emerging and neglected sandfly-borne phleboviruses around

Emerging and neglected sandfly-borne phleboviruses around the Mediterranean

:

UMR_D 190 – Emergence des Pathologies ViralesAix-Marseille Univ - IRD - EHESP – IHU Méditerranée Infection

Marseille, FRANCE

Pr. Rémi CHARREL --- [email protected]

ü Arthropod-borne viruses transmitted by sandflies :

à Reoviridae (Changuinola virus)

à Rhabdoviridae (Irririvirus, vesicular stomatitis virus …)

à Bunyaviridae : genus Phlebovirus

Viruses

ü The genus Phlebovirus

à Enveloped virus

à Size is 85-120 nm

à Trisegmented single stranded RNA genome, negative polarity

Phleboviruses

ü Segmented RNA genome

à 3 different sized segments

à S = 1.7 kb, Nucleocapsid and NS

àM = 3.2 kb, Glycoproteins GN & GC + NS

à L = 6.4 kb, RNA polymerase

• Bujaru virusü BujaruüMunguba

• Chandiru virusü Alenquerü Chandiruü Itaitubaü Niqueü Oriximinaü Turuna

• Chilibre virusü Cacaoü Chilibre

• Frijoles virusü Frijolesü Joa

• Punta Toro virusü Buenaventuraü Punta Toro

• Rift Valley fever virusü Belterraü Icoaraciü Rift valley fever

• Salehebad virusü Arbiaü Salehebad

• Sandfly fever Naples virusü Karimabadü Sandfly fever Naplesü Sabinü Tehranü Toscana

• Uukuniemi virusü EgANü Fin Vü Grand ArbaudüManawaüMurreü Oceansideü Pontevesü Precarious pointü RMLü St Abbs headü Tunisü Uukuniemiü Zaliv Terpeniya

9 species

Phlebovirusesgrouping 37 viruses

• Tentative speciesü Sandfly fever Sicilianü Aguacateü Anhangaü Arboledasü Arumowotü Caimitoü Chagresü Corfouü Gabek Forestü Gordilü Itaporangaü Odrenisrouü Pacuiü Rio Grandeü Saint-Florisü Urucuri

+ 16 unclassified viruses

+ 10 new unclassified viruses

• Bujaru virusü BujaruüMunguba

• Chandiru virusü Alenquerü Chandiruü Itaitubaü Niqueü Oriximinaü Turuna

• Chilibre virusü Cacaoü Chilibre

• Frijoles virusü Frijolesü Joa

• Punta Toro virusü Buenaventuraü Punta Toro

• Rift Valley fever virusü Belterraü Icoaraciü Rift valley fever

• Salehebad virusü Arbiaü Salehebad

• Sandfly fever Naples virusü Karimabadü Sandfly fever Naplesü Sabinü Tehranü Toscana

• Uukuniemi virusü EgANü Fin Vü Grand ArbaudüManawaüMurreü Oceansideü Pontevesü Precarious pointü RMLü St Abbs headü Tunisü Uukuniemiü Zaliv Terpeniya

9 species

Phlebovirusesgrouping 37 viruses

• Tentative speciesü Sandfly fever Sicilianü Aguacateü Anhangaü Arboledasü Arumowotü Caimitoü Chagresü Corfouü Gabek Forestü Gordilü Itaporangaü Odrenisrouü Pacuiü Rio Grandeü Saint-Florisü Urucuri

+ 16 unclassified viruses

+ > 10 new unclassified viruses

Old World sandfly-transmitted viruses

ü In the Old-World before 1970

Phlebovirus infections

ü Clinical picture

à Summer fever

àMild febrile syndrome

à No fatality recorded

Sandfly Sicilian serocomplex

Sandfly Naples serocomplex

ü Vector = P. papatasi

ü In 1971 : Toscana virus

Phleboviruses infection

Sandfly Naples serocomplex

Sandfly Sicilian serocomplex

ü First isolated in Italy, 1971

ü From P. perniciosus & P. perfiliewi

ü During 80’s & 90’s : Toscana virus


ü Human disease demonstrated in 1985

ü Tropism for the CNS

àMeningitis and encephalitis

ü 1st cause of summer meningitis in Italy in 80’s

ü Serological data and virus isolation

à Portugal, France, Spain, Greece, Cyprus, Turkey, Croatia

The only sandfly-transmitted virus that demonstrates neurotropic activity

• Spain 1988 & 2003: serologic and molecular evidence:

