Mosquito-borne viral infection

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Human Pathogen 2

Structure and properties of virionTransmission and epidemiologyClinical features Laboratory diagnosis Treatment & control

Poonsook Keelapang, Ph.D.Department of Microbiology, Faculty of Medicine,Chiang Mai University

• Viruses that maintained in nature through biological transmission between susceptible vertebrate hosts by hemotophagous arthropods (mosquito, tick, sandfly) or through transovarian and possible sexual transmission in arthropods

• Viruses multiply and produce viremia in vertebrates, multiply in the tissues of arthropods and are passed on to new vertebrate by the bites of arthropods after a period of extrinsic incubation.

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Arthropod-borne viruses (Arboviruses)

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1. Plague2. Smallpox3. Crimean ‐ Congo hemorrhagic fever4. West Nile Fever5. Yellow fever6. Lassa fever7. Nipah virus disease8. Marburg virus disease9. Ebola virus disease10. Handra virus disease11. Severe Acute Respiratory Syndrome 12. Middle East Respiratory Syndrome 

ประกาศกระทรวงสาธารณสขุโรคตดิตอ่อนัตราย พ.ศ. ๒๕๕๙๓ มถิุนายน ๒๕๕๙

Family Flaviviridae (Flavus = yellow)

• Enveloped, spherical RNA virus (50-60 nm in diameter)

• Linear positive-sense ssRNA genome• Genus Flavivirus (53 species)

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EM: Dengue virus100 nm

3 structural proteins: Capsid (C), premembrane (prM/M) and envelope (E) (receptor binding, fusion, induce neutralizing Ab, hemagglutinating activity (goose RBCs)

7 nonstructural proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5

Replicate in cytoplasm, bud into ER

C prM E NS1 NS3 NS5NS2A NS2B NS4A NS4B

3’UTR5’UTRStructural proteins Nonstructural proteins

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Yellow fever virus Dengue virus

Murray Valley encephalitis virus

St. Louis encephalitis virus Japanese encephalitis virus

West Nile virus

Jawetz, Melnick, & Adelberg’s Medical Microbiology 27th Edition

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Major geographicdistribution

Primary vector

Reservoir Disease

Yellow fever virus Africa, South America

Aedes (Human, Monkey)

Hepatic necrosis,hemorrhage

Dengue virus Tropical and subtropical regions

Aedes (Human, Monkey)

Hemorrhagic fever

Zika virus Africa, Asia, South America

Aedes (Human, Monkey)

Fever+ rash

Japanese encephalitis virus

East and SoutheastAsia

Culex Bird, pig Encephalitis

West Nile virus Africa, Eastern Europe, Middle East, Asia, North America

Culex Bird Encephalitis

Murray Valley encephalitis virus

Australia Culex Bird Encephalitis

St. Louis encephalitis virus

America Culex Bird Encephalitis

Louping ill Britain Tick Sheep Meningitis Powassan Canada Tick Rodent EncephalitisRussian spring-summer encephalitis

Central Europe, USSR

Tick Rodent, bird Encephalitis

Yellow Fever Virus (YFV)• Mainly found in tropical Africa, south America, Caribbean • WHO estimates about 200,000 cases occur annually

• One antigenic type

Aedes africanus Aedes aegypti

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2015-2016AngolaCongo

Haemagogus ssp

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Outcome of YFV infection: Asymptomatic or a mild undifferentiated febrile illness: Classic yellow fever (incubation 3-6 days): sudden onset of fever,

headache, chills, generalized myalgia, nausea, vomiting

Severe cases (15-20%): fever, liver damage (jaundice), renal damage (oliguria, proteinuria), coagulation defects (nosebleed, bleeding gum, black vomit), hypotension seizures, coma, shock, and death.

