Nipah virus (1998/99), Hendra virus (1994) (Henipavirus, Paramyxoviridae)

Disease in humans, pigs and horses. Virus transmitted from Pteropus bats to humans by contaminated sap, from pigs or horses.

Up to 90% C/F, no treatment, no vaccine for NiV HeV vaccine for horses NiV disease in humans during the outbreak in Malaysia: primarily encephalitis (40% cases pulmonary involvement) epidemic in humans masked by Japanese encephalitis outbreak 40% C/F; handling of pigs a critical factor

NiV disease in pigs highly contagious (100%) “general clinical signs” - respiratory, some CNS signs mortality during the outbreak max.5%

NiV disease in humans in Bangladesh/India outbreaks: Primarily pulmonary, high % of encephalitis 70% C/F drinking contaminated date palm sap and fruit critical factor

Human HeV disease: flue-like symptoms respiratory and renal failure, C/F 50% relapsing encephalitic disease

Current veterinarian cases 2008/2009/2010 – vaccine for horses

Acute equine respiratory syndrome two days from the onset of the disease to death

Other species susceptible to henipaviruses cats, dogs, goats Nipah virus, Hendra virus, Cedar virus (Henipavirus, Paramyxoviridae)

Negative single strand RNA viruses, nonsegmented genome (15 – 18kb), enveloped

5’ L G F M P/V/W/C N 3’

Equine morbillivirus – Morbillivirus (measles virus) –most related virion proteins (Mononegavirales, Paramyxoviridae, Henipavirus)

L - “polymerase” P - polymerase associated protein (V/W/C) G - glycoprotein F - fusion protein N - nucleocapsid protein M - matrix protein

N+ P+ L + RNA

Wang et al., 2001 Life Virus is genetically conserved Domain

ONE serotype Kingdom

NiV two genotypes, HeV one Phylum

Class NiV and HeV use same receptor, & are antigenically cross-reacting Order

Family

Henipaviruses infect Genus different orders of mammals (Chiroptera, Artiodactyla, Carnivora Species Perissodactyla, and Primates. Nipah virus replication cycle

Virus entry and uncoating Virion assembly, virus egress

Protein processing Virus attachment and entry

Receptor: ephrin B2 and ephrin B3 – G protein (attachment protein) highly conserved among species

Ephrin B2 contributes to signalling events during inflammation, angiogenesis, etc. endothelial cells, epithelial cell (incl. olfactory and respiratory ) immune cells (lymphocytes, monocytes, macrophages, osteoclasts, dendritic cells)

Ephrin B3 Neurons ( brain, primary sensory neurons) Syncytial respiratory epithelial cells (HeV) staining for HeV antigen

F (fusion) protein: Virus envelope – cell membranes fusion Cell - cell fusion

Syncytia

syncytial cells a hallmark of paramyxovirus infection

syncytial macrophage in lung (bronchiole), attenuation and necrosis of the epithelium Nipah virus replication cycle Virus entry and uncoating

Receptor: ephrin B2, ephrin B3

Macropinocytosis

Receptor mediated endocytosis (caveolae; clathrin coated vesicles?)

Fusion with cell membrane Mitogen Activated Protein Kinases (MAPK)

NiV ephrin Eph4

Phosphorylation of ERK1/2

MRC5, IPAM Urushihhara & Kinoshita, 2011 Early activation of ERK pathways in NiV infected cell lines 15 30 1h MR 15 30 1h Immediate C5 ST

IPA M Delayed ERK (two stage - ERK signalling through nucleus) macropinocyctosis pH independent endocytosis fusion Nipah virus replication cycle

Primary transcription (RdRp) independent of cellular factors Translation, Transcription Replication in cytoplasm

RNP: RNA, L, N, P RNA synthesis: L+P requirement, RNA has to be encapsidated (N) Sequential transcription: STOP - START

End gene – Intergenic region – Start gene

In 20-30% polymerase falls of the template: most abundant N mRNA mRNA is capped (L) and polyadenylated

