Flavivirus

Flavivirus

1. Replication

   • 1. Virus attachment to plasma membrane
     • Adhesion
   • 2. Entry to cytoplasm/nucleus
     • Endocytosis (clathrin)
     • Low pH/ fusion nucleocapsid release
   • 3. Un-coating of virus particle
     • Synthesis of polyprotein
     • Golgi associated protein cleavage (host and virus proteases)
     • Long protein formed, which is then cut up into smaller parts
   • 4. Synthesis of mRNA and protein
     • Genome replication via RNA intermediate
   • 5. Replication of nucleic acid
   • 6. Virus assembly
     • Budding from ER (peplomers)
   • 7. Exit of virus from cell
     • Maturation and exocytosis, need not be cytopathic
   • Louping ill virus replication
     • Receptors: sialic acid on glycolipids /gangliosides, found on a wide variety of cells including nerves
       • Hence can cause encephalitis
         • Encephalitis is a sudden onset inflammation of the brain
     • Virus enters and replicates well in a variety of mammalian and avian cell types.
       • Therefore disease not species restricted.
       • Pig = intermediate host
     • Double stranded RNA intermediates, potentially good interferon inducers.
     • Virus does not usually shut of host cell protein synthesis or only does so very late in replication cycle.
     • Virus does not lyse the host cell: exit by exocytosis.
     • Variably cytopathic to mammalian or avian cells
     • Non cytopathic in insect cells.
   • Bovine Viral Diarrhoea replication
     • Receptor CD46 (complement control protein)
       • Located on surface of most cells
     • Virus Polyprotein cleaved by Host protease and virus NSP2/3 protease
     • Does not shut down host protein synthesis
     • Blocks Type 1 interferon production
     • Non lytic infection with exocytosis
     • Most strains non cytopathic
     • Persistent infection of cell lines in vitro, cattle in vivo
     • Disease associated with conversion to cytopathic strain with NSP3 mutations
       • Will start to destroy other cells inside the cow
     • The N-terminal protease Npro catalyses its own separation (grey arrow)
       • Npro also binds to Interferon regulatory factor-3 (IRF3) leading to proteasomal degradation thereby inhibiting the transcription of IFN.
         • Mechanism inside cell blocking interferon production
     • Structural glycoprotein protein Erns (rns = RNAse) is released from infected cells
       • Intrinsic RNase activity - degrades extracellular viral ss-and dsRNA
         • Prevents activation of interferon responses by TLR3 in the endosome of plasmacytoid dendritic cells.
           • Suppress monocytes response to anything else – blocks interferon response and causes animal to be susceptible to other diseases/viruses

2. Characteristics

   • Genome
     • Flaviviridae small +ve RNA virus
   • Structure
     • Spherical capsid (icosahedral symmetry)
       • Surrounded by a tight lipid envelope which has indistinct peplomeres
     • Double layer – has an envelope
   • Genus: Flavivirus
     • (Arboviruses – mosquitoes in the tropics and ticks in temperate areas)

3. Louping ill

   • Pathogenesis - infection from bite of carrier tick
     • Replication at site of entry in leucocytes/macrophages DCs
     • Spread to local lymph node where more cells are infected
     • 1o Fever associated with interferon response and viraemia spread to other lymphoid organs
     • Further replication and continuing prolonged viraemia (not a true persistent infection).
     • Opportunity for infection of ticks (extended duration of feeding)
     • In some animals prolonged viraemia allows spread to CNS and onset of neurological signs.
       • 2o fever (ataxia, tremors, louping, paralysis, death)
       • 1. Blood: viraemia and infected lymphoid cells Virus attached to RBCs
       • 2. Infection of endothelium
       • 3. Infection of neurones microglia astrocytes: Death of nerve cells Pass across endothelial cells + BBB → binds to gangliocytes (high levels in neurones)
     • Perivascular cuffing: infiltration of lymphocytes, monocytes and dendritic cells across inflamed endothelium
       • Stained for macrophage /dendritic cells
       • Virus encephalitis – leukocytes clumping around the edge of blood vessels
     • Cerebellum: Purkinje cells infected with Louping Ill
       • Stained for virus antigen
   • Epidemiology
     • Tick borne disease: Ixodes ricinus
     • Reservoir hosts many birds and small mammals
     • Red grouse, Voles, Hares etc. with sporadic epidemics in livestock and humans
       • Moorland – can be quite pathogenic in grouse
     • Can infect sheep, goats, cattle, deer, horses
     • Louping ill virus in Ticks (non pathogenic)
     • Virus first acquired by blood feeding on viraemic host
   • Immunology
     • More severe disease associated with Immunosuppression by Ehrlichia Phagocytophillia (cow infection)
     • Rise in Antibody titre prior to 2o vireamia is protective
     • Lambs are protected by maternal antibodies; most disease seen in sheep from 6 months -2yrs of age
       • Born in spring – high tick burden, protected by maternal antibodies
       • By next spring – turned back out on the moor – then develop disease