Case mortality rate: 20-50%https://brianaltonenmph.files.wordpress.com/2013/02/12cb1852-ca52-a385-4df510507aee6c19_whitesofeyesturnyellow.jpg

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Prevention:Live-attenuated yellow fever vaccine YF-17D provides protection for >10 years; used for persons living in or traveling to endemic areas

Contraindications: • Allergy to a vaccine component • Age <6 months (8 months)• Immunocompromised (HIV with low

CD4 T cells, thymus disorder, abnormal immune function, malignant, transplantation, immunosuppressive)

(Age >60 years, pregnancy, breastfeeding)

Dengue virus (DENV)• 4 serotypes: DENV-1,-2,-3,-4 (40% difference)• Tropical and sub-tropical regions

2013: 390 millions infections/year; 96 millions casesThailand: 6-29% per year

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Guzman MG et al. 2014

DENV in Thailand

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Daytime biting, Breeds in clean water in household container

Aedes aegypti Aedes albopictus

Transmission • Sylvatic cycle (mosquito ↔ monkey)• Urban cycle (mosquito ↔ human)

• Female mosquito takes up DENV in infected blood meals midgut ovaries, brain and fat body salivary glands (~5-10 days) chronic infection (vertical/sexual transmission)

Viremia in human ~ 4-5 days (fever phase)

Vectors : Aedes ssp.

.

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DENV infection in human skin: immature dendritic cells, Langerhans cells lymphoid organs, blood: monocytes, macrophages,

B lymphocytes liver, spleen, kidney, lung, bone marrow, thymus, brain etc.Immune response• Primary infection (first serotype): CMI, HMI

• Serotype – specific : prolonged • Cross – reactive : short - term (3 - 9 months)

• Secondary infection - tend to be more severe ? Antibody-dependent enhancement ? Original antigenic sin

Fc-bearing monocytes or macrophages

• Severity: age (children), gender (female), nutrition (well-nourished), race (Asian, Caucasian), genetics, virus strain

Dengue Classification

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1. Severe plasma leakage2. Severe hemorrhage3. Severe organ impairment

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with warning signs

Dengue + Warning signs

without

Severe Dengue

Probable dengueLive in/travel to dengue endemic area. Fever and 2 of the following criteria:• Nausea, vomiting• Rash• Muscle & bone pains• Tourniquet test positive• Leukopenia• Any warning signs

Warning signs• Abdominal pain or

tenderness• Persistent vomiting• Clinical fluid accumulation• Mucosal bleed• Lethargy, restlessness• Liver enlargement > 2cm• Hematocrit• Platelet count

Laboratory-confirmed dengue

DENGUE GUIDELINES FOR DIAGNOSIS,TREATMENT, PREVENTION AND CONTROL (WHO,2009)

Severe plasma leakage• Shock (DSS)• Fluid accumulation with respiratory distressSevere bleedingSevere organ involvement• Liver: AST or ALT > 1,000 IU/ml

• CNS: impaired consciousness

• Heart and other organs

Outcomes of DENV infection Asymptomatic (majority) Symptomatic (incubation 4-7 days)• Undifferentiated fever

Fever without specific symptoms• Dengue fever (DF)

Fever with +2 of the following: headache, retro-orbital pain, arthralgia/bone pain, myalgia, rash, hemorrhagic manifestations, leukopenia

• Dengue hemorrhagic fever (DHF) without/with shock (DSS)Fever, hemorrhagic manifestations, thrombocytopenia, plasma leakage

• Expanded dengue syndrome/isolated organopathy(unusual manifestations e.g. hepatic and brain dysfunction)

http://naturalunseenhazards.files.wordpress.com/2010/07/dengue-shoulder.jpg

15Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever (WHO, 2011)

Hepatomegaly Increased vascular permeability

Hemorrhagic manifestations:(+) tourniquet

petechiae

FeverAnorexiaVomiting

Dehydration

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Thrombocytopenia

Leakage of plasmaHemoconcentration

HypoproteinmiaPleural effusion/ascites

Coagulopathy

Major manifestations/Pathophysiological changes of DHF

DHFSeverityGrade

I

II

III

IV

Hypovolemia

Shock

Death

Disseminated intravascular clot (DIC)

