Sonia Mara Raboni2005

Laboratório de Virologia – Hospital de Clínicas da UFPR

IBMP – Instituto de Biologia Molecular do Paraná

Genus Genus Human diseaseHuman disease

BunyavirusBunyavirus LaCrosse encephalitis, LaCrosse encephalitis, othersothers

PhlebovirusPhlebovirus Rift Valley fever, sandfly Rift Valley fever, sandfly feverfever

NairovirusNairovirus Crimean-Congo Crimean-Congo hemorrhagic feverhemorrhagic fever

TospovirusTospovirus Plant virus, no known Plant virus, no known human diseasehuman disease

HantavirusHantavirus Hemorrhagic fever with Hemorrhagic fever with renal syndromerenal syndrome

Hantavirus pulmonary Hantavirus pulmonary syndromesyndrome

5 genera, 250 species5 genera, 250 species

Family Family BunyaviridaeBunyaviridae

HANTAVIRUS

80 – 120 nm Genoma s/s – Capacidade de

codificação L – 8,5 kb / L, ? M – 5,7 kb / G1, G2 S – 1,9 kb / N

Linear RNA genômico não

infecioso

No arthropod vector establishedNo arthropod vector establishedUnique among genera of BunyaviridaeUnique among genera of Bunyaviridae

Rodent hostsRodent hostsGenus and possibly species specificGenus and possibly species specific

TransmissionTransmissionAerosolization of rodent excretaAerosolization of rodent excreta

Characteristics of Characteristics of HantavirusesHantaviruses

Chronically infected Chronically infected rodentrodent

Virus is present in Virus is present in aerosolized excreta, aerosolized excreta,

particularly urineparticularly urine

Horizontal transmission of Horizontal transmission of infection by intraspecific infection by intraspecific

aggressive behavioraggressive behavior

Virus also present in Virus also present in throat swab and fecesthroat swab and feces

Secondary aerosols, mucous Secondary aerosols, mucous membrane contact, and skin membrane contact, and skin

breaches are also a breaches are also a considerationconsideration

Transmission of HantavirusesTransmission of Hantaviruses

Sin NombreSin NombrePeromyscus maniculatus

Rio SegundoRio SegundoReithrodontomys mexicanusReithrodontomys mexicanus

El Moro CanyonEl Moro CanyonReithrodontomys megalotisReithrodontomys megalotis

AndesAndesOligoryzomys longicaudatusOligoryzomys longicaudatus

BayouBayouOryzomys palustrisOryzomys palustris

Black Creek CanalBlack Creek CanalSigmodon hispidusSigmodon hispidus

Rio MamoreRio MamoreOligoryzomys microtisOligoryzomys microtis

Laguna NegraLaguna NegraCalomys lauchaCalomys laucha

MuleshoeMuleshoeSigmodon hispidus

New YorkNew YorkPeromyscus leucopusPeromyscus leucopus

JuquitibaJuquitibaUnknown HostUnknown Host MacielMaciel

Necromys benefactusNecromys benefactus

Hu39694Hu39694Unknown HostUnknown Host

LechiguanasLechiguanasOligoryzomys flavescensOligoryzomys flavescens

PergaminoPergaminoAkodon azaraeAkodon azarae

OrOránánOligoryzomys longicaudatusOligoryzomys longicaudatus

CCañaño Delgaditoo DelgaditoSigmodon alstoniSigmodon alstoni

Isla VistaIsla VistaMicrotus californicus

Bloodland LakeBloodland LakeMicrotus ochrogasterMicrotus ochrogaster

Prospect HillProspect HillMicrotus pennsylvanicusMicrotus pennsylvanicus

New World HantavirusesNew World Hantaviruses

BermejoBermejoOligoryzomys chacoensisOligoryzomys chacoensis

Peromyscus maniculatusDeer mouse

Sigmodon hispidusCotton rat

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromePathogenesisPathogenesis

Funcional derangement of vascular endotheliumNo cytopathic effectsImmunopathologic

Mediated by cytokine responses Pulmonary and cardiac effects

Thrombocytopenia consumptionsequestration (RE system)