- 15 strains isolated from patients,

- 26% seroprevalence rate in Granada (Mendoza-Montero et al 1998 Clin Infect Dis)

• France 2004: serologic and molecular evidence (Hemmersbach-Miller et al 2004 Eur J Intern

Med, Peyrefitte et al 2005 Emerg Infect Dis).

• France 2005-2009- seroprevalence 12% in healthy blood donors southeastern France- detection of TOSV RNA

- in patients with meningitis- in P perniciosus in Marseille and Nice- in Sergentomyia minuta

è Toscana virus is in the top-3 of causes of meningitis in France and Spain

Toscana virus outside of Italy

• Spain 1988 & 2003: serologic and molecular evidence:

- 15 strains isolated from patients,

- 26% seroprevalence rate in Granada (Mendoza-Montero et al 1998 Clin Infect Dis)

• France 2004: serologic and molecular evidence (Hemmersbach-Miller et al 2004 Eur J Intern

Med, Peyrefitte et al 2005 Emerg Infect Dis).

• France 2005-2009- seroprevalence 12% in healthy blood donors southeastern France- detection of TOSV RNA

- in patients with meningitis- in P perniciosus in Marseille and Nice- in Sergentomyia minuta

è Toscana virus is in the top-3 of causes of meningitis in France and Spain

• Turkey 2009: serologic and molecular evidence (Ergunay et al Clin Microbiol Infect 2010).

• Greece 2010 (Papa et al 2010)

• Elba island 2010 (Gabriel et al 2010, Sonderegger et al 2009)

• Morocco 2012 (Es-Sette et al) / Tunisia 2013 (Bichaud et al) / Croatia 2012 (Punda-Poli• et al)

Toscana virus outside of Italy

Portugal / Spain /

France / Morocco

France / ItalyTurkey / Tunisia

Croatia

A

C

B

Bichaud et al, in press

• meningitis: brutal onset (70%), headache (100%), fever (76-97%), nausea and vomiting (67-88%), myalgias (18%). neck rigidity (53-95%), Kernig sign (87%), poor levels of consciousness (12%), tremors (2.6%), paresis (1.7%), nystagmus (5.2%)

CSF > 5-10 cells, normoglyco- proteinorachia leucocytosis(29%) or leucopenia (6%)

mean duration of the disease is 7 daysoutcome is usually favorable.

- meningoencephalitis (Baldelli et al 2004)- encephalitis (Dionisio et al 2001)- deafness

• other disease manifestations not involving the CNS- no published data exist to suggest that TOSV could cause other clinical syndromes

Disease in humans and Toscana virusclinical forms of the infection

• febrile illnessself-limiting febrile illness without CNS manifestationusually neither hospitalized nor investigated further

• asymptomatic or pauci-symptomaticseroprevalence studies suggest that a large proportion of infections by TOSV are a- or pauci-symtomatic

CNS forms

Febrile forms

Non symptomaticforms

???

ü Since 2000

à Several teams studied phleboviruses


Sandfy Fever Sicilian Virus

(SFSV)

Sandfy Fever Naples Virus

(SFNV)

Toscana Virus (TOSV)

Vector(s) Clinical Syndromes

P. papatasi Sandfy fever (Papatacci fever, 3-day fever)

P. papatasi Sandfy fever (Papatacci fever, 3-day fever)

P. perniciosusP. perfiliewi

Sandfly feverAseptic meningitis

ü Progressivelybecome extinct in western Europe

ü Currently Toscana virus

à In the top 3 viruses causing meningitis during summertime

ü Phlebovirus infections :