4. Pestivirus: Bovine Viral Diarrhoea/mucosal disease

   • Infection of pregnant cows
     • (by non-cytopathic strain)
     • Trans-placental infection of foetus
       • 1st 30 days early embryonic death
       • Cow mounts an immune response and clears the virus
     • 30-90 days embryonic/ foetal death
       • Cow mounts an immune response and clears the virus
     • Trans placental infection > 125 days
       • Cow and calf mount immune response and calf reaches term
       • Some Foetal defects may occur, retinal dysplasia, cerebellar hypoplasia, cerebral cavitation low birth weight, neonatal death.
     • 90-120 days gestation
       • Infection during a critical period of calf’s immune system development
         • Learning what is self and what is non self
       • Cow mounts immune response and clears virus
       • Calf survives, but is immuno-tolerant to virus
       • Lifelong persistent infection with no antibodies or immune response
   • Mucosal disease
     • Animal that have become persistently infected (tolerant) with a non cytopathic strain may go on to develop mucosal disease
       • This is associated with the appearance of cytopathic variants of the virus in the blood of infected animals
       • These arise from virus mutants of the original strain (deletions, duplications, rearrangements, re-combinations or insertion of cellular sequences)
       • All lead to production of an 80kd. NS3 that mediates the cytopathic effect
       • Virus acquires a tropism for the mucosal immune system e.g. peyers patch, lamina propria and MLN
       • Onset may be acute or gradual, pyrexia, anorexia, profuse watery diarrhoea, nasal discharge erosive stomatitis, dehydration emaciation and death
     • Pathology
       • Erosions in mouth, oesophagus, forestomachs
       • Inflammation, hyperemia, intestines
       • Necrosis of peyer’s patches and lymphoid tissues
   • Endemic herds
     • High level of immunity (80% seropositive) virus infection of susceptible adults is rare
     • Newborn calves are protected by maternal antibody
     • Heifers are most likely to become infected when they join the main herd (i.e. during second pregnancy)
     • Persistent infection of cows at around 1% of animals
   • Common virus infection of cattle throughout the world
   • Doesn’t often cause diarrhoea – rota and corona viruses are more common causes of diarrhoea
   • Only a proportion of infected animals show any signs of disease
   • Many different isolates mainly non cytopathic
     • Can be divided into 2 serologically distinct biotypes.
       • Biotype 1 – most common in UK, conversion to cytopathic form causes “typical” mucosal disease.
       • Biotype 2 – less common in UK also non cytopathic
         • But conversion to cytopathic form causes thrombocytopaenia and haemorrhagic disease
   • Conversion to Cytopathic strains arise de novo by mutations to NSP3 (protease) and other virus genes
     • Including point mutations, duplication of virus genome segments and incorporation of host RNA (many cases 80kD NSP3)
   • One virus, many diseases
     • BVD: acute infection of susceptible cattle.
     • Infection via nasal route replication in epithelium, viraemia associated with T and B cells leukopenia (decrease in number of WBCs)
     • Disease usually mild (non cytopathic virus): high morbidity, few clinical signs (if any), transient fever and recover, antibody response to virus.
       • May be off colour for a bit, may have fever
     • Immuno-suppressive: blocks interferons, leukopaenia, alters monocyte function, promotes T-helper 2 response (exacerbation of concurrent respiratory disease)
       • Will worsen any other concurrent diseases
     • Occasionally moderate/severe outbreaks
       • Fever depression, diarrhoea, erosions in mouth some deaths.
       • + immuno-suppression may exacerbate respiratory disease.
       • Recovered animals seroconvert to BPV

5. Control

   • Louping ill
     • Inactivated cell culture vaccines given at 1 yr. annual boosters given to breeding flock
       • Bird populations moving around and very high tick burdens can give rise to peaks in disease
       • Tend to be well vaccinated
   • BVD Control Programme
     • Drawbacks
       • Testing and removal to generate BVD free herd
       • Risk of heard breakdown
       • Vaccine not all totally effective, may not protect from all strains of virus and are not certain to prevent foetal infection
       • Some evidence that attenuated vaccines may contribute to disease onset in tolerant animals (BVDV can be spread by dirty needles)
     • 1. Testing and removal of persistently infected animals
       • Virus capture assay +ve/antibody –ve
       • Difference between inactivated vaccine and non-cytotoxic disease? Probably not much
     • 2. Vaccination of remaining animals
       • 1st dose inactivated vaccine (no response to NSP3)
       • 2nd booster dose (live attenuated vaccine)
     • 3. Continue to monitor and remove of virus +ve animals
     • 4. Vaccination of replacements @ 6 months
     • 5. Maintain bio-security

6. Diagnosis

   • Louping ill
     • Virus isolation, serology, (IgM), immunohistology
       • IgM – response at the present – first immunoglobulin produced by antibodies
       • IgG – may have been a historical infection, and is just antibody left over
   • BVD
     • Tissue culture (plus Immunofluoresence, non cytopathic).
     • Detection of viral RNA by RTPCR. (ear punch test)
     • Serology by ELISA
       • Remember no antibody in tolerant carriers
       • Milk antibody for herd test
     • NS3 antigen commonly used highly conserved
       • Not induced by inactivated vaccine.
     • Antigen capture ELISA for persistent infection.

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