5’ L G F M P/V/W/C N 3’ NiV C, V and W proteins – co-transcriptional editing (RNA editing)

Results in proteins that have common N terminal and unique C terminals

Rodriguez and Horvath, 2004. Host Evasion by Emerging Paramyxoviruses: Hendra Virus and Nipah Virus V Proteins Inhibit Interferon Signalling. Viral Immunology. Vol 17:210-219. Protein translation

Structural proteins: F0, G, N, P, L, M Nonstructural proteins: V, W, C

Secondary transcription (N, P, L) mRNA cRNA, or replicative intermediate (RI)

Switch from transcription to replication (N?) Nipah virus replication cycle

Protein processing

Glycosylation of the F0 and G proteins in Golgi apparatus Phosphorylation of the P (and N protein)

Cleavage of the F0 into functional F (F1-F2)

F0 migrates to a plasma membrane, and is re-internalized by Endocytosis. In the early endosome Cathepsin L/B cleaves

the F0 into functional F1/2 NiV activates the p38 MAPK pathway, and needs phosphorylation of MAPK p38 for replication:

Required for viral protein phosphorylation?

1.0E+07

1.0E+06 MRC5

ST

1.0E+05 IPAM

Viral Viral titres PFU/ml NiV 1.0E+04

1.0E+03 12 24 48 hours post inoculation

Comparison of viral titres (PFU/ml) in NiV infected human and porcine cell lines, with the addition of p38 MAPK inhibitor at (A) 25 µM and (B) 50 µM concentrations. Nipah virus replication cycle Virion assembly, virus egress

Although G and F1/2 localize to the cell Plasma membrane

Assembly driven by the M protein – recruits RNP

In polarized cells: Infection via apical and basal membranes Budding via the apical membrane

Immunogold label – N protein exposure viremia

Infection of polarized epithelial cells (respiratory)

shedding (OR into the lumen)

Driven by the crossing the epithelial barrier - viremia, infection M (matrix) protein of underlying cells

Pathogenesis

wildlife reservoir flying foxes (fruit bats) Pteropus spp. no disease, no pathology, 47% seroprevalence Virus shedding associated with stress Disease in humans Original outbreak: Malaysia 265 encephalitic cases, 105 fatal fever, sever headache, myalgia, signs of encephalitis or meningitis handling of pigs a critical factor

Subsequent: India and Bangladesh 70% case fatality ratio Fatal encephalitis Infections linked to contaminated sap and fruit Human to human transmission

Other species cats, dogs, horses, goats

Photos courtesy of Dr. S. Luby Acute Respiratory Distress Syndrome

Male, 40 yrs, day 6 of Male, 35 yrs, 5th day of illness, died 2 days later illness, died next day

Hossain MJ, et al Clin Inf Dis. 2008;46(5);977-84.

Pathogenesis

NiV disease in pigs in the Malaysian outbreak (porcine respiratory and encephalitic syndrome)

Common: invasion of CNS and lung, viremia Different: direct invasion of brain via cranial nerves transient immunosuppression

(secondary bacterial infections) KT Wong

Human brain

Neuron Staining in a gray m. Glial cell Oro-nasal cavity Virus antigen detected in: olfactory and respiratory epithelial cells endothelial and perithelial cells cranial nerves U of Guelph (primary sensory neurons) immune cells (lymphocytes, monocytes, macrophages, osteoclasts) Inhalation/ingestion Oro-nasal cavity (mucosal exposure) Cranial nerves

Olfactory and respiratory epithelial cells Cranial nerves (primary sensory neurons) Cells of the immune system Endothelial and perithelial cells (smooth muscle cells of tunica media)

Viremia (cell-associated/ cell-free)

Small blood vessels and lymphatic vessels in different organs (endothelial cells and smooth muscle cells) > Vasculitis and fibrinoid necrosis Lung (trachea) Brain Pathogenesis Lymhoid organs and lymphoid tissues human infections (swine) (other organs)