à Known for a long time

à But remained neglected

à Few data available on their epidemiology

ü Bibliographic research in PubMed for the last 10 years :

à "Toscana virus" = 234 references

à "Leishmaniasis" >10,000 ref

à Other emerging viruses : "Chikungunya" = 955 ref"West Nile" = 3 500 ref"Dengue" = 4 000 ref

Analysis of the peer-reviewed literature« Toscana virus »

Analysis of the peer-reviewed literature« Toscana virus »

10655TOTAL

1021basic science

910diagnostics

246seroprevalence and seroepidemiology

4817human cases and series

151Virus discovery and detection

2003 --- 20131971 --- 2002

10655TOTAL

1021basic science

910diagnostics

246seroprevalence and seroepidemiology

4817human cases and series

151Virus discovery and detection

2003 --- 20131971 --- 2002

• Co-circulation of different phleboviruses in the same geographic area raises the question of respective impact on human/animal health

The antigenic cross-reactivity between many phleboviruses renders the answer to this problem more complex than initially beleived

• The lack of available full-length genome sequences of phleboviruses hampers the development of real-time RT-PCR tests for diagnostic purpose of known phleboviruses

• The absence of field studies (such as supported by the Rockefeller foundation after WWII) has left a large number of phleboviruses undiscovered: these virus are likely to play an important role in ecology and human and veterinary medicine

Questions to be addressed

èVirus discovery using field studies

Data of distribution of known viruses are up-to-date ?Missing viruses in the phylogeny?

New phleboviruses

Sandfly trapping

ü CDC miniature light traps

à Inside house, animal housing facilities (sheep, goat, rabbits…)

à From dusk to down

Field Work

ü Each morning, sandflies were :

à Collected and identified

à Pooled by species, gender and trapping place, max 30

à Preserved in dry ice, liquid nitrogen, - 80°C Viral infectivity

ü Need for an entomologic plateform to perform field samples

à Defrost samples only one time = preserve viral infectivity

Lab work

ü Virus detection

= molecular biology tools, to detect the presence of phlebovirus RNA

ü Virus isolation

= using cells cultures

ü Genetic characterization of newly discovered phleboviruses

= Sequencing and phylogeny analysis

ü 12 sandfly trapping campaigns :

à Summers 2007, 2008, 2009, 2010

à France, Tunisia Algeria

à Turkey in 2012 and 2013

Field Work

Aim : to detect, to isolate and to characterize existing and/or new phleboviruses in sandfly populations

