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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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)