Anna Papa, MD, PhDAristotle University of Thessaloniki, Greece

Clinically important arboviruses in Europe -2

CCHFVGenus Nairovirus

Family Bunyaviridae

Symptoms: High fever, hemorrhagic manifestations.Fatality rate: up to 30%.

Pathophysiology: increased vascular permeability.Cytokine storm.

Crimean-Congo hemorrhagic fever

Routes of transmission to humans

1. bites of infected Ixodid ticks2. contact with blood or tissues from

viremic patients or livestock.

Seasonal distribution: from March to October.Groups at risk: farmers, shepherds, woodcutters, veterinarians, military personnel, health-care workers.

barrier nursing

precaution measures

Bente et al 2013

• 2-host tick• Capable to survive in a wide range oftemperature and humidity conditions.Prefers the Mediterranean climate of N.Africa and S. Europe with low tomoderate levels of humidity and a longdry and hot season during the summermonths.• Both immature and adult stages arecharacteristically found in steppe,savannah and scrubland hill and valleybiotypes.

Hyalomma marginatumMediterranean Hyalomma

Distibution of Hyalomma marginatum (main vector of CCHFV)

50o North latitude: limit for geographical distribution of Hyalomma ticks

Geographic distribution of CCHF

Sargianou M, Papa A. Expert Rev Anti Infect Ther. 2013

The most extensive distribution among human tickborne viruses.The most widely distributed agent of severe HF known.

88%

Since 2002 >9000 reported casesFatality 5%

CCHF in Turkey

CCHF in India

Pakistan, Iran, and Afghanistan are endemic to CCHF. The active areasare those in the border between the three countries. The movements ofthe nomads across the borders with their animals including thoseinfested with Hyalomma ticks are responsible for transmitting the virusto both the animals and the humans.

2016: CCHF in Spain1 fatal case + 1 nurse who survived

Pathogenesis of viral hemorrhagic fevers

Course of the disease

Ergonul et al. Lancet 2006

Cytokines: IL-6, IL-10, TNF-α, and IP-10, MCP-1

Similarities with Ebola virus disease

• Supportive medical management: monitor fluid and

electrolyte balances, administration of fresh frozen plasma or

products.

• Ribavirin (not FDA approved). Early in the course of the

disease.

Systematic review and meta-analysis revealed that the available data in the literature are inadequate to support a claim of efficacy of ribavirin in CCHF. Need to conduct a randomized placebo-controlled trial.

Ascioglu et al. JAC 2011

Treatment of CCHF

The utility of ribavirin in CCHF is highly debated !

T-705 is highly potent against CCHFV in vitro exceeding the efficacy of the current standard drug, ribavirin. Co-administration of ribavirin and T-705 yields beneficial rather than adverse effects.

It acts as nucleotide analog that selectivelyinhibits the viral RNA polymerase or causeslethal mutagenesis upon incorporation intothe virus.

Animal models

1.

2.

3.

Vaccine in Bulgaria: formalin-inactivated suckling mouse brain-derived virus suspension.

Vaccines

Hewson et al, 2007

Geographic correlation of genotypes

The Greek AP92 strain

1975. Isolation of AP92 from R.bursa ticks, collected from goats inVergina village.The veterinarian working with thisstrain presented high titers ofantibodies against CCHFV, with nosymptoms of the disease.

1980-81. seroprevalence 6% in the area. None of the seropositivepersons recalled any symptoms resembling CCHF, leading to theproposal that probably AP92 strain is not a human pathogen.

Patient: a 46-year-old woman living 2 km N. of Komotini city. No travel abroad. Agricultural worker (tobacco plantation). Few goats nearby her home. Tick bite.