Done by other groups

Done by our group

Sample crushing + centrifugation

600µL EMEM + Tungsten bead

SN Aliquoting- 80 °C

Virus Isolation

Virus detection

Electron microscopy

PCR, Q-PCR

Geneticcharacterization

Sanger sequencing

Cytopathic effect

Immunofluorescence

Next GenerationSequencing

Next GenerationSequencing

Automation

From 504 sandflies / day

To 15,120 sandfies / dayNucleic acidextraction

Inoculation onto vero cells

Sandfly Fever Naples Serocomplex

TOSV

SFNV

Sandfly Fever SicilianSerocomplex

SFSV

Sandfly-borne phleboviruses in the

old worldBefore 2008

L-segment, AA

TOSV France2004 H/IMTSSA FJ153281

TOSV Spain2005 EsPhGR40 FJ153280TOSV Spain2004 ESPhGR79 GU183147

TOSV SLP008 1 Morocco 2008 JN8TOSV France2006 AK DQ656070

TOSV France2006 AR DQ656071TOSV Italy1993 ISS Phl 3 NC006319

TOSV France2006 1500590 DQ975233

TEHV I47 GQ165522SFN 30451 GQ165528

SFNV Poona EF095548

RVFV Smithburn DQ375430KAR I58 GU143712

AGUV SSMP

Cyprus virus AY962268SFSV Turkey2008 Izmir19 GQ847513

SFSV Sabin EF095551Chios A virus AY293623

CFUV PA Ar 814 GQ165521

Arumowot AR 1284 64 GU143714Adria Albania2005 ALB1 HM043725

Salehabad I 81 GU143716ARBV Ph 1 35 M6 DQ862467

Gouleako virus F23 K1 EF423167UUKV NC 005214

99

100

87

71

65

69

100

99

99

68

98

100

84

71

80

60

55

99

0.05

SalehabadSerocomplexARBV


TOSV

SFNV


SFSV

L-segment, AA





TOSV France2006 1500590 DQ975233

TEHV I47 GQ165522

SFN 30451 GQ165528

SFNV Poona EF095548


AGUV SSMP







99

100

87

71

65

69

100

99

99

68

98

100

84

71

80

60

55

99

0.05

TOSV Tunisia2010 T166 JX867537TOSV Tunisia2010 T152 JX86753493

PUNV Tunisia2008 P6B2 GQ165519PUNV Tunisia2010 T114

PUNV Tunisie2010 T122PUNV Tunisia2008 P1B4 FJ848989

PUNV Tunisia2009 T101

100

Unknown Phlebovirus Tunisia2010 T98

Tunisia2010 T91Tunisia2010 T13199

81


Algeria 2007 A5 GU183867Algeria 2007 A6 GU18386899

Kabylia Algeria2007 F16 GU183869

Algeria 2006 Ph ariasi EU240882

PUNV

TOSV France2010 Corse A1

Utique Tunisia2010 T2Utique Tunisia2008 P15 B1 GU233647 Utique Tunisia2008 P6 B1 GU233649

Utique Tunisia2010 TG1Utique Tunisia2010 TG37

Utique Tunisia2008 P21 B1 GU233651 Utique Tunisia2008 P13 B2 GU233653

Utique Tunisia2008 P14 B1 GU233650 Utique Tunisia2008 P4 B4 GU233648Utique Tunisia2008 P13 B4 GU233646 Utique Tunisia2008 P23 B3 GU233652

63

58

77

100

Utique virus

MASV France2009 Nice NA14MASV France2005 Marseille W EU725771

MASV France2005 Nice BP DQ656073MASV France2005 Nice BM DQ656072

Grabai Spain2004 Gr36 GU183150

MASV France2010 Brive L41MASV France2009 Marseille M43MASV France2009 Marseille M51Granada Spain2010 B43-02 GU143721

100

MASV

Damyeri C21Damyeri C253

Camili C1-C12

Sandfly-borne phleboviruses in the

old worldToday

Turkey

Algeria

Tunisia

France

Spain

Tunisia

Algeria

Turkey

Tunisia

Tunisia

FranceSpain

Cyprus

Greece

ItalyFranceSpainPortugalTurkeyTunisia


TOSV

SFNV


SFSV

Now …

L-segment, AA





TOSV France2006 1500590 DQ975233

TEHV I47 GQ165522

SFN 30451 GQ165528

SFNV Poona EF095548


AGUV SSMP







99

100

87

71

65

69

100

99

99

68

98

100

84

71

80

60

55

99

0.05

TOSV Tunisia2010 T166 JX867537TOSV Tunisia2010 T152 JX86753493

PUNV Tunisia2008 P6B2 GQ165519PUNV Tunisia2010 T114

PUNV Tunisie2010 T122PUNV Tunisia2008 P1B4 FJ848989

PUNV Tunisia2009 T101

100

Unknown Phlebovirus Tunisia2010 T98

Tunisia2010 T91Tunisia2010 T13199

81


Algeria 2007 A5 GU183867Algeria 2007 A6 GU18386899

Kabylia Algeria2007 F16 GU183869

Algeria 2006 Ph ariasi EU240882

PUNV

TOSV France2010 Corse A1

Utique Tunisia2010 T2Utique Tunisia2008 P15 B1 GU233647 Utique Tunisia2008 P6 B1 GU233649

Utique Tunisia2010 TG1Utique Tunisia2010 TG37

Utique Tunisia2008 P21 B1 GU233651 Utique Tunisia2008 P13 B2 GU233653

Utique Tunisia2008 P14 B1 GU233650 Utique Tunisia2008 P4 B4 GU233648Utique Tunisia2008 P13 B4 GU233646 Utique Tunisia2008 P23 B3 GU233652

