hantavirus christian a. garcía sepúlveda md phd laboratorio de biología molecular facultad de...

HantavirusChristian A. García Sepúlveda MD PhDLaboratorio de Biología MolecularFacultad de Medicina Universidad Autónoma de San Luis Potosí

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Hantavirus

Introduction

• Hantaviruses are rodent-borne viruses which may be transmitted to humans in aerosolized urine, feces, or saliva, and occasionally by bite.

• Hantaviruses cause three diseases in humans:

Hemorrhagic Fever with Renal Syndrome (HFRS)Hantavirus Pulmonary Syndrome (HPS) Nephropatia epidémica (NE)

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Hantavirus

History

• While HPS has only been identified since 1993, HFRS was known to Russian doctors since 1913.

• HFRS recognized in China as early as 1000 years ago (Liao Dynasty 907-1125).

• Japanese troops in Manchuria (1934).

• Russian physicians first to implicate mice in transmission (1939).

• Japanese physicians compile clinical and pathological description of “Epidemic Hemorrhagic Fever” (1940).

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Hantavirus

All in a name

• In 1951 hemorrhagic fever outbreak in Korea during world war II affected 3,200 soldiers and resulted in a 7-15% mortality rate.

• Outbreak occurred along Hantaan River (tributary of the Imjin).

• Korean hemorrhagic fever (KHF) drew widespread international attention and was reclassified as HFRS in 1983 by the WHO along with “Epidemic Hemorrhagic Fever”and Russian Hemorrhagic fever outbreaks.

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DNAVirus

RT

ssDNA

dsDNA

Virus

ssRNA+

dsRNA

ssRNA-

RNAVirus

Hantavirus

Virology - Species

Rabies

Ebola

Flu

Lassa

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Hantavirus

Virology - Species

• Bunyaviridae family 5 genera, 250 species

Genus Human disease

Bunyavirus LaCrosse encephalitis, others

Phlebovirus Rift Valley fever, sandfly fever

Nairovirus Crimean-Congo hemorrhagic fever

Tospovirus Plant virus, no known human disease

Hantavirus Hemorrhagic fever with renal syndrome

Hantavirus pulmonary syndrome

More than 20 types are human pathogens.

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Hantavirus

Virology - Species

Prospect Hill virus (PHV)

Puumala virus (PUUV)

Rio Mamore virus (RIOMV)

Rio Segundo virus (RIOSV)

Seoul virus (SEOV)

Sin Nombre virus (SNV)

Thailand virus (THAIV)

Thottapalayam virus

(TPMV)

Topografov virus (TOPV)

Tula virus (TULV)

Akabane virus

Bakau virus

Bunyamwera virus

Andes virus (ANDV)

Bayou virus (BAYV)

Black Creek Canal virus (BCCV)

Cano Delgadito virus (CADV)

Choclo virus (CHOV)

Dobrava-Belgrade virus (DOBV)

Hantaan virus (HTNV)

Isla Vista virus (ISLAV)

Khabarovsk virus (KHAV)

Laguna Negra virus (LANV)

Muleshoe virus (MULV)

New York virus (NYV)

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Hantavirus

Virology - Species

• Hantavirus are similar to other Bunyaviridae members in:

– Negative sense RNA genome (ssRNA-)

– Lipid membrane

– Tri-segmented genome

• Hantavirus are different to other Bunyaviridae members in:

– Hantavirus are transmitted through aerosolized rodent urine, feces and saliva.

– Others genera transmitted through arthropod vectors.

• ARBOVIRUS = Arthropod Borne Virus

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Hantavirus

Virology – Physical properties

• Spherical or oval-shaped.

• 80-120 nm diameter.

• Grid-like surface pattern.

• Lipid bilayer envelope

• Granulofilamentous interior

• Survive 12 hours at 4°C, high salt concentration and non-physiological pH and 1-3 days after drying.