Dugbe virusU04958, AP92, Greece

U75674, IbAr10200, NigeriaDQ211646, SPU97/85, S. Africa

U88415, SPU415/85, S. AfricaU88416, UG3010, Uganda

AJ538196.Baghdad 12, IraqDQ211645, Oman, Oman

AF527810, Matin, PakistanAY366375, 729/02, Iran

AY297692, TAJ/HU8975 AF481799, Uzbek/TI10145, Uzbekistan

AJ010648, BA66019, ChinaAF481802, STV/HU29223, Russia

EF432640, Kelkit S/hu1/2004, TurkeyDQ211643, Drosdov, Russia

DQ211644, Kashmanov, RussiaDQ206447, ROS/HUVLV-100, Russia

AY550256, BUL6, BulgariaAF449482, Al/kukes3/01, Albania

EU871766, Rodopi, 2008, GreeceFJ160262, Gotze Deltsev, 2008, Bulgaria

AY550255, BUL3, BulgariaAY550254, BUL2, Bulgaria

AY550258, BUL10, BulgariaAY550253, BUL1, BulgariaEU727456, GOU-OT07, TurkeyEF012361, 2005-54, TurkeyAY277676, Bul/Hu517, Bulgaria

DQ211649,200310849, TurkeyDQ133507, Hoti, KosovoAF404507, Kosovo

EF432649, Kelkit S/hu11/2005, TurkeyAF428144, 9553/01, KosovoDQ211640, ArD15786, Senegal

U88411, DAK8194/ArD8, Senegal

EUROPE 2

AFRICA 3

AFRICA 2

ASIA 2

ASIA 1

EUROPE 1

AFRICA 10.1

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Two CCHFV lineages are present in Greece

Europe

2nd International Conference on Crimean-Congo Hemorrhagic Fever

10-12 September 2017Makedonia Palace hotel, Thessaloniki, Greece

The Conference is organized under the auspices of the Medical School, Aristotle University of Thessaloniki

www.med.auth.gr/cc-conference-2017

Bunyaviridae family

Main vectors of phleboviruses: phlebotomine sandflies

EtymologiaPhlebotomus: from the Greek words phlebo + tomi=opening a vein

Only females bite!

70 antigenically distinct serotypes:

• Sandfly Fever group – 55 serotypes (most transmitted by sandflies, few by mosquitoes, e.g. Rift Valley fever)

•Uukuniemi group – 13 serotypes (transmitted by ticks).

• Severe fever and thrombocytopenia syndrome (SFTS) virus (transmitted by ticks).

TAXONOMY

VIRION

Enveloped, spherical. Diameter 80-120 nm. Glycoproteins serve as neutralizing and hemagglutinin-inhibiting antibody targets and are exposed to selective pressure.

GENOME: RNA

S : N protein and a NSs. Uses an ambisense coding strategyM : precursor of the viral glycoproteins Gn and Gc, and NSm. L : viral RNA polymerase.

Segmented negative-stranded RNA genome. Encodes for 6 proteins.

• > 500 different species

• Widely distributed in Med countries from May to September. The number increases after rainy season.

• Abundant in peri-urban and rural environments, close to domestic animals and human populations.

• A cool, shaded, slightly damp environment is ideal for the sandfly life.

• Active during night - silent flight - short flight range.

The sandfly becomes infected when biting an infected human in the period between 48 h before the onset of fever and 24 h after the end of the fever, and remains infected for its lifetime

Phlebotomine sandflies (Psychodidae)

1908: Austrian military commission (Doerr et al) working on the Dalmatian coast investigated the etiology and transmission of sandfly fever. Vector: P. papatasi .

Before World War I: British investigators in Malta.

During World War II: 8,500 cases among US soldiers in the Sicily campaign (summer 1943), Naples outbreak (summer 1944).

1943-44: Isolation of Sicilian and Naples strains from patients in Palermo, Sicily and Naples (Sabin).

August 1944: First use of DDT for the control of malaria vectors, decrease of SF cases.

1948: the largest outbreak occured in Serbia (>1 million cases).

1971: Isolation of TOSV from P. perniciosus in Tuscany, Italy.