63

58

77

100

Utique virus

MASV France2009 Nice NA14MASV France2005 Marseille W EU725771

MASV France2005 Nice BP DQ656073MASV France2005 Nice BM DQ656072

Grabai Spain2004 Gr36 GU183150

MASV France2010 Brive L41MASV France2009 Marseille M43MASV France2009 Marseille M51Granada Spain2010 B43-02 GU143721

100

MASV

Damyeri C21Damyeri C253

Camili C1-C12

Isolated

Full genetic sequence

Done

Ongoing

IRAN

Qume

Garmsar

Dashliborun

Esfarayen

IRAN

3794TOTAL

1P.caucasicusEsfarayen

49SergentomyiaEsfarayen

59P.papatasiEsfarayen

1P. sergentiDashliborun

5P.mongolensisDashliborun

6P.caucasicusDashliborun

1449SergentomyiaDashliborun

893P.papatasiDashliborun

3P.alexandriGarmsar

197SergentomyiaGarmsar

2P.sergentiGarmsar

5P.tobbiGarmsar

685P.papatasiGarmsar

1P.mascitiQume

10P.alexandriQume

181SergentomyiaQume

247P.papatasiQume

numberspeciescity

Phlebovirus detection fromSergentomyia spp. pools from

Iran29.06.2013

IRAN

• 1 positive among 164 pools• From Dashliborun (Shordakesh village)• Sergentomyia (female pool of 30) • Collected on 2011.7.6

IRAN

Flavivirus detection fromLutzomyia Lutzomyia

(Psathyromyia) abonnenci sandflies from Ecuador

29.06.2013

ECUADOR

• 1 positive among 16 pools• Lutzomyia (Psathyromyia)

abonnenci(female pool)

ECUADOR

Complete genome sequence usingNext Generation Sequencing

ü … also a new tool for direct microorganism discovery!

à Directly using mix of insects

à e.g. : Paraiso Escondido, flavivirus in sandfly from Ecuador

à Possibility to sequence non isolated viruses

ü To obtain full genetic sequence from isolated viruses

à Classic PCR = 1 year of work for 1 virus

à Full sequence of TOSV Tunisia = 1 NGS run

à but big amount of data to manage

ü Better understanding of phleboviruses phylogeny with full-lenghgenome sequences


• seroprevalence studies with the capacity to discriminate between the differentviruses (neutralization tests) because other tests are cross-reactive

• Naples / Toscana / Tehran / Massilia / Punique / Granada• Sicilian / Utique / SFSV Turkey / SFSV Algeria

Question

?

?

F

M

• How to distinguish different phleboviruses from each others using antibody studies ???

• The affinity / avidity of the antibodies is proportional to the antigenic closeliness of the virus: the more similar the viruses, the more cross-reactive the antibodies

• However techniques such as IF, ELISA are not discriminative enough

- therefore the only possibility is to use Neutralisation tests

France & Tunisia

Tunisia

France

Toscana virus versus Punique virus in Tunisia

• Questions :

– is Punique virus capable to infect humans ???

– What are the respective proportions of seroprevalence against Toscana & Punique in humans


• Questions :

– is Punique virus capable to infect humans ???– What are the respective proportions of

seroprevalence against Toscana & Punique in humans

• First step : Elisa tests

– 1 273 human sera – From different regions of Tunisia– Tested by Elisa for the presence of antibodies against

phleboviruses

516 sera Elisa positive

Microneutralisation tests

• Results

216 sera without any antibodies

7 sera with antibodies as specific for both viruses

21 sera with antibodies more specific for Punique

509 sera with antibodies more specific for Toscana

N 1/10 1/20 1/40 1/80

N 216 18 2 0 1 237

1/10 87 7 0 0 0 94

1/20 103 10 0 0 0 113

1/40 141 21 2 0 0 164

1/80 115 25 5 0 0 145

662 81 9 0 1 753

Punique virusTotal

Toscana virus

Total

• Results

On the 537 (753 – 216) reactive sera :