Severe bleeding(GI, brain, etc)

DFDHF/DSS

Nimmanitya, 1978Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever (WHO, 2011)

Spontaneous bleeding

Circulatory failure – weak pulse, narrow pulse pressure, hypotension, restlessness

Profound shock –undetectable BP and pulse

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http://www.cdc.gov/dengue/training/cme/ccm/images/tourniquet_test1.png

>10 จุด ตอ่ตารางนิ้ว

Inflate the cuff to a pressure exactly halfway between systolic and diastolic pressures for 5 minutes

Tourniquet test

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Phases of infection: DHFFebrile2-7 days

Convalescent2-4 days

Critical 24-48 hours

No aspirin

↑> 20%

< 100,000/mm3

Plasma leak to pleural or peritoneal spaces Improve appetite and

sense of well being

Intravascular fluid therapy

Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever (WHO, 2011)

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Diagnosis of dengue virus infection

1° infection 2° infection

IgG

IgM cut-off

IgG cut-off1:2560 HI

tite

r

IgM IgM

Onset of symptoms

viremia

Virus isolation

RNA, NS1

• Virus isolation (1-2 wks)Toxorhychites mosquito (intrathoracic), Mosquito cell lines (C6/36, AP-61), mammalian cell lines (Vero, BHK-21)

• Detection of viral genomeRT-PCR (1-2 days)

• Detection of viral antigen NS1 Ag (immunochromatographic test, ELISA) (15 min -3 hr)

Detection of antibody (2 sera – acute, convalescent) -IgM/IgG rapid test • Increased IgM titer (IgM detectable from d5-21 and persist for 2-3 months) • >4-fold rising IgG titer (HI, ELISA, NT) (IgG detectable from d6-30; 1°type-

specific, 2°cross-reactive)• IgM/IgG: 1°> 1.2 ; 2°< 1.2

viremia

Virus isolation

RNA, NS1

NS1antigen appears as early as day 1 after the onset of the fever and declines to undetectable levels by5–6 days

Dengvaxia® (CYD-TDV)(First approved human vaccine: Mexico, Brazil, Philippines, Thailand)

Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial Capeding et al., Lancet 2014; 384:1358-65 22

live recombinant tetravalent dengue vaccine

prME YF17D

0/6/12 month schedule

For use in individuals 9-45 years of age living in endemic areas

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Zika virus 1947 : First isolated from a sentinel monkey in Zika forest in Uganda 1954 : First report case in human in Nigeria

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Adapted from https://wwwnc.cdc.gov/travel/files/zika-areas-of-risk.pdf

11947 First documented in monkey in Uganda

1954 First human case in Nigeria

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2007 Outbreak in Yap185 cases

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2013Outbreak in French polynesia8510 cases

5 2014Outbreak in New Caledonia114 cases

62014-2016Outbreak in Central and South Americaestimated > 1.5 million cases

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Zika virus: 1 serotype, 2 lineages (African and Asian)Transmission:

Vector : Aedes ssp. (Ae. aegypti, Ae. albopictus, Ae. Africanus, etc.) Sexual transmission : semen, female genital tract (Penot et al., 2016, Moreira

et al. 2016) Platelet transfusion (Motta et al., 2016)Perinatal transmission (Besnard et al., 2014)

http://www.vox.com/2016/1/20/10795562/zika-virus-cdc-mosquitoes-birth-defects

Consequent of Zika virus infection• Asymptomatic : 80%• Symptomatic

– Incubation period: not clear (4-7 days?)– Symptoms: fever, skin rash, conjunctivitis, muscle and joint pain,

malaise and headache; usually mild and last for 2-7 days– Complications:

• Guillain-Barré syndrome• Zika virus infection during pregnancy may cause congenital brain

abnormalities such as microcephaly, metal retardation, and visual and auditory impairment; greatest risk if mother is infected during the first trimester