1983: Association of TOSV with neurologic disease.

Sandfly fever viruses: Historical perspectives

Although sandflies are widely distributed, most sandfly fever cases occur around the Meditteranean Basin.

The incidence of SF in the Americas is relatively low. The sandflies there are sylvan and do not enter houses to bite people.

Meditteranean Sea

• Sandfly fever Sicilian virus• Sandfly fever Naples virus• Toscana virus

• Asymptomatic infections• Mild disease, flu-like symptoms• CNS infections (meningitis, encephalitis)

Phleboviral infections

Phlebotomus fever, papataci fever, 3-day fever

A few days (2-5) after the infective bite, a feeling of lassitude, abdominal distress and chills develop followed by high fever, severe frontal headaches, muscle and joint pain, flushing of the face, conjunctiva injection, and tachycardia. After 2 days the fever begins to subside and the temperature returns to normal. Fatigue, depression, bradycardia and subnormal blood pressure may persist from few days to several weeks.Recovery is usually complete.

Similar symptoms to dengue fever, but without rash.

Sandfly fever

Toscana virus infections

- Asymptomatic- Influenza-like syndrome- Meningitis or meningoencephalitis in summer

May to October, with peak in August. Central Italy: 80% of acute CNS viral infections in children and 50% of cases in adults during summer. Central Anatolia, Turkey: 15.7% of aseptic meningitis.Portugal: 5.6% of meningitis cases.

Seroprevalence varies among countries and among regions of a country.

Italy: 3% in northern Italy (Torino), to 16% in Umbria and 22% in central Italy. Spain: 5% to 26%. France (south-eastern ):12% in blood from donors.

Turkey: 17.8%

Cyprus: 20%, Greece 50% in Ionian islands

Virus isolation and RT-PCR in blood or cerebrospinal fluid is only possible in early stages of infection i.e. the first 1-2 days after symptom onset and before the IgM detection.

In most cases the diagnosis is based on serological testing (ELISA, IFA) of acute and early convalescent sera.

Serological cross reactions exist within the antigenic complex. Neutralization assays using early convalescent sera are the reference method to identify the viruses or to assess the antibody response specificity.

Laboratory diagnosis of phleboviral infections

* The patient has lifelong immunity to the infecting phlebovirus but not to heterologous serotypes.

Prevention of sandfly bites (insect repellents)

Control of sandflies and their breeding grounds with insecticides

Prevention

Ordinary mosquito nets and screening are not sufficient to prevent sandfly bites: unfed female flies can pass through.

SF outbreak in Cyprus, 2002

Time: May to September 2002

Place: Cyprus, Athalassa National Forest Park, 5km SE of Nicosia

Patients: 256/581 (44%) soldiers of the Greek Army forces, 17-22 y.

Signs and Symptoms: high fever (39-41oC), headache, rigors, arthralgia, myalgia,

lower back pain.

Duration of symptoms: 60-72 h. Post infectious asthenia sydrome (2 weeks)

Laboratory findings: leucopenia, monocytosis, 2X elevated transaminases

(mainly ALAT)

Laboratory diagnosis:

Genetic detection of a SFSV-like strain (Cyprus virus)

Isolation in Vero E6 cells

Serological cross-reactivity for phleboviruses

0 24h 48h

δ=100 nm

Cyprus virus isolated from patient’s blood on the 1st day of illness

Cyprus – Sicilian:6.7% at nt level1.5% at aa level

Phylogenetic tree including Cyprus virus (partial L segment)

