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Nipah virus ()

Zoonotic cross inter-species barrier

Hana Weingartl Adjunct professor, Medical Microbiology, University of Manitoba

Head, Special Pathogens Unit National Centre for Foreign Animal Disease Canadian Food Inspection Agency Canadian Science Centre for Human and Animal Health It is not in virus interest to kill the host /cell

Zoonotic viruses often cause diseases with high fatality rate in humans with short clinical phase - survival of a human host is irrelevant

Productive infections of multiple species, and successful intra- and inter- (optional) species transmission without a requirement for adaptation in a “new” host Interspecies barriers

geographical, ecological (Wallace’s line)

- Entry into the host cell: attachment to the host cell = interaction with a specific host cell receptor ability to enter the host cell to deliver the genetic information

- Replication within the host cell: interaction with cell factors (molecules) in the nucleus and/or cytoplasm virus needs to gain (partial) control over specific cell functions, such as metabolism and replication to produce progeny

host defence mechanisms Reservoir host

Amplifying host

Enzootic x Epizootic

Sporadic x Endemic x Epidemic x Pandemic

Dead-end host Goals of (zoonotic) viruses

1. Find/encounter a new host

2. Enter the cell by a pathway that ensures replication

3. Produce mRNA, replicate the , generate virus proteins, evade host defences – stage I

4. Assemble progeny virus, leave the cell

5. Evade host defences – stage II, re-infect other cells

6. Transfer to a new host or persist in the environment until 1. (Or stay within the host) Rift Valley fever virus (Phlebovirus, Bunyaviridae)

disease in domestic ruminants (sheep, goats, cattle, camels) and humans (transmission from mosquitoes or due to contact to blood and infected tissues)

Rift Valley fever first described in 1915, virus isolated in 1930 More then 10% Case/Fatality in humans, no treatment, no vaccine Recognition of RVF outbreak

Clinical signs in animals are nonspecific, epizootics usually recognized due to 90% mortality in newborn ruminants (calves, lambs), and abortions in adult animals Innaparently infected animals high viremia - transmitting species

accompanied by concurrent outbreak of febrile disease in humans: influenza-like >5% serious; >1% fatal (but could be 10 – 35%) high viremia - transmitting species (amplifying host) Rift Valley fever virus (Phlebovirus, Bunyaviridae)

• L segment • Transcriptase (RdRp) • M segment • Glycoprotein (Gn) • Glycoprotein (Gc) • Nonstructural NSm1 = large 78kDa glycoprotein • Nonstructural (NSm2) • S segment • Nucleocapsid (N) • Nonstructural (NSs)

Glycoprotein Gn/Gc heterodimers in lipid envelope (lipid composition) Genome (negative and ambisense coding strategy)

6404 Nt

3885 Nt

1690 Nt

(Flick and Bouloy, 2005)

Consensus terminal nucleotide sequences: 3’ UGUGUUUC- 5’ ACACAAAG- Segment reassortments occur in nature Life

Domain

Virus is genetically conserved Kingdom

Phylum 72 different strains/isolates – L segment – one real time RT-PCR assay Class

ONE serotype Order

Family Arthropode-borne virus (Arthropoda) infecting mammals (Chordata): Genus

ruminants, humans, etc. Species

RVF ENZOOTIC CYCLE RVF EPIZOOTIC CYCLE

1. Find/encounter a new host LIVESTOCK

EPIDEMIC CULICINE CULICINE MOSQUITOES, MOSQUITOES, BITING FLIES BITING FLIES

HUMANS

LIVESTOCK LIVESTOCK

AEDINE AEDINE AEDINE AEDINE MOSQUITOES MOSQUITOES MOSQUITOES MOSQUITO ES

EGGS EGGS

DAMBO DAMBO

R. Swanepoel 2. Enter the cell by a pathway that ensures replication

Virus attachment and entry, preferred target cell

Gn/Gc virus attachment proteins (high-mannose N-glycans on the viral glycoproteins produced in mosquitoes – DC-SIGN binding)

Receptor unknown, likely a protein dermal Dendritic Cells C-type-lectin (DC-SIGN) – attachment Virus entry and uncoating

Virus egress

RNA synthesis Virion assembly

?

