Flavi and Pestiviruses

October 12, 2010

Flaviviridae

Pestiviruses

FlavivirusesHepacvirusesHepatitis C virus

BVD, Hog cholera, Border disease

Yellow feverJapanese encephalitisSt. Louis encephalitisDengueWest Nile virus

(arthropods, biological vectors)

West Nile OutbreaksWest Nile Outbreaks• Israel - 1951-1954, 1957• South Africa - 1974• Romania – 1996• Italy – 1998, 2008• Russia - 1999 (human)• United States –1999-2009 (equine, human)• Canada - 2001-2009 (equine, human)• Israel – 1998, 2000 (human)• France (Rhine delta) - 2000 (equine)

springearly summer

return from south

overwinteror

eggs

amplification in birds

late summer andfall

dead-end hosts

Saskatchewan mosquito species shown to be capable of transmitting

WNV• Aedes vexans (spring to fall)• Ochlerotatus flavescens,

spencerii(July-August)• Culex restuans*, tarsalis* (July-

August)• Culiseta inornata*• Coquillettidia perturbans

How does the virus overwinter and spread?

• migratory birds?

• overwintering mosquitos?

• bird to bird transmission?

– Komar et al. EID March 2003

in the mosquito

1. virus ingested

with blood meal

2. virus multiplies in gut epithelial cells

3. virus leaks from gut and infects salivary glands

4. virus released in saliva during feeding

(sufficient amount of virus must be ingested - > 105 infectious units/ml)

in mammalsvirus transmitted in mosquito

saliva during probing

virus deposited in extra vascular tissue

replication in skin and lymph nodes

amplification in extra-neural tissues

viremia (secretion in milk)

crosses blood/brain barrier (repl’n in vascular endothelium, exacerbated by concurrent infections)

viremia terminated by immune response

viral damage to neurons and glia or dysfunction

inflammation perivascular infiltration (plasma cells and macs), cerebral edema.

“flu” likesymptoms

“neurological”signs

IgM,

CSF IgM, pleocytosis,PCR

PCR

equine cases of WNV neurological disease

• Ataxia 86%• Depression 51%• Hind limb weakness 49%• Difficulty or inability to rise 46%• Muscle tremors 41%• Fever only 24%• Differentials:

– rabies, EHV 1, EEE, WEE, botulism

• 10% to 50% of horses with neurological signs die

clinical signs in people

• most asymptomatic• fever, “flu” like symptoms (fatigue, anorexia, nausea,

vomiting, arthralgia, rash, lymphadenopathy)• encephalitis, meningoencephalitis - ataxia, painful

eyes, seizures, change in mental status (confusion)case fatality rate in hospitalized patients - 10-12%

risk factor for severe disease (age 50-60 yr are 10 times and >80 yr are more than 40 times likely)

Petersen and Margin, WN virus: a primer for the clinician 2002. Ann Int med 137:173

unusual cases in USA

• infant infected through breast milk

• 2 people infected through blood transfusion

• 2 laboratory workers while dissecting infected animals

the Canadian experience

• 2000 - 2,288 birds examined, 185 tested - no positives

• 2001 - 2,807 bird carcasses from NF to Sask tested– 128 WNV infected birds from 12 health

dist. in Ontario– no disease in horses or humans

human cases 2003-2005

May June July Aug Sept Oct

http://www.phac-aspc.gc.ca/wnv-vwn/mon-hmnsurv-archive-eng.php

http://www.phac-aspc.gc.ca/wnv-vwn/mon-hmnsurv-eng.php

West Nile virus in domestic birds

• geese

• ducks

• chickens and turkeys

• ostriches, emus

testing for WNV - serology

strong + cont

weak + cont

samples from 45 horses, Virden Manitoba (Aug-Sept, 2002)

IgM captureELISAPDS immunol.lab

vaccines

Intervet – PreveNile Live Flavivirus chimera

Fort Dodge

West Nile Virus – Common Client Questions

• Should we vaccinate our horse – is it safe and does it work?

• Can I catch WNV from a horse?• What signs might a horse show early on?• Is there a treatment?• What can we do to limit the risk to our horse?• Can our other pets get it?• When should we vaccinate our horses?

Pestiviruses

• Systemic haemorrhagic disease - pigs (USA)

• Enteric disease - calves (USA)

• Congenital, neurological “hairy-shakers” (England/Wales)

1940sHCV

BVDV

BDV

FlavivirusesFlaviviruses

Pestiviruses Hepatitis CLouping ill

Yellow feverDengue

Pestiviruses of Artydactyla

bovine viral diarrhoea(BVDV)

hog cholera(HCV)border disease

(BDV)

Japaneseencephalitis

Murray ValleySt. Louisencephalitis

West Nile

Wide host range (camelids, deer etc)

BVDV genome and gene products

UTR structural nonstructural

Regions that show the most variation

gp53 p125(p54+p80)

Biotypes of BVDV

NON-cytopathic(natural state)

Cytopathic(mutant)

Based on effect on cells in tissue-culture

CP vs NCP – genetic differences

UTR structural nonstructural

p125(p54+p80)

Biotypes - implications

• Non-cytopathic– Implications for vaccines and research

• Cytopathic– Mucosal disease

BVDV “types” Not different serotypes!!

• BVDV-1

• BVDV-2

Based on:1. sequence differences In non-translated region of genomeDoes not imply differences in pathogenicity2. Antigenic differences

UTR structural nonstructural

Antigenic differences between BVDV-1 and BVDV-2

Serum against

BVDV-1strains

BVDV-2strains

VN titre againstBVDV-1strains

BVDV-2strains

800->12,800 100->3,200

50->400 3,200->51,200

Pellerin et al.(1994) Virology 203:260-268

Pathogenesis - infection

• Intra-species– PI carriers

• Inter species• Vaccine related?• Artificial breeding programs• Blood• Persistence in acutely infected animals

Pathogenesis

Pathogenesis - disease in imm.competent, non-pregnant

animals• Sub-clinical• Mild fever, leukopenia, decreased milk

production• Mild BVD - mild erosive lesions, ulcerative

stomatitis, diarrhoea, respiratory• Severe disease - lesions mimic MD,

thrombocytopenia, haemorrhagic syndrome, hyphemia

Pathogenesis - pregnant animals

• All syndromes described above

• Embryonic death

• Abortions

• Birth defects

• Persistently infected calves

Pathogenesis - PI animals

• Healthy, normal

• “poor-doers”

• Mucosal disease

Guess which one is persistently infected?

Calves of the same age. From Lee et al. CVJ 38:29

consequences of having a PI animal

• Loneragan et al. JAVMA, Feb 15, 2005– PI animals more likely to be ill, require

treatment or die– Incontact animals more likely to be sick,

require treatment

• Dieguez et al. Res Vet Sc, Aug 2009• Correlation BVD status and respiratory

disorders, mortality

Mucosal disease

• NCP->CP (p80)• Infection with antigenically related CP virus

(mutation, herd-mate, vaccination?)• Low morbidity high mortality• High fever, depression, anorexia, diarrhoea• Ulcerative mucosal lesions• Death 2 days -> 3 weeks

Mucosal Disease (by mutation)

MD

Mucosal Disease (by infection or injection)

MD

No Mucosal Disease if CP virus antigenically different

Immune response

No MD

Diagnostic procedures• Virus isolation

– Small numbers ($31.50)– Herd screening - microtitre ($10 -2->10, $6 >10)

• Antigen capture ELISA– Herd screening

• Serology– VN ($14)– ELISA ($5/animal)

• PCR– genotyping ($62)– pooled samples ($30)

Diagnostic procedures

• PCR (BVDV-1 vs BVDV-2)• Immunohistochemistry or IFA

– Abortions ($45)– PI animals (approx. $6/animal) BVDV-2

BVDV-1a

BVDV-1b

Immunohistochemistry - PI vs acute

Persistently infected calf:Antigen in hair follicle epithelium

Acutely infected calf:Antigen in superficial layerof epidermis (foci)

From Brad Njaa et al. 2000. J. Vet. Diag. Invest. 12:393-399.

Diagnostic Parameters (acute infection)

infectionincubation period

clinical disease

virus

infect. virus detectable in serum

infect. virus detectable in WBC

Virus det.by PCR

antigens in biopsy

antibody5-7 days

Diagnostic parameters (abortion)

• Fetus– Often no infectious virus– Submit liver, kidney, spleen, thymus for IH

or IFA– Fetal antibody if late term

Diagnostic parameters (PI)

• Large amounts of virus in blood, serum and secretions (103-107 TCID50/ml)

• Maternal antibody interferes with isolation• <3 months - submit blood• >3 months - serum • Repeat isolation in 3 months• Immunohistochemistry - follicle epithelium

Herd screening

Management of BVD

• Test and remove PI animals

• Test all new born calves for 9 months

• For 9-12 months segregate age groups

• Quarantine replacements

• Vaccination with MLV BVDV

• BVD infections may persist for some time after removal of PI animals (Collins et al. 2009)

Vaccines

• Inactivated or attenuated

• Most (all) contain CP BVDV

http://www.cattlenetwork.com/content.asp?contentid=119624

Interspecies transfer

• Sheep

• Wild ruminants– Natural infections (caribou: 40-100%)– Transfer (llamas, alpacas)