Most FrequentMost Frequent OtherOther RareRare

FeverFever DizzinessDizziness RhinorrheaRhinorrhea

MyalgiaMyalgia ArthralgiaArthralgia Sore Sore ThroatThroat

Nausea/Nausea/VomitingVomiting

CoughCough

Shortness Shortness ofofBreath Breath (late (late in the course in the course

of disease)of disease)

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeClinical Clinical PresentationPresentation

TachypneaTachypnea

TachycardiaTachycardia

HypotensionHypotension

Crackles or rales on lung Crackles or rales on lung examination examination

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndrome

Physical ExaminationPhysical Examination

Bilateral interstitial Bilateral interstitial infiltratesinfiltrates

moderate to rapid progressionmoderate to rapid progression

Bilateral alveolar Bilateral alveolar infiltratesinfiltrates

Pleural effusionPleural effusion

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndrome

Radiographic FindingsRadiographic Findings

May 27, 1993May 27, 1993

May 30, 1993May 30, 1993

May 31, 1993May 31, 1993Source: Dr. L. Ketai

Radiographic Progression of Radiographic Progression of HPS HPS

in the Lungsin the Lungs

ChemistryChemistryLow albuminLow albumin

Elevated LDHElevated LDH

Elevated AST (SGOT)Elevated AST (SGOT)

Elevated ALT (SGPTElevated ALT (SGPT))

HematologyHematologyLow platelet count Low platelet count

Atypical lymphocytes Atypical lymphocytes

(immunoblasts)(immunoblasts)

Left shift on WBC differentialLeft shift on WBC differential

Elevated hematocritElevated hematocrit

Elevated WBCElevated WBC

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeCommon Laboratory FindingsCommon Laboratory Findings

+++

FeverFeverPulmonaryPulmonaryedemaedemaShockShock

DiuresisDiuresis

ProdromeProdromeCardiorespiratoryCardiorespiratoryConvalescenceConvalescence

ImmunoblastsImmunoblasts

HCTHCTASTASTLDHLDH

3-6 days3-6 days 7-10 days7-10 days

PlateletsPlatelets +

+++

+

++

+

++

++

+

+

++

+

+

++

+

+

++

+

+

+++++++

++++

Clinical Progression of HPSClinical Progression of HPS

Early aggressive intensive Early aggressive intensive carecare

Early use of inotropic agents Early use of inotropic agents (Dobutamine)(Dobutamine)

Early ventilationEarly ventilation Careful monitoringCareful monitoring

OxygenationOxygenation Fluid balanceFluid balance Blood pressureBlood pressure

HPS HPS ManagementManagement

SerologySerologyIgMIgM

IgGIgG

ImmunohistochemistryImmunohistochemistry

Reverse transcription Reverse transcription and polymerase chain and polymerase chain reaction (RT-PCR)reaction (RT-PCR)

Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeLaboratory-confirmed Laboratory-confirmed

DiagnosisDiagnosis

Peridomestic exposurePeridomestic exposure

Peridomestic & occupational Peridomestic & occupational

exposureexposure

Peridomestic & recreational Peridomestic & recreational

exposureexposure

Occupational exposure Occupational exposure

Entering/cleaning rodent-infested Entering/cleaning rodent-infested structuresstructures

Armstrong, L.R. et al., JID 1995; 172 (October)Armstrong, L.R. et al., JID 1995; 172 (October)

69% (48/70)69% (48/70)

19% (13/70)19% (13/70)

9% (6/70)9% (6/70)

4% (3/70)4% (3/70)

9% (6/70)9% (6/70)

Rodent ExposureRodent Exposure70 confirmed HPS 70 confirmed HPS

casescases

Risk groupRisk groupForest workersForest workers11

Health care workersHealth care workers22

Prodromal HPSProdromal HPS33

ContactsContacts44

Rural OCCRural OCC55

Rodent workersRodent workers66

TotalTotal

Location/timeLocation/timeSW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1994SW US, 1994US, 1994US, 1994

Positive/tested (%)Positive/tested (%)0/1430/1430/3960/3963/299 (1.0%)3/299 (1.0%)3/239 (1.3%)3/239 (1.3%)1/522 (0.2%)1/522 (0.2%)8/932 (0.9%)8/932 (0.9%)15/2531 (0.6%)15/2531 (0.6%)

1. Vitek et al, 19961. Vitek et al, 1996 2. Vitek et al, 19962. Vitek et al, 1996 3. Simonsen et al, 19953. Simonsen et al, 19954. Zeitz et al, 19954. Zeitz et al, 1995 5. Zeitz et al, 19955. Zeitz et al, 1995 6. Armstrong et al, 19956. Armstrong et al, 1995

Prevalence of SNV IgG Prevalence of SNV IgG Antibodies in Select U.S. Antibodies in Select U.S.