Protein synthesis Protein processing

Virus entry and uncoating

Endocytosis Virus egress via non-coated (smooth surfaced) vesicles (2/3) and via clathrin-coated vesicles (1/3)

early endosomes dissociation from the receptor

RNA synthesis late endosomes (& lysosomes)Virion assembly - acidification pH dependent fusion Gc postulated as a fusion protein based on similarity to fusion proteins of viruses in and Togaviridae – or is it necessary to have the herodimer? ? Ribonucleoprotein, Protein synthesisRNP = RNA, L, NProtein processing uncoating

TubularVirus entry viral andfactories uncoating (Golgi connected to mitochondria and RER) connect replication with assembly Virus egress RNPs accumulate in Golgi and form tubular Golgi stacks Mammalian L (polymerase) Globular part of the stack = site of cell mRNA, cRNA + vRNA synthesis already in forms of S, M , L RNP

RNA synthesis Virion assembly

?

Protein processing Virus entry and uncoating RNA synthesis only within RNP = ribonucleocapsids (requires RNA, L and N proteins) Virus egress

L polymerase protein (RNA dependent RNA polymerase; RdRp):

Endonuclease activity cap snatching from host mRNA (10-18 Nt primers) = primed mRNA synthesis RNA synthesis mRNA does not have poly-A tail (transcription termination signal)

switch to replication (trigger unknown; encapsidation by N as antitermination signal?) priming not required for cRNA and vRNA synthesis Virus entry and uncoating First 10-13 Nt on the 3’end = genome promoters in vitro; (contain signals for mRNA and cRNA synthesis) requirement in vivo (cells) for RNA synthesis – aroundVirus 100 egress Nt, on both 3’ and 5’ ends of the RNA prime and re-align model (transcription and replication)?

NSs mRNA synthesis some antigenomes are encapsidated NSs primary mRNA synthesis RNA synthesis from the cRNA

Primary transcription (mRNA)

Protein synthesis required for genome replication and

Protein synthesis secondary transcription (and virion formation) Virus egress

Virion assembly

Golgi Protein processing

Tubular viral factories (Golgi connected to mitochondria and RER) connect replication with assembly Virus assembly

RVFV assembles in the Golgi apparatus Budding into the Golgi cisternae Transport to the cell membrane within vesicles similar to those in secretory pathways Exocytosis (lysis of mammalian cells, in insect cells the CPE is non-cytolytic)

(Gerrard and Nichol 2007) Pathogenesis Can mosquitoes clear the virus? DRAIANAGE/DRYING OR FLOODING OF DAMBOS: EFFECTS ON FLOODWATER-BREEDING AEDES MOSQUITOES If not: in long term, all the infected FLOODING LEVELS mosquitoes die & RVFV with them

POROUS SOIL

MOSQUITO EGGS

IMPERVIOUS LAYER

uninfected C6/36 mosquito cells

transovarial transmission (Aedes) – virus survives in eggs periods of draught up to several years; however drop in egg numbers and survival, and the overall reproductive fitness RVFV infected C6/36 mosquito cells Disease in humans Virus is genetically conserved incubation period: 2 – 6 days eye disease – 1 – 3 weeks after the first symptoms (retinal lesions; some permanent visual RVFV needs a loss (5%), death uncommon) mammalian host acute neurological disease - 1 – 3 weeks after the first symptoms (meningo-encephalitis; death uncommon) hemorrhagic fever – 2 – 4 days after the first symptoms (severe liver disease, jaundice, vomiting blood, passing blood in feces, purpuric rash (bleeding in the skin), bleeding from gums; patients viremic for 10 days; case-fatality rate 50%

