabdoviridaeRhabdoviridae
FamilyFamily
OrderOrder
ParamyxovirinaeParamyxovirinae PneumovirinaePneumovirinae
RespiroviusRespirovius MorbillivirusMorbillivirus HenipahvirusHenipahvirusPneumovirusPneumovirus MetapneumovirusMetapneumovirus
GenusGenus
(Newcastle (Newcastle disease virus)disease virus)
(Measles)(Measles) (Nipah virus)(Nipah virus)
(Hendra virus)(Hendra virus)
SpecieSpecies
Sub-familySub-family
RublavirusRublavirus
(Mumps)(Mumps)
Nipah Virus GenomeNipah Virus Genome
3’ Leader 5’ Trailer
N P M F G L
Intergenic region
3’ and 5’ UTR**
~18 kb1.6 kb 2.2 kb 6.8 kb
Multiple open reading frames
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)
NH2
COOH
F2 F1
TM
F0 anti-AU1
NH2
TM
TM
COOH
anti-AU1 ExtracellularIntracellular
*** **
*******
F
G
anti-F2
anti-G
G1G2G3G4G5G6G7
F5F4F3F2F1
NiV also infects
NeuronsSmooth Muscle Cells(surrounding small arteries)
Fusion of ectodomain of NiV-Gopt allows for immunoadhesin that binds to NiV receptor
020406080100
AA BB
4
103
102
101
100
10
70
60
50
40
30
20
10
0
4
103
102
101
100
10
70
60
50
40
30
20
10
0
4
103
102
101
100
10
80
70
60
50
40
30
20
10
0
4
103
102
101
100
10
70
60
50
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30
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10
0
293T293T
HeLaHeLa
PK13PK13
VeroVero
FcFcFcFc--NiVNiV-G-GNoNoTreatmentTreatment
P = 0.45P = 0.45P = 0.01P = 0.01
Nuclei in Nuclei in
syncytiasyncytia
//
100X field100X field
Cell numberCell number
Fluorescence IntensityFluorescence Intensity
Fusion
Permissive
Permissive
Permissive
Non-Permissive
Fc-NiV-G blocksfusion mediatedby NiV F&G
NiV-Gecto
COOH NH2
TM
Extracellular Intracellular
******* GG1G2G3G4G5G6G7
ectodomain
huIgG1-Fc
Fc-NiV-G can IP cognate NiV receptor
• Biotinylate cell surface proteins
• Pre-clear with Fc-only coated Protein-G Dynal beads
• IP pre-cleared supernatant with Fc-only construct or Fc-NiV-G
• Blot IPed lysate with Streptavidin-HRP
Fc-only Pre-clear
Fc-only IP
Fc-NiV-G IP
64
48
kDa
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)
All RNA viruses
Other “exotic” emerging viruses (hemorrhagic fevers)