PopulationsPopulations

CountryCountry Positive/tested (%)Positive/tested (%) TimeTimeParaguayParaguay11 44/345 (12.8%)44/345 (12.8%) 19951995Western ParaguayWestern Paraguay2 2 78/193 (40.4%)78/193 (40.4%) 19931993

(Indian Population)(Indian Population)

ArgentinaArgentina33 <1%<1% 19961996 Salta ProvinceSalta Province22 38/222 (17.1%)38/222 (17.1%) 19931993(Indian Population)(Indian Population)

ChileChile33 2-13%2-13% 19971997

1. Williams, 19971. Williams, 1997 2. Ferrer, 1998 3. Peters, 1998; Weissenbacher, 1996 2. Ferrer, 1998 3. Peters, 1998; Weissenbacher, 1996

Prevalence of SNV IgG Prevalence of SNV IgG Antibodies in Select South Antibodies in Select South

American PopulationsAmerican Populations

Control Mice InsideControl Mice Inside

Control Mice OutsideControl Mice Outside

Use Safety PrecautionsUse Safety Precautions

HPS PreventionHPS Prevention

TRANSMISSÃO TRANSMISSÃO AÉREAAÉREA

SafetySafety Label as infectious substance and/or Label as infectious substance and/or human human blood precautions blood precautions Double container with absorbent material Double container with absorbent material sufficient sufficient for volume being sentfor volume being sent Plastic tubes preferable over glassPlastic tubes preferable over glass

ConditionsConditions Sera -- room temperature or cold pack Sera -- room temperature or cold pack Clot or buffy coat -- dry iceClot or buffy coat -- dry ice Fresh tissues (1-cm cubes) -- dry iceFresh tissues (1-cm cubes) -- dry ice Formalin-fixed tissue and blocks -- room Formalin-fixed tissue and blocks -- room temperature (don't freeze)temperature (don't freeze)

Safety PrecautionsSafety Precautions

HANTAVIROSES NO BRASIL

HPS

Síndrome Pulmonar por Hantavirus 1993 Vírus Sin Nombre

Brasil Novembro/93 – juquitiba(SP) Sazonalidade: roedores

Paraná Maior numero de casos identificados

Hantavirose: Casos e Óbitos, Brasil

1993-2004*

8 2 10 8

166

83 8541

233

79

502

213

0

100

200

300

400

500

600

Norte Nordeste Sudeste Centro-Oeste

Sul Total

Casos Obitos

*Dados parciais Dez/2004. Fonte: Ministério da Saúde

Hantavirose: Série Histórica do Paraná,

1992-2004*

4 42 2

85

26

6

32

1315

6

14

6

10

5

0

5

10

15

20

25

30

35

1992 1998 1999 2000 2001 2002 2003 2004

Casos Obitos

*Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações

N=111

Map of Brazil showing the state of Parana where the HPS cases have occurred

Hantavirose: Dados Demográficos, Paraná

1992-2004*Distribuição de Casos

por Sexo

M94%

F6%

0

5

10

15

20

25

30

35

0-9a 10-19a

20-29a

30-39a

40-49a

50-59a

60-69a

Distribuição por Faixa Etária

* Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações

Hantavirose: Ambiente de Contaminação, Paraná, 1992-2004*

6% 3%

90%

1%

Domiciliar

Lazer

Trabalho

Ignorado

* Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações

Hantavirose: Evolução das Ocupações, Paraná, 1992-2004*

0

5

10

15

20

25

1992 1998 1999 2000 2001 2002 2003 2004

Agricultura Relacionado ao Pinus

* Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações

Hantavirose – Coleta de Roedores

N = 181 (+ 13,2%)

Oligoryzomys sp (19 – 79,1%)

Akodon sp (4 – 16,6%)

Trichomys sp (1 – 4,1%)

Fonte SESA;CSA;DV zoonoses e intoxicações

Hantavirose - Inquérito Soroepidemiológico

General CarneiroN = 14219,7%(+)