26https://static01.nyt.com/images/2016/07/23/nyregion/23ZIKA2/23ZIKA2-master675.jpg

https://med.stanford.edu/news/all-news/2016/09/how-zika-affects-cranial-precursor-cells/_jcr_content/main/image.img.620.high.jpg

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No specific treatmentNo vaccinePrevention• Protection against mosquito bites• For regions with no active transmission of Zika virus,

WHO recommends practising safer sex or abstinence for a period of 6 months for men and women who are returning from areas of active transmission

Diagnosis• RT-PCR (serum, plasma, urine*, saliva, CSF)• Zika-specific IgM , PRNT – difficult to interpret in endemic areas and

in travellers with a previous history of flavivirus infection or vaccination against flavivirus

Japanese Encephalitis Virus (JEV)

• First recognized in Japan (1871) • South Asia, Southeast Asia,

East Asia and the Western Pacific

• 67,900 cases/year• 15,000 human deaths/year

• Temperate area : epidemic, summer

• Tropical area : sporadic, rainy season,

• Children (<15 years) are principally affected

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JEV: 1 serotype, 5 genotypes

Vector: Culex tritaeniorhynchus, Cx. gelidus, Cx. vishnui, Cx. Annulus, Cx. pseudovishnui, Cx. fuscocephalus

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- Bite at night time- Breed in rice fields, irrigation canals and water pools

filled with stagnant water

Dead end (incidental) hosts: human, horse

Natural cycle

• JE virus is maintained in a cycle involving mosquitoes and vertebrate hosts, mainly pigs and wading birds

Amplifying hosts/Reservior: pig, birds (egrets & herons)

Clinical spectrum of JEV infection in human

• Asymptomatic:symptomatic = 25-1000:1

• Incubation period : 5 to 15 days1. Prodrome : sudden onset of severe headache, high fever,

nausea, vomiting2. Encephalitis stage: altered sensorium, convulsion, neck

stiffness, muscular rigidity, mask-like facies, acute flaccid weakness, movement disorder; Case fatality rate : 8.5-72%

3. Late stage: recovery or persistence of neurologic, cognitive, or psychiatric sequelae (45-75% of survivors)

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Anterior horn cells of spinal cord

Thalamus

Midbrain

Basal ganglia

Cerebral cortex

Cerebellum

31U.K. Misra, J. Kalita / Progress in Neurobiology 91 (2010) 108–120

severe neck (retrocollis-คอแอน่ไปดา้นหลงั) upper limb dystonia (กลา้มเนื้อแข็งเกร็งผดิรูป)severe wasting of both

the legs

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Laboratory:• RT-PCR (CSF) • CSF : Anti-JE IgM > 40 IU/ml• Serum: Anti-JE IgM > 40 IU/ml

JE IgM:dengue IgM ratio > 1

Clinical: fever + conscious change

Diagnosis

Treatment : no specific antiviral drug

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Personal protective measures to prevent mosquito bites

Human vaccine

- Killed, bivalent vaccine (Beijing-1 & Nakayama): prepared in mouse brain, 3 doses with 91% protection

- Inactivated Vero cell culture-derived JE vaccine (IXIARO)

- Live-attenuated, monovalent vaccine (SA14-14-2): propagated in primary hamster kidney cells; > 95% protection

- Live-attenuated, chimeric JEV/YFV vaccine (IMOJEV): propagated in Vero cells ; 95% protection

Prevention and control

Outbreak of West Nile Virus in the US.