• Exposure to lipid solvents and nonionic detergents destroys viral envelope

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Hantavirus

Virology – Structure & morphology

Membrane Glycoproteins

• G1: 64-67kDa

• G2: 54 kDa, highly conserved

• Integral membrane proteins

• G1-G2 heterodimers form 7-8 nm projections on virion surface

• Cysteine-rich

• Contain asparagine-linked sugar groups

• Important in cell entry and pathogenesis

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Hantavirus


Nucleocapsid protein (N)

• 48 kDA

• Complexes with genomic vRNA in virus, as well as with cRNA after infection, but not with mRNA

• Necessary for virus replication and packaging

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Hantavirus


Polymerase (L)

• 247 kDA

• RNA-dependent RNA polymerase (RdRp)

• Complexed with ribonucleocapsids in virion

• Endonuclease activity to cleave host mRNA

• Transcriptase activity for making cRNA and mRNA from vRNA

• Helicase activity to unwind vRNA during transcription

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Hantavirus

Virology – Genome

• Bunyavirus-like

• ssRNA(-)

• Trisegmented

Large (L) segment, 6.5 kb, codes for polymerase

Medium (M) segment, 3.6-3.7kb, codes for G1 & G2 glycoproteins

Small (S) segment: 1.7-2.1kb, codes for nucleocapsid protein (NP)

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Hantavirus

Virology – Genome

• Panhandle structure

Conserved repeated complementary sequences at 5’ and 3’ ends form panhandle structures

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Hantavirus

Virology – Genome

REPLICATION

•Viral polymerase transcribes negative-strand vRNA to sense cRNA

•cRNA is used as template to make more negative-strand vRNA

•pppG is used to prime cRNA and vRNA synthesis

•Same “prime and realign” strategy

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Hantavirus

Virology – Life cycle

Attachment

• G1 & G2 glycoproteins interact with cell surface receptors

• Pathogenic hantaviruses bind β3 integrins

• Non-pathogenic hantaviruses bind β1 receptors

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Hantavirus


Integrins

• Heterodimeric receptors composed of α and β subunits.

• Involved in endothelial cell adhesion, platelet aggregation, cell migration, Ca++ channel activation & extracellular matrix interactions.

Expressed by endothelial cells, macrophages & platelets (cells infected by Hantavirus)

Attachment of G1/G2 proteins of virion to integrin initiates endocytosis & also activates the receptor.

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Hantavirus


Entry and uncoating

• Virus particles bound to integrin receptors are taken in by receptor mediated endocytosis

• Newly formed vesicles are acidified

• Acidic environment changes conformation of G1 & G2

• Viral and cell membranes fuse

• vRNA & polymerase are released into cytoplasm

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Hantavirus


Primary transcription

• Viral polymerase transcribes nucleoprotein-coated negative-sense vRNA to mRNA.

• Polymerase acts as endonuclease and cleaves host mRNAs 7-18 nt from the 5’ cap.

• Capped oligonucleotides from cell’s own mRNA are used to prime transcription.

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Hantavirus


Secondary transcription

• Extra vRNA synthesized during replication is used as template to make mRNA.

• Since more template is present after vRNA is replicated, more mRNA can be transcribed and more viral proteins can be made

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Hantavirus


Translation

• L & S segment mRNA is translated on free ribosomes.

• M segment mRNA translated on ER-bound ribosomes.

• G1 & G2 peptides produced from M are cleaved cotranslationally

• Separate signal sequences for G1 and G2 cause ER attachment and embed the peptides in ER membrane.

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Hantavirus


Replication

• Viral polymerase transcribes negative-strand vRNA to sense cRNA.

• cRNA is used as a template to make negative-strand vRNA

• More genetic material means more virions produced

• pppG is used to prime cRNA and vRNA synthesis

• “Prime and realign” strategy.

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Hantavirus


Assembly

• Membrane-bound G1 & G2 peptides are transported to Golgi

• Carbohydrates are attached by N-linked glycosylation

• vRNA complexes with nucleopcapsid.

• vRNA forms panhandle structure.

• vRNA complexes with polymerase

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Hantavirus


Virion release

By either Golgi budding or membrane budding

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Hantavirus


Virion release


• Virion particle is formed inside the Golgi

• Virions are transported to cell membrane by vesicles and released by exocytosis, just like in secretion

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Hantavirus


Virion release


• Sin Nombre & Black Creek Canal

• G1 & G2 embedded into cell membrane through Golgi vesicles

• Virions bud from cell membrane, not through Golgi

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Hantavirus

Virology – Pathogenesis

• Virus replication typically halts host macromolecule synthesis.

• Hantavirus release does not require host cell lysis.

• Hantavirus is able to establish a persistent infection in rodent host cells.

• Hantavirus replication does not affect reservoir cell’s natural functions.