HANTAVIROSESPropostas

Investigar sorologias de contatos com pacientes soropositivos para analisar transmissão inter-humana;

Caracterização genômica dos hantavirus detectados em pacientes e camundongos;

Produção de antígeno N-recombinante para o desenvolvimento de ensaio para detecção sorológica desta infecção;

Objetivos

Epidemiologia Molecular

Correlação Clínica x Molecular Produção de antígeno recombinante para diagnóstico

Desenvolvimento de métodos de diagnóstico laboratorial

Inicio 0ut/2002

Clinical Survey of Hantavirus in Southern Brazil and the Development of an Specific Molecular Diagnosis Tools

Sonia M Raboni1, Gisélia Rubio2, Luana de Borba1, Aurélio Zeferino1, Irene Skraba3, Samuel Goldenberg1 and Claudia N

Duarte dos Santos1 1 Instituto de Biologia Molecular do Paraná, IBMP

/Fiocruz/Brazil 2 Secretaria Estadual de Saúde do Paraná, Brazil 3 Laboratório Central do Estado do Paraná, Brazil

Am J Trop Med Hyg, 2005, in press

Table 1. Clinical and Laboratory Records from Brazilian HPS Patients

Clinical or Laboratory Data Frequency n/N %

Fever 72/82 87.8

Headache 70/82 85.3

Cough 67/81 82.7

Myalgia 66/81 81.4

Thoracic pain 49/81 60.4

Dyspnea 54/82 65.8

Vomiting 39/82 47.5

Hypotension (SP <100 mm Hg) 19/38 50

Interstitial infiltrate 38/47 80.8

Pleural effusions 1/38 2,6

Hematocrit >45% 52/71 73.2

Thrombocytopenia (<150,000/mm3)

45/63 71.4

Leukocytosis (>10,000 cells/mm3)

22/54 40.7

Creatinine > 2,0mg/dl 19/29 65.5

0

5

10

15

num

ber o

f cas

esFigure 2. Seasonal Distribution of

Hantavirus Pulmonary Syndrome, 1998-2004, Paraná State, Brazil

1998 1999 20022000 2001 2003 2004

nRT-PCR S Segment

434 pb

RT-PCR Nucleoproteína

434 pb

Table 3: nRT/PCR for hantavirus detection from blood and clot fractions from patients with hantavirus pulmonary

syndrome

N Patient* nRT-PCRPrimers Johnson

et al.

nRT-PCRBrazil-specific Primers

N Patient* nRT-PCRPrimers Johnson

et al.

nRT-PCR

Brazil-specific Primers

1 BR/01-50 - + 12 BR/02-85 - +2 BR/01-51 - - 13 BR/02-86 NT -3 BR/01-52 - - 14 BR/03-91 NT -4 BR/01-55 - + 15 BR/03-92 NT -5 BR/01-60 - - 16 BR/03-95 NT +6 BR/02-67 - - 17 BR/03-97 NT +7 BR/01-69 - - 18 BR/03-98 NT +8 BR/02-71 - + 19 BR/03-99 NT +9 BR/02-72 - + 20 BR/03-100 NT +10

BR/02-73 - + 21 BR/03-101 NT +

11

BR/02-74 + - 22 BR/04-102 NT +

60%

Hantaviruses in Central South America: Insights from the Phylogenetic Analysis of

the S Segment from HPS Cases in Paraná, Brazil

Sonia M Raboni, MD, MSc;* Christian M.Probst, MD,MSc.*; Juliano Bordignon, MSc;* Aurélio Zeferino;* Claudia N.

Duarte dos Santos, PhD*#.