34http://wwwnc.cdc.gov/eid/images/05-0289a-F1.gif

New York

West Nile Virus (first detected in Uganda 1937)(Middle East, Australia, Africa, Europe, America)

• Vector: Culex ssp. Reservoir: Bird

• Incidental hosts: human, horse

• Asymptomatic: 80%

• West Nile fever: fever, vomiting, skin rash with swollen lymph glands

• West Nile encephalitis (1 in 150): high fever, neck stiffness, disorientation, coma, tremors, convulsions, muscle weakness and paralysis. High risk: elderly (> 50 years) and immunocompromised persons (mortality rate: 3-15%)

• IgM antibody-capture ELISA (CSF, serum), no vaccine35

Family Togaviridae (toga=cloak)2 Genera: Alphavirus, Rubivirus

• E1 spikes act as attachment proteins, induce neutralizing activity and possess hemagglutinating activity

• Replicates in cytoplasm

E1(59 kDa)

E2C

• An enveloped, icosahedral RNA virus (70 nm in diameter) with a linear single-stranded, positive-sense RNA (11.8 kb)

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Major geographicdistribution

Primary vector

Reservoir Disease

Eastern Equine Encephalitis virus

North America Culex,Anopheles

Birds Encephalitis (mortality rate 30‐70%)

Western Equine Encephalitis virus

North America Culex,Anopheles

Reptile,(Bird?)

Encephalitis(mortality rate 3‐7%)

Venezuelan Equine Encephalitis virus

Central and SouthAmerica

Culex,Anopheles

Rodents Encephalitis

Chikungunya virus Africa, Asia Aedes Human Fever + rash + arthralgia

Onyong‐nong virus Africa Anopheles ? Fever + rash + arthralgia

Ross River virus Australia Culexannulirostris

Kangaroo, wallabies

Fever + rash + arthralgia

Mayaro virus South America Haemagogous Monkeys Febrile illness

Alphaviruses

Chikungunya virus (G. Alphavirus)(Makonde )= that which bends up

• First isolated in 1952 from the serum of a febrile patient during a dengue epidemic in Tanzania (Lumsden, 1955).

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3 genotypes: West African, Asian, East-Central-South African

Weaver et al., N Engl J Med 2015;372:1231-9.

Transmission - mainly through mosquito bites

Viremia persistsfor upto 5 days (up to 109 copies/ml)

Madariaga et al., Braz J Infect Dis 2016;20:91-8.

Extrinsic incubation period:7- 10 days

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Others: rodent, birdPrimary reservoir: human

40Couderc T & Lecuit M. Antiviral Research 2015;121:120–131.

Pathogenesis

Outcome of chikungunya virus infection• Asymptomatic infection: 3-25%• Incubation: 2-4 days (range 1-12 days)• High fever (>39°C), arthralgia (joint pain), headache, rash,

back pain, myalgia, nausea, vomiting and photophobia

• Migratory polyalthalgia, bilateral and symmetric. Most commonly in hands and feet joints. Joint swelling.

WHO SEAROBurt et al., Lancet 2012; 379: 662–71

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Infant (intrapartum transmission): neurologic disease, hemorrhagic symptoms, and myocardial disease

50-60% persistent joint pains for months or years

Case fatality rate = 1:1000

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Schwartz O & Albert ML. Nature Reviews Microbiology 2010; 8: 491-499.

Diagnosis• Virus isolation: < 3 days• Genome detection (RT-PCR): < 8 days• Anti-CHIKV IgM: > 5 days, peak at weeks 3 - 5 after the onset of

illness and persist for about 2 months• Anti-CHIKV IgG: (paired sera) 4 fold rising titer by HI or ELISA no specific treatment no vaccine

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Bunyaviruses

• Enveloped RNA virus (Ø 100-120 nm)

• Tripartite, linear negative-sense ssRNA genome

Major geographicdistribution

Primary vector

Reservoir Disease

California encephalitis virus

North America Aedesmelanimon

Rodent Encephalitis 

Bunyamwera Africa Aedes Rodent, rabbit Febrile illness

La Crosse America (Midwestern states)

Aedestriseriatis

Rodent Encephalitis(Mortality rate 1%)

Rift Valley fever Africa Aedes Sheep, Cattle Hemorrhagic fever (Mortality rate 50%)

Crimean ‐ Congo hemorrhagic fever  Africa, Asia , Europe     Tick  

Recommended readings

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