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Hantavirus

Virology – Pathogenesis

Systemic Inflammatory Response

• Immune system activated against Hantavirus epitopes

• Virus epitopes expressed on surface of host cells triggers cytotoxic T-cell attack on host tissues

• Symptoms are consistent with inflammatory response

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Hantavirus

Virology – Host specificity

• Each strain of hantavirus has a specific rodent host

• Hantavirus species appear to have co-evolved with host rodent species

• Rodents carrying hantavirus are asymptomatic

• 3 main groups:– sigmodontinae – new world mice and rats– murinae – old world mice and rats– arvicolinae – voles and lemmings

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Hantavirus

Epidemiology – Reservoirs

• Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter.

• A primary host or definitive host is a host in which the parasite reaches maturity and, if applicable, reproduces sexually.

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Hantavirus



• A secondary host or intermediate host is a host that harbors the parasite only for a short period, during which (usually) some developmental stage is completed.

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Hantavirus



• A paratenic host is similar to a secondary host, only that it is not needed for the parasite's development cycle to progress.

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Hantavirus



• A reservoir host is a primary host that can harbor a pathogen indefinitely with no ill effects, may be reinfected several times.

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Hantavirus



• A dead-end host is an intermediate host that does not allow transmission to the definite (primary) host.

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Hantavirus

Epidemiology – Reservoir distribution

• Striped field mouse (Apodemus agrarius)Hantaan Virus

• Brown or Norway rat (Rattus norvegicus)Seoul virus

• Bank vole (Clethrionomys glareolus)Puumala virus

• Yellow-necked field mouse (Apodemus flavicollis)Dobrava virus.

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Hantavirus

Epidemiology – Geographical distribution

• New World Hantaviruses

Sin NombreSin NombrePeromyscus maniculatus

Rio SegundoRio SegundoReithrodontomys mexicanusReithrodontomys mexicanus

El Moro CanyonEl Moro CanyonReithrodontomys megalotisReithrodontomys megalotis

AndesAndesOligoryzomys Oligoryzomys longicaudatuslongicaudatus

BayouBayouOryzomys Oryzomys palustrispalustris

Black Creek CanalBlack Creek CanalSigmodon hispidusSigmodon hispidus

Rio MamoreRio MamoreOligoryzomys Oligoryzomys

microtismicrotis

Laguna NegraLaguna NegraCalomys lauchaCalomys laucha

MuleshoeMuleshoeSigmodon hispidus

New YorkNew YorkPeromyscus leucopusPeromyscus leucopus

JuquitibaJuquitibaUnknown Unknown HostHost

MacielMacielNecromys benefactusNecromys benefactus

Hu39694Hu39694Unknown Unknown HostHostLechiguanasLechiguanas

Oligoryzomys flavescensOligoryzomys flavescens

PergaminoPergaminoAkodon azaraeAkodon azarae

OrOránánOligoryzomys longicaudatusOligoryzomys longicaudatus

CCañaño Delgaditoo DelgaditoSigmodon alstoniSigmodon alstoni

Isla VistaIsla Vista Microtus californicus

Bloodland LakeBloodland LakeMicrotus ochrogasterMicrotus ochrogaster

Prospect HillProspect HillMicrotus pennsylvanicusMicrotus pennsylvanicus

BermejoBermejoOligoryzomys Oligoryzomys

chacoensischacoensis

CalabazoCalabazoZygodontomys Zygodontomys

brevicaudabrevicaudaChocloChoclo

Oligoryzomys Oligoryzomys fulvescensfulvescens

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Hantavirus


The Deer Mouse is a deceptively cute animal, with big eyes and big ears. Its head and body are normally about 2 - 3 in long and the tail adds another 2 - 3 in in length. The underbelly is always white and the tail has sharply defined white sides. The deer mouse is found almost everywhere in North America. Usually, the deer mouse likes woodlands, but also turns up in desert areas.