Parenchymal and epithelial cells, endothelial and perithelial cells, resident and infiltrating/transmigrating immune cells Direct invasion of CNS via cranial nerves

Trigeminal nerve Glial cells in the brain and neurons)

Olfactory nerve (axon, nasal turbinates Olfactory bulb (brain; neurons)

J. Neufeld, S. Czub Invasion of CNS due to viremia

Detection of virus by real-time RT-PCR in serum and PBMC of NiV infected pigs Ependyma (lining of ventricles of the brain)

Figure 3

Blood vessels in the brain (endothelial cells) Meninges Inoculation of porcine peripheral blood mononuclear cells with NiV

productive infection monocytes

?

anti-NiV guinea pig polyclonal antibodies IPAM

Nipah (guinea pig polyclonal antibody) CD6+ sorted T cells stained intracellularly with NiV- Nucleocapsid (N) antigen at 48 hpi

Confirmed by Intracellular staining for NiV non-structural C protein

NiV yield in supernatants of sorted

7000 T cells at 24 hpi

6000

5000

4000 pfu/ml 3000

2000

1000

0 T cells T CD4+ T CD8+ Henipaviruses target endothelial cells of SMALL blood vessels, preferentially in brain and in lungs

T cells CD6 marker strong ligand for ALCAM (CD166) Activated leukocyte cell adhesion molecule Henipaviruses target immune cells

Passive vehicle for dissemination (human) = viremia

Infection of a specific subtypes of immune cells (= viremia + transient immune suppression in swine)

NiV infects and replicates in: porcine monocytes (likely macrophages and dendritic cells) NK cells CD8+ T lymphocytes (cytolytic T cells, CD8+ subset of gamma/delta T cells)

CD8+ CD4+ T lymphocytes cytolytic and memory helper T cells (located in large portion in lymph nodes and tonsils of pigs)

Lymphoreticular system: lymph nodes (SM) and spleen, tonsils -lymphocytes and macrophages/dendritic cells surrounding blood vessels

Normal lymph node

Lymphocyte necrosis

Lymphocyte depletion

endothelial cells blood/lymphatic vessels and follicular cells – lymph nodes 4 weeks post inoculation with NiV Changes in population frequencies of CD8+ and CD4+ following NiV inoculation of PBMC

CD8+ CD4+ A & D PBMC controls

CD8+ CD4+ B & E 24hrs pi

CD8+ CD4+ C & F 48 hrs pi Comparison of T cell subpopulations between pigs that died during acute infection versus survivors

Survivor

Died CD4+ T lymphocytes (helper T cells) Bystander death? Population frequency appear resistant to NiV infection Decreased (life/death)

B lymphocytes, CD4-CD8- T lymphocytes (CD8- gamma/delta cells) are resistant to NiV infection, and not reduced during NiV infection

Humoral response appears to be functional (but delayed?)

1400

1200 influenza 1000 Recovery 800

600

400

200

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Swine Inhalation/ingestion Cranial nerves Oronasal cavity

Viremia Encephalitis/meningitis “early” and “late” Damage of the vascular system Infection of immune cells - necrosis (transmigration = Lymphoid organs Infiltration/recruitment) organs/ specific tissues

Lung (respiratory disease) Transient immunosuppression/ immunomodulation

Secondary bacterial infections Pathogenesis – underlying mechanisms

Henipaviruses target IFN-type I system:

IFN induction (downregulation of expression of IFN-alpha, BLOCK?) IFN signalling (interference with STAT1 signalling pathway; human) Partial block of the antiviral state (human >> swine) (establishment of the antiviral state in human cells requires higher dose of IFN-alpha)

Impaired immune cell signalling NiV C, V and W proteins – co-transcriptional editing (RNA editing)

Shaw, M.L., et al., 2004. Nipah Virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively.