*Instituto de Biologia Molecular do Paraná, IBMP /Fiocruz/Brazil

Universidade Federal do Paraná, BrazilJ Med Virol, 2005, in press

0.1

SEO

DOB

ILV1

MO46

PHV

TUL

PUU

TOP

KHU

BAY

MUL

BCC

PRG

MAC

OWR

ACRF

ACB

LPM

JM

AS

LNA

CAN

DA

GFE

APM

VS

BER

LEC

Hu39694

ORN

AND Nort

9717869

9718133

CHI 7913

AH 1

LN

OM 556

RM 97

RMx 1

CC107

CC074

SN 77734

NM H10

NM R11

MON

H NY1

RI 1

NYa

Paraná

HantaanSeoulDobrava

PuumalaProspect Hill

PrgMac

BerLec

Andes

LNV

SNV

Brazil Hantavirus Recombinant Nucleoprotein

Objectives

Produce a recombinant antigen to detect IgM antibodies against hantavirus by immunoblot and/or capture ELISA

The development of region-specific antigens should be undertaken to improve serological reactivity

-hantavirus -histidina

BM 1 2 3 4 5 6

48kDa

6 5 4 3 2 1 BM 6 5 4 3 2 1 BM

50kDa

40kDa

1. NI

2. 0,5mM IPTG

3. 1mM IPTG

4. NI

5. 0,5mM IPTG

6. 1mM IPTG

30oC

37oC

50kDa

40kDa

Ensaio ImunoenzimáticoIgG

EIE IgG: AMOSTRAS IgM POSITIVAS

5

4

5 5

10

9

0

2

4

6

8

10

12

PCR + PCR -

Positivo NegativoTotal

N = 19

47% de positividade

Soroteca: IBMP

IgG: AMOSTRAS IgM POSITIVAS

PositivasColeta (dias)

Media: 4 dias

NegativasColeta (dias)

Media: 5,4 dias

ObservarAmostras com

resultados de IgG desconhecidos

Períodos de coleta variados

Sem coleta de segunda amostra para avaliar soroconversão

Maior parte das amostras: coágulos ou material hemolisado

IgG: AMOSTRAS IgM POSITIVAS

IgG: Estudo Soroepidemiológicode General Carneiro

9

99

010

71

10

20

40

60

80

100

Kit IBMP Kit Focus

reagente não reagente indeterminado

N = 107

N = 82

IgG: Estudo Soroepidemiológicode General Carneiro

82 Amostras testadas pelos dois kits

14 amostras tiveram resultados discordantes

Repetido estes testes pelo kit IBMP e pelo kit Pergamino (Argentina)

7 7

0

7 7

00

2

4

6

8

kit IBMP kit PERG

reagente não reagente

indetem

N = 14

IgG: Estudo Soroepidemiológicode General Carneiro

11 amostras foram selecionadas para teste de imunoblotting Todos os

resultados foram concordantes com os testes IBMP e Pergamino

IgG: Estudo Soroepidemiológico

de General Carneiro - Conclusão Gold Standard

EIEIBMP

Positivo

Negativo

Total

Positivo 5 0 5

Negativo

0 77 77

Total 82

PELO TESTE IBMP:

Prevalência de 8,4% (9/107) de anticorpos IgG anti-hantavirus na população de General Carneiro.CONSIDERANDO COMO VERDADEIRO POSITIVO OS RESULTADOS CONCORDANTES EM 2 TESTES E O WESTERN BLOT PARA OS RESULTADOS DISCORDANTES TEM-SE: (Em 82 amostras analisadas por pelo menos 2 testes)

)

Neste estudo:

S = 100% e E = 100%

IgG: Doadores de Sangue

1 2 21 2 3

81

98

115

83

102

120

0

20

40

60

80

100

120

data 23/nov data 26/nov data 29/nov

reagente indeterminado não reagente total

Soroteca Hospital de Clínicas - UFPR

Cut off: Média DO + 3x Desvio padrão

Indeterminado: 10% acima ou abaixo do Cut off.

N = 305

IgG: Doadores de Sangue

30 soros que tiveram absorbância acima de 0,2 foram repetidas com o kit IBMP 18 mantiveram absorbância acima de

0,2 na repetição. 17 destas foram avaliadas pelo teste de

imunoblotting 3 amostras foram positivas

IgG: Doadores de Sangueimunoblotting

Ensaio ImunoenzimáticoIgM (em fase de padronização)

IgM: Comparação de amostras da Soroteca do

IBMP25

13

0

5

10

15

20

25

kit IBMP

reagente não reagente

Das 13 amostras negativas: 5 amostras eram de casos

negativos As outras 8 amostras

negativas foram repetidas com kit IBMP, Pergamino e Focus:

6 foram negativas 1 negativa IBMP e positiva

pelos outros 2 kits 1 negativa IBMP e

Pergamino, mas indeterminada com kit Focus

N = 38