1. Emerging Viral Pathogens The Nipah Virus Experience

2. 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

3. 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

4. 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(?)

5. Mononegavirales FiloviridaeParamyxoviridaeBornaviridaeRhabdoviridae Family Order Paramyxovirinae Pneumovirinae RespiroviusMorbillivirusHenipahvirus PneumovirusMetapneumovirus Genus (Newcastle disease virus) (Measles) (Nipah virus) (Hendra virus) Specie Species Sub-family Rublavirus (Mumps)

6. Nipah Virus Genome 3’ Leader 5’ Trailer NPMFG L Intergenic region 3’ and 5’ UTR** ~18 kb 1.6 kb2.2 kb 6.8 kb Multiple open reading frames

7. 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 (10 11 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)

8. 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

9. Classsical “herringbone” morphology of paramyxoviral nucleocapsid

10. Virus Emergence (El Nino)

11. 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)

12. NiV also infects Neurons Smooth Muscle Cells (surrounding small arteries)

13. Fusion of ectodomain of NiV-Gopt allows for immunoadhesin that binds to NiV receptor Fusion Permissive Non- Permissive Fc-NiV-G blocks fusion mediated by NiV F&G NiV-G ecto ectodomain huIgG1-Fc

14. 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 64 48 kDa

15. 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)

16. All RNA viruses Other “exotic” emerging viruses (hemorrhagic fevers)