Sites of phosphorylation – Ser 240 and Ser 472

P58 Mab binding site (211-221 aa) Common N Editing Site (1325nt) terminal to P/V/W

N 5’ P 3’ C Terminal Terminal STAT1 binding site (50 to 150 aa) Unique C terminal +2G +1 Reading of NiV P Frame +1G

5’ C 3’ 5’ V 3’ 5’ W 3’ Begins at nt 128 downstream from the P Unique C terminal Unique C terminal reading frame of NiV V of NiV W Pathogenesis – underlying mechanisms Evade host defences (stage I – cellular): V – W - (P) proteins – STAT1 • V protein localizes to the cytoplasm • W localizes to the P P nucleus STAT2 STAT1 V

• Both proteins bind

X X

to STAT 1 and interrupt with the protein shuttling back and forth W

STAT2 STAT1 P P Evasion of antiviral state and immune cell signalling

Shaw, M.L., Garcia-Sastre, A., Palese, P., Baster, C.F. 2004. Nipah Virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, Respectively. Journal of Virology. Vol. 78: 5633-5641 work with recombinant proteins V and W; confirmed for infected cells (W does not translocate into nucleus in all types of cells during in vitro infections )

MRC5 ST

MRC5 ST 100 100

75 75 50 50

37 MW V C NM N U UC UN 37 MW V L C NM N U UC UN

Figure 2 (A) Co-localization of STAT1 and NiV P/V/W proteins within MRC5 and ST cell line infected with NiV 24 hrs post infection at an MOI of 0.1. Fold change in p-eIF2α / total eIF2α

hours post inoculation

ST MRC5 p-eIF2α p-eIF2α 6 8 12 24 48 M + 6 8 12 24 48 M + total eIF2α total eIF2α

IPAM p-eIF2α 6 8 12 24 48 M + Phosphorylation of eIF2α in cells total eIF2α infected with NiV - indication of establishment of antiviral state alternative IFN signalling pathway?

Mitogen Activated Protein Kinase (MAPK) signalling

Virus infection

Antiviral state Fold in p-p38 – total p38 1 3 6 12 24 48 M 48 24 12 6 3 1 change IPAM ST 1 3 6 12 24 48 M 48 24 12 6 3 1

hours post inoculation p

- Total p38 Total p38

Total p38 Total p - p38

in NiV infected cells Phosphorylation p38 of

MRC5 1 3 6 12 24 48 M M 48 24 12 6 3 1

Total p38 Total p

- p38 MRC5 IPAM ST

Cells have capability to establish antiviral state

120 Exogeneous IFN-alpha 100 induced antiviral state against NiV 80 (compared to VSV) SJPL/VSV 60 SJPL/NiV MRC5/NiV

40

20

0 10 20 40 80 160 320U

Cells treated with human or porcine IFN-alpha, respectively, for 24hrs prior to virus challenge IFN induction PBMC harvested from compromised? NiV infected pigs: ELISA no upregulation of IFN-alpha

3 3 Relative cytokine 2.5 2.5 mRNA expression 2 2 in T lymphocytes

1.5 1.5

1 1

0.5 0.5

0 0 INF alpha INF TNF alpha IL-8 INF INF TNF IL-8 gamma alpha gamma alpha Mitogen induced Mitogen Induced T cells and NiV infected T cells Virus entry – first step in evasion of immune response? in porcine dendritic cells and plasmacytoid dendritic cells ?

macropinocytosis

? pH-independent fusion endocytosis

Enhancement of early endosomes Block of recognition by Toll like receptors? Gerber et al., 2002 New disease agent

Laboratory characterization of the agent (virus)

Zoonotic?

Mode of transmission

Source of the agent Proving a causal relationship [virus] (reservoir) between the agent/ virus and the disease 1840: dr. Henle Koch’s Postulates

Organism is associated with disease if: 1. The organism is present in the lesions 2. Can be isolated from the clinical cases 3. Inoculation with the organism causes similar disease in the host 4. Organism can be recovered from the experimentally infected animals

Basic questions: virus replicates in the experimental host infection causes clinical disease shedding – transmission detection –development and evaluation of diagnostic tools vaccine efficacy testing: (vaccine development) development of a challenge model pathogenesis