Total case/fatality rate 1-3% Sheep and goat fever, inappetence, listlesness mucopurulent nasal discharge bloody diarrhea 90 - 100% of pregnant animals abort 90% mortality in lambs/kids within 36hrs after the onset of signs 20 - 60% mortality in adult animals

if the animals survive the hepatic infection, some of them develop encephalitis Cattle less severe clinical signs, dysgalactia 10 - 30% mortality in calves 90 - 100% of pregnant cows abort Camels high levels, viremia, 90-100% abortion rates some early post-natal deaths Infections in healthy (not pregnant) adult animals – innaparent sheep

Disease time course) and clinical manifestation

human

Bird et al, 2009 Calf (infected at 10 days of age), 28 dpi

Experimental Infection With RVFV

lamb, infected at 14 days of age, 2 dpi LESIONS IN LIVESTOCK

LIVER – NECROTIC FOCI HAEMORRHAGE INTO ABOMASUM/ INTESTINES

PATHOGNOMONIC LIVER NECROSIS WITH PRIMARY FOCI

LIVER – INTRANUCLEAR INCLUSIONS neglected tropical disease

Pathogenesis – not well known Underlying cellular mechanisms - not well known

evade host defences = switch from arthropode host to mammalian host

Other ()

Infection of dendritic cells with “insect” virions: no IFN- type I induction – facilitates first round of infection in the mammalian host (linked to a lipid composition of the “insect” virion)

This step occurs already prior to virus replication in the cell C6/36 * *

Vero E6

RVFV – in addition to insect cell derived envelope, large glycoprotein (Nsm1, LGp) in present in the virions Pathogenesis – molecular/cellular mechanisms

Non-structural protein NSs

For Bunyaviridae, unique nuclear localization of NSs in mammalian cells

Bouloy and Weber, 2010 Cellular DNA mostly excluded from the filaments BUT Nss interacts with pericentromeric gamma satelite sequences

chromosome cohesion and segregation defects

NSs expression linked to cell cycle arrest (G0/G1 or S)

fetal and newborn death? Nonstructural protein NSs

– Inhibits host cellular transcription, impact on production of interferon α/β, IFN signaling as well

NSs (has to be in form of filaments) interacts with SAP30 belongs to repressor complex intervening in regulation of gene transcription

Insect cells express significantly less of the NSs protein antiviral state(???) Nonstructural protein NSs – Inhibits host cellular transcription in general way

– NSs sequesters host cell protein p44 into the filaments - (subunit of TFIIH basal transcription factor) –TFIIH cannot assemble – targeted for degradation --- reduced transcription – TFIIH is involved in transcription activity of Pol II and Pol I NSs specifically targets antiviral dsRNA-dependent protein kinase (PKR) for degradation

RVFV protein synthesis NSs cellular protein synthesis impaired via interference with transcription

RVFV RNA PKR replication

Insect cells express significantly less of the NSs protein antiviral state(???) NSs Inhibits host cellular transcription in nucleus

Blocks production of Interferon

Targets host antiviral protein(s) for degradation Nonstructural protein NSm(2) Inhibits host caspase activation (MP12 x ZH501)

Blocks stimulation Suppresses apoptotic pathway of development and differentiation of embryonic cells, monocytes, NSm suppresses host Caspase-8 and -9, initiators of the extrinsic and intrinsic death T and B cells effector pathways

Caspase-8 deficiency may be less severe in humans because humans possess semi-redundant Caspase-10 10 RVFV immune response pattern for goats, 8 acute phase

6

4

2 Viremia Viremia 0 0 1 2 3 4 5 6

NSs

LGp DC NSs Immune cell differentiation NSm Reverse genetics: Forward genetics: gene physical trait (phenotype)

function (phenotype) gene Tools – systems in virology (may include specific cells) Recombinant viruses with directed deletion or point mutations

Recombinant expression of proteins

Gene silencing (RNA interference)

Interference using transgenes Alonso JM, Ecker JR (2006) Nature Reviews Genetics 7: 524 – 536 Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis