Clinically Important Vector-Borne Diseases of Europe

Home , Banna virus

Natalie Cleton, DVM Erasmus MC, Rotterdam Department of Viroscience [email protected]

No potential conflicts of interest to disclose Erasmus Medical Centre Department of Viroscience

Laboratory Diagnosis of Arboviruses

Natalie Cleton Marion Koopmans Chantal Reusken

[email protected] Distribution

Arboviruses with public health impact have a global and ever changing distribution

Notifications of vector-borne diseases in the last 6 months on Healthmap.org Syndromes of arboviral diseases

1) Febrile syndrome: – Fever & Malaise – Headache & retro-orbital pain – Myalgia

2) Neurological syndrome: – Meningitis, encephalitis & myelitis – Convulsions & coma – Paralysis

3) Hemorrhagic syndrome: – Low platelet count, liver enlargement – Petechiae – Spontaneous or persistent bleeding – Shock

4) Arthralgia, Arthritis and Rash: – Exanthema or maculopapular rash – Polyarthralgia & polyarthritis Human arboviruses: 4 main virus families

Family Genus Species examples

Flaviviridae flavivirus Dengue 1-5 (DENV) West Nile virus (WNV) Yellow fever virus (YFV) Zika virus (ZIKV) Tick-borne encephalitis virus (TBEV) Togaviridae alphavirus Chikungunya virus (CHIKV) O’Nyong Nyong virus (ONNV) Mayaro virus (MAYV) Sindbis virus (SINV) Ross River virus (RRV) Barmah forest virus (BFV) Bunyaviridae nairo-, phlebo-, orthobunyavirus Crimean-Congo heamoragic fever (CCHFV) Sandfly fever virus (SFV, TOSV) Rift Valley fever virus (RVFV) Oropouche virus (OROV) 5 Reoviridae Seadorna- en coltivirus Colorado tick-borne fever (CTFV) Banna virus (BANV)

8 maart 2016 5 8 maart 2016 Overview of spread & syndromes of vector-borne diseases

Many diseases display overlapping symptoms and geographical distribution

Europe AR NS HS WNV* TBEV* DENV^ East Asia North America DENV^ WNV* CCHFV AR NS HS AR NS HS TAHV LIV DENV^* JEV* DENV^* WNV* CEV/LCV* DENV^ SINV* TOSV* §SFV* DENV^ WNV* WNV TBEV OHFV CHIKV BATV CHIKV* WNV SFTSV CHIKV POWV TAHV SLEV TAHV BANV CCHFV EEEV West and Central Asia TAHV WEEV AR NS HS CTFV DENV^* CHIKV* RVFV* North Africa WNV* WNV* CCHFV AR NS HS TAHV TBEV DENV^* Caribbean and Central America DENV^* TOSV* RVFV* SINV BANV OHFV South and Southeast Asia AR NS HS WNV* RVFV* CCHFV* TAHV AHFV AR NS HS DENV^ OROV* DENV^* CHIKV* TAHV YFV* §SFV* RVFV* DENV^ JEV* DENV^* * SINV DENV^* * WNV WEEV TAHV §SFV* WNV* WNV* KFDV ZIKV* EEEV BUNV ZIKV* TBEV SFTSV OROV* VEEV Sub-Saharan Africa TBEV BANV CCHFV CHIKV* ILHV AR NS HS CHIKV* TAHV WNV DENV^ WNV* DENV^* TAHV Flaviridea SLEV * WNV* RVFV* RVFV* Togaviridae South America YFV* BUNV NRIV ZIKV TAHV ILEV AR NS HS CHIKV* BWA CCHFV Bunyaviridae DENV^* OROV* DENV^* SINV BUNV ZIKV* WEEV YFV ONNV ILEV Reoviridae WNV EEEV Oceania BWA CHIKV* VEEV TAHV AR NS HS MAYV* SLEV ILEV RRV* MEV* DENV^ OROV* WNV TATV §WSBV BFV* JEV ILHV NRIV CHIKV WNV ROCV SINV WNV DENV^ ZIKV

Fig 1. Geographical distribution of medically important arboviruses that cause febrile disease in humans.

All arboviruses cause febrile symptoms, but symptoms more specific to certain viruses are represented in three columns: 1) Arthralgia-Rash (AR); 2) Neurological symptoms (NS), and 3) Hemorrhagic symptoms (HS). Virus name is written in the color of its corresponding (and potentially cross-reactive) family. Arboviruses not known to cause more than febrile symptoms are preceded with a §-sign. Arboviruses more likely to be diagnosed in travelers are followed by *.

AKHV = Alkhurma hemorrhagic fever virus; BANV = Banna virus; BFV = Barmah Forest virus; BWAV = Bwamba virus; BUNV = Bunyamwera virus; CEV = California encephalitis virus; CHIKV = Chikungunya virus; CTFV = Colorado tick fever virus; CCHFV = Crimean-Congo hemorrhagic fever; DENV = Dengue virus; EEEV = Eastern equine encephalitis virus; GROV = Guaroa virus; ILEV = Ilesha virus; ILHV = Ilheus virus; JEV = Japanese encephalitis virus; KFDV = Kyasanur Forest disease virus; LCV = La cross virus; LIV = Louping Ill virus; MAYV = Mayaro virus; MURV = Murray Valley virus; NRIV = Ngari virus; OHFV = Omsk hemorrhagic fever virus; ONNV = O’Nyong Nyong virus; OROV = Oropouche virus; RVFV = Rift Valley fever virus; ROCV = Rocio virus; RRV = Ross river virus; SFV = Sandfly fever (Naples / Sicilian / other); SFTS V = Severe Fever with Thrombocytopenia Syndrome Virus; SINV = Sindbis virus; SLEV = St. Louis encephalitis virus; TAHV = Tahyna virus; TATV = Tataguine virus; TBEV = Tick-borne encephalitis virus; TOSV = Toscana virus; VEEV = Venezuelan equine encephalitis virus; WEEV = Western equine encephalitis virus; WNV = West Nile virus; YFV = Yellow fever virus; ZIKV = Zika virus. Cleton et al 2012 Journal of Clinical virology & Cleton et al 2015 PNTD Testing algorithm

Section based on

- Travel history

- Clinical symptoms

- Potential cross-reactivity / vaccination

- Virus kinetics (Days post onset symptoms)

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- Exposure history

- Risk factors Testing algorithm and Information provided

Information provided to LIMS

Travel history 75%

General symptoms 42% ?

Vaccination history 0.5%

Days post onset 0.5% Sample possibilities

- Serum / plasma => Virus detection / Antibodies

- CSF => Virus detection / Antibodies

- Urine (WNV, DENV, ZIKV) => Virus detection

- Tissues (post mortem) => Virus detection

Cleton et al, Clinical Virology, 2012 Testing method options

Viral detection • Virus isolation => Vero cells => BSL3/4!!!!

• Detection of viral genome => RT-PCR / IHC

• Detection of viral antigen => DENV NS1

1 8 maart 2016 0 Testing method options

Antibody detection • IgG/IgM

- Seroconversion

- IgG/IgM 4x titer increase =>2 samples needed with 14 day interval

- IgM in liquor in case of neurological disease => confirmative

- IgG saliva (experimental)

ELISA

Immuno flourenscence Plaque reduction Neutrilization

Haemagglutination inhibition 1 8 maart 2016 1 Test selection dependent on disease kinetics

Humans are often dead-end hosts in these cases • West Nile virus • Japanese encephalitis virus • Tick-borne encephalitis virus • Sandfly fever virus

Onset of NS symptoms

L. Peterson, proceedings on Arboviral Management 2012 WNV, TBEV, TOSV sensitivity type of tests

Sensitivity Molecular Serology

0-3 days POS Fever => 56% in serum Fever => 54% in serum NS => 10% serum / 55% CSF NS => 75% serum/CSF

4-7 days POS Fever => 10% in serum Fever => 98% in serum NS => 10% serum / 55% CSF NS => 100% serum/CSF

>7 days POS Fever => 4% in serum Fever => 98% in serum NS => 1% serum / 10% CSF NS => 100% serum/CSF

Urine: +/- 14 days POS RT-PCR pos

POS = Post Onset Symptoms NS = Neurological symptoms Disease kinetics CHIKV, ZIKV, DENV

Day 1-2

Onset of NS symptoms

Guzman, M. G. et al. Nature Reviews Microbiology 8, S7–S16 (2010). DENV, (ZIKV), CHIKV sensitivity type of tests

Sensitivity Molecular Serology

0-3 days POS Fever => 50% in serum Fever => 50% in serum

4-7 days POS Fever => 50% in serum Fever => 75-100% in serum

>7 days POS Fever => <10% in serum Fever => 100% in serum

DENV NS1-antigen-capture: 2-9 days POS (primary) or 1-4 days POS (secondary) • not replacement for PCR! • low sensitivity….. • cross-reactivity…..(minimal) • Aspecific reactions (snap-test)

POS = Post Onset Symptoms NS = Neurological symptoms Disease kinetics CCHFV

Ergonul, LID 2006 CCHFV sensitivity type of tests

Virus isolation Molecular Serology

0-3 days POS 67% serum 0% serum

4-7 days POS 71% serum 65% serum

>7 days POS 61% serum up to 98% serum 16 days POS

POS = Post Onset Symptoms Interpretation of serological results

Results dependent Health status of patient

• Vaccination history: Cross-reactivity in serological tests due to flavivirus vaccines • Yellow fever virus (South Americas, Africa) • Japanese encephalitis virus (Asia) • Tick-brone encephalitis virus (Central and Eastern Europe) • Dengue virus (Central and South Americas)

• YOPI’s => low or no immune response (extended viremia or IgM response)

• Previous infections => antigenic sin

• Cross-reactivity & sensitivity differ dependent on viral antigen used (or whole virus used) => in-house validation essential for interpretation!

18 1 8 maart 2016 8 Antigenic sin

 Memory B-cells developed against epitopes of primary pathogen

 Secondary infection with closely related pathogen (Influenza, Dengue, HIV, Cowpox)

 Memory B-cells activated to primary pathogen

 Reducing production of b-cells to secondary pathogen

http://nfs.unipv.it/nfs/minf/dispense/immunology/lectures Interpretation of arbovirus diagnostic results

Acute sample

Antigen- Virus IgM IgG PCR capture isolation Pos Pos Pos Pos Neg Neg Pos Neg No Vaccination Neg in CSF? history? IHC Pos Yes No Neg Pos interpretatio interpretatio interpretatio ID Virus n not n not ID Virus n not possible possible possible Convalescent serum sample with 2wk interval needed

Seroconversion or 4-fold titre increase (VNT) Pos Neg

ID Virus No diagnosis Interpretation of results dependent on test specifics

Sensitivity / Specificity provided by manufacturer

● Analytical sens/sepc dependent on: - cross-reactivity - lab variation - robustness test - a-specific reactions (Malaria in snap-tests)

● Diagnostic sens/sepc dependent on: - Sampling day post onset disease => viral kinetics

- Comparison method used (how solid is the Gold Standard?)

- Background of validation samples used (predictive value):  Endemic / sporadic / disease free area  Healthy / disease population  Vaccination status / previous exposure Important terms in interpretation of (all!) diagnostic tests

Bayesian Clinical Diagnostic Model

True presence of disease

Total positive for Total negative for Total population disease (a+c) disease (b+d)

Positive predictive value Predicted positive True positive (a) False positive (b) = for diseases (a+b) True positive (a) Predicted positive (a+b) Test outcome Negative predicitive value Predicted negative False negative (c) True negative (d) = for disease (c+d) True negative (d) Predicted negative (c+d) Sensitivity Specificty = = True positive (a) True negative (d) Prevalence =? total positive (a+c) total negative (b+d) The influence of selected testing population

Low prevelance population (1%) High sensitivity (99%) High specificity (99%)

Usefullness of a diagnostic test for prediciting disease (>1) or predicting absennce of disease (<1)

= the probability of a person who has the disease testing positive the probability of a person who does not have the disease testing positive

https://kennis-research.shinyapps.io/Bayes-App/ The influence of selected testing population

High prevelance population (10%) High sensitivity (99%) High specificity (99%)

https://kennis-research.shinyapps.io/Bayes-App/ OIE diagnostic method validation steps

http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/1.01.05_VALIDATION.pdf Overview of spread & syndromes of vector-borne diseases

Many diseases display overlapping symptoms and geographical distribution

Fig 1. Geographical distribution of medically important arboviruses that cause febrile disease in humans.

Virus detection Antibody detection • Multi-plex PCR • Luminex • Microarrays • Microarrays • Multiplex IFA (commercial) Protein microarrays

• high throughput • simultaneous detection of multiple IgG and IgM antibody profiles (hundreds of proteins) • adaptable for region, syndrome and (multiple species) diagnostics

Flavivirus protein Immunological Specificity Print viral proteins on slides reactive Envelope High Low

Domain III envelope Low High

NS1 High High

Aruna Sampath & R. Padmanabhan 2009 Based on ELISA method

Neutrocellulose

www.microarray.prostatecentre.com Slides scanned for fluorescence Koopmans et al, 2011 Validated for use in travelers

Dengue virus 1 (DENV1)

Dengue virus 2 (DENV2)

Dengue virus group Dengue virus 3 (DENV3)

Dengue virus 4 (DENV4)

Dengue virus 5 (DENV5)

Japanese encephalitis virus (JEV)*

Mosquito-borne virus West Nile virus (WNV) Japanese encephalitis group Usutu virus (USUV) Examples of patient profiles on protein microaray St. Louis encephalitis virus (SLEV)

Yellow fever virus group Yellow fever virus (YFV)* Flavivirus genus Spondweni virus group Zika Virus (ZIKV)

Mammalian tick-borne Tick-borne encephalitis Tick-borne virus viruses virus (TBEV)*

Current validated proteins (grey) Limited cross-reactivity

Patients living in dengue endemic areas

Patients with multiple serocomplex exposures Conclusions

 Increase and change in arbovirus activity in and beyond Europe

 Clinical symptoms of arboviruses overlap and are not restricted to a viral family

 Geographical distribution of arboviruses with comparable syndromes overlap

 Within genus (specifically flaviviruses) a large amount of serological cross-reactivity occurs

 Also, flavivirus vaccines (TBEV, YFV & JEV) cause false positive serological tests

 Therefore have a clear substantiated diagnostic algorithm if vector-borne are considered! – Multiplex! – General clinical syndrome – Travel- and exposure history – Vaccination history – Potential cross-reactivity – Days post onset symptoms! (sensitivity PCR vs serology differs significantly per group) Questions for further research discussion

● How much diagnostics is enough? Cost versus Benefit

› What are the costs of (extra) testing?

› How many (extra) diagnoses will this achieve?

› What is possible/reliable in the lab?

› What is the added value to patient care?

› What is the added value to future patient care?

› What is the role/responsibility of clinician in Public Health?

› How’s paying? Thank you for your attention