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?