ü 4% have antibodies against Punique virus

ü 96% have antibodies against Toscana virus

N 1/10 1/20 1/40 1/80

N 216 18 2 0 1 237

1/10 87 7 0 0 0 94

1/20 103 10 0 0 0 113

1/40 141 21 2 0 0 164

1/80 115 25 5 0 0 145

662 81 9 0 1 753

Punique virusTotal

Toscana virus

Total

Microneutralisation tests


• Conclusions

– 96 % of infections are due to Toscana virus

– Toscana virus = main pathogen with an impact on public health in Tunisia

– Punique virus, with only 4% of infections, does not have an important public health impact in Tunisia: the question of its capacity to causedisease in humans remains and should be addressed by testingpatients with neuro-invasive diseases and with acute fever of unknownorigin through real-time RT-PCR assay in hospitals and in patients visiting general practitionners

F

In Tunisia

In Franceü Isolation of to viruses in south of France

à TOSV and MASV

à Distinct but antigenetically and genetically closely related

àWithin the Sanfdly fever Naples serocomplex

Seroprevalence study using comparative VNT(i) To determine wether MASV is able to infect humans

(ii) to estimate and to compare the respective involvement of TOSV and MASV in human infections in South of France

ü TOSV = recognised human pathogen

üMASV : no evidence suggesting that it is capable

à To infect humans

à To cause a disease

ArboMED / PRIAM project: arboviruses in the MediterraneanRisk perception of arboviral diseases in the Mediterranean

• Blood donor samples

• 3 study regions / 1 control region

• 14,000 sera collected in 2-weeks

• IgG seroprevalence

• tested for different arbovirusesincluding phleboviruses

• tested for Leishmania Ab

• tested for saliva Ab

• collection period: Sept-Oct 2012

studied regionscontrol region

PRIAM / ArboMED project

?

F

M

• Toscana virus and Massilia virus are present in France (Charrel et al Emerg Infect Dis 2007,

Charrel et al Vector borne zoonot dis 2009)

• what is the respective role of Toscana virus and Massilia virus ???

• ELISA results

-

France

France

Same methodology applied for sera from France

PRIAM project

0.37%

7.3%

2.5%

0.75%

1.7%

3%

PRIAM project

0.37%

7.3%

2.5%

0.75%

1.7%

3%

6.3%

5.5%

10.2%

9.7%

1.8%

7.9%

4.3%

3.7%

0.8%

2.8%

2.5%

1.2%

1.7%

2.5%

0.0% 0.7%

0.8%

0.0%


• seroprevalence studies with the capacity to discriminate between the differentviruses (neutralization tests) because other tests are cross-reactive

• Naples / Toscana / Tehran / Massilia / Punique / Granada• Sicilian / Utique / SFSV Turkey / SFSV Algeria

• impact on human health è specific PCR tests to be used in diagnostics in virology labsMassive sequencing using NGS is necessary to increase the number of sequences available in the databaess

• Continue and Extend virus discovery programs based on multidisciplinary teams(entomologists, ecologists, virologists, veterinarian, …)

Questions to be solved and Future directions

• Unité des Virus Emergents UMR190- Laurence Bichaud- Gregory Moureau- Cigdem Alkan- Xavier de Lamballerie- Geraldine Piorkowski- all members of the group

• Unité d'entomologie médicale IP Tunis- Elyes Zhioua- Ifhem Chelbi- all members of the group

• Unité d'entomologie médicale IP Alger- Idir Bitam

• Laboratoire de Parasitologie, Paris 13, Bobigny- Arezki Izri

• Tehran, Iran- Vahideh Moin-Vaziri

• MIVEGEC- Anne-Laure Banuls- Denis Sereno- François Renaud- Didier Fontenille

• Turkey universities- Bulent Alten- Yusuf Ozbel- Koray Ergunay- Ozge Kasap

Acknowledgements

Thank you for your attention

Contact: [email protected]

emerging and neglected sandfly-borne phleboviruses around

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