Summer of 2007 and 2008

Turkey

France

Tunesia

Spain

Algeria

D10759, Uukuniemi virus

DQ862467, Arbia virus, Italy

Adria virus, sandflies pool 5, Albania

Adria virus, sandflies pool 1, Albania

X56464, Rift Valley Fever virus, Egypt

AY293623, Chios virus, Greece

EF095551, Sicilian virus, Italy

AY962268, Cyprus virus, Cyprus

DQ656072, Massilia virus, France

EF095548, Naples virus, strain Poona, France

FJ153281, Toscana virus, France

FJ153280, Toscana virus, Spain

FJ153279, Toscana virus, Spain

X68414, Toscana virus, Italy

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ADRV vs ARBV: 22.9% and 6.7% at nt and aa level

0.1

Adria virus

2005

D10759, UUKV

AY293623, Chios virus, Greece

GQ165521, Corfou virus, Greece

GU233649, Utique virus, Tunesia

GU233647, Utique virus, Tunesia

AY962268, SFSV, Cyprus

EF095551, SFSV, Italy

DQ375430, RVFV

X56464, RVFV

DQ862467, ARBV, Italy

Adria-1, Albania

Adria-5, Albania

Adria-15, Greece

DQ656072, Massilia virus, France

DQ975233, TOSV, Italy

FJ153281, TOSV, France

FJ153280, TOSV, Spain

FJ153279, TOSV, Spain

GQ165522, Tehran virus, Iran

GU183868, SFNV, Italy

EF095548, SFNV, France

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Salehabad

serocomplex

0.1

23 Sept. 2009.

2.5-y boy admitted to a hospital in

Thessaloniki, Greece, because of a first

episode of febrile seizures. No underlying

disease.

Fever (38.2˚C) and vomit while in the

nursery school. Sudden adherence of eye

gaze, peroral cyanosis, masseter muscle

spasm, tonic convulsions of the body and

extremities and involuntary loss of urine.

The episode lasted 3 minutes. He

presented irritability and felt sleepy. By the

time he entered the hospital, he was

apyretic.

Clinical examination normal. leukocytosis

(22.600/μl) - 85.7% neutrophils.

Full recover, discharge 2 days later.

Adria is pathogenic to humans

30.6.2012. Female, 49 y.

PCR in CSF: Toscana virus lineage C.

Corfu virus

Chios virus*

Lefkas virusAdria virus*

Phleboviruses in Greece

Cyprus virus*

TOSV*SFSV*

Phlebovirus infections in Greece, 2010-2014

Time: April to October 2010-2014Tested: 494 patients with febrile syndrome (50% with CNS infection)Results: 43 (8.7%) patients had a phlebovirus infection.Phleboviruses account for 7-17% of CNS infections.

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Phlebovirus infections 2010-2014

Total Phlebo pos

CNS phlebo pos

SFTSV or Huaiyangshan virus

Since 2007 many cases of a life-threatening disease with sudden fever, gastrointestinal symptoms, thrombocytopenia, and leukopenia were reported in Henan and Hubei provinces, China. After that the virus was detected in additional provinces. Fatality 30%.Patients reported of tick bites. A novel phlebovirus was identified designated SFTS bunyavirus. Although closely related to Uukuniemi virus, this virus was distinct.

KF892040, LEIV-21C

KF767460, LEIV-279Az

HM566191, Zaliv

KF767463, LEIV-13841Ka

NC 005214, UUKV

HM566159, EgAN

JF838324, Chize

JF838327, Grand Arbaud

HM566181, Precarious Point

JF838333, RML-105355

KF892052, Rukutama

KF892046, Khasan

KM048315, H5

KJ746874, RTS2

KM048313, H12

KJ746873, RTS1

KM048311, FI6

KJ746901, ADTPV-1

Antigone2-Imathia-Greece-2012

Antigone1- Pella-Greece-2013

Antigone1-Ioannina2-Greece-2012

Antigone1-Imathia-Greece-2012

Antigone1-Ioannina1-Greece-2012

JQ956376, Bhanja

JQ956379, Palma

KF848980, HIGV

HQ830166, JS26

JQ670934, AHL/China/2011

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0.00.10.20.30.4

Tick-borne phleboviruses in the Mediterranean countries

Greece