• Peromyscus maniculatus (Sin Nombre Virus)

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Hantavirus

Four Corners Disease and HPS

– Hantavirus Pulmonary Syndrome

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Hantavirus

HPS & Four Corners Outbreak

• May 1993– First clinical case– Abrupt fever, myalgia, pulmonary edema– Otherwise healthy individuals who enjoyed outdoor activities

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Hantavirus


• May 1993

N = 465 100%

Gender Male 292 64%Female 173 37%

Race White 355 78%American indian 87 19%Black 7 2%Asian 3 1%

Ethnicity Hispanic 65 14%

Case fatality 165 35%

Age (years) Mean 38 (10-83)

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Hantavirus


• June 1993– 12 fatalities – Unexplained Adult Respiratory Distress Syndrome (ARDS)– Sera cross-reacted with Hantaan, Seoul, Puumala virus (CDC)– Rodents trapped - deer mouse main reservoir

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Hantavirus


• Winter and spring 1993– Drought for several years followed by

snow and rain– Vegetation blossomed and rodent

population grew tenfold

• Virus isolated and named– Sin Nombre Virus (SNV)

• Newly emerging virus has been present since 1959

– 38 year old Utah man– Events inspired 1995 film, Outbreak

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Hantavirus


• Sin Nombre virus caused approximately 200 confirmed cases of HPS during the six month outbreak and led to a 50% mortality rate.

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Hantavirus

HPS & Non-SNV strains

• Black Creek Canal Virus -FL

• Bayou Virus - TX

• New York-1 Virus -NY

Sin NombreSin NombrePeromyscus maniculatus

El Moro CanyonEl Moro CanyonReithrodontomys megalotisReithrodontomys megalotis

BayouBayouOryzomys Oryzomys palustrispalustris

Black Creek CanalBlack Creek CanalSigmodon hispidusSigmodon hispidus

MuleshoeMuleshoeSigmodon hispidus

New YorkNew YorkPeromyscus leucopusPeromyscus leucopus

Isla VistaIsla Vista Microtus californicus

Bloodland LakeBloodland LakeMicrotus ochrogasterMicrotus ochrogaster

Prospect HillProspect HillMicrotus pennsylvanicusMicrotus pennsylvanicus

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Hantavirus

Epidemiology – Geographical distribution

• New World Hantaviruses

Brazil (168)

Paraguay (74)

Uruguay (23)

Argentina (404)

United States (335)

Canada (36)

Panama (31)

Chile (273)

Bolivia (20)

Countries with reported cases of HPS

Countries with no reported cases of HPS

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Hantavirus

HPS– Seasonal variations

Most cases reported during the late spring and early summer

– 1993 HPS outbreak followed a dramatic increase in precipitation (92-93 el niño).

– Similar pattern of rainfall followed by drought during 95-96Paraguay HPS outbreak.

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Hantavirus

HPS– Seasonal variations

Above normal precipitation leads to abundance in food resources for rodents (increase in rodent population).

When food sources are depleted, rodents move into more permissive environments (human establishments).

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Hantavirus

Epidemiology – Transmission

• Infectious virus is shed through host saliva, urine and fecal matter.

• Humans are infected by inhaling the virus from aerosols produced while host is excreting waste.

• Initially only HFRS strains known to transmit human to human.

• Human to human transmission of HPS only documented in argentina (andes virus).

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Hantavirus

Epidemiology – Transmission

Peridomestic exposure

Peridomestic & occupationalExposure

Peridomestic & recreationalexposure

Occupational exposure

Entering/cleaning rodent-infested structures

69% (48/70)

19% (13/70)

9% (6/70)

4% (3/70)

9% (6/70)

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Hantavirus

Clinical characteristics

Three different clinical manifestations of hantavirus infection caused by different viral strains:

Hemorrhagic fever with renal syndrome (HFRS)Found in Europe and Asia

Nephropathia Epidemica (NE)Found in Europe

Hantavirus pulmonary syndrome (HPS)Found in north and south America

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Hantavirus

Hantavirus Pulmonary Syndrome (HPS)

• HPS has a 14 to 17 day incubation period.

• At onset patients will have a headache, fever, myalgias and a general feeling of discomfort.

• Most patients present with abdominal pain, nausea vomiting and a low platelet count.

• Patients seek medical attention until their cardiopulmonary system becomes compromised.

• Severe lung edema sets in 48 hours after cardiopulmonary involvement.

• Death usually occurs due to shock and cardiac complications (40%)

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Hantavirus

Hantavirus Pulmonary Syndrome (HPS)

• The discovery of HPS in North America led to retrospective studies in South America.

• More than 140 cases of HPS confirmed in Argentina.

• The Andes hantavirus was discovered in long tailed pygmy rice rats in southern Argentina.

• Andes Virus is the only known hantavirus to be transmitted person-to-person.

• Prior to the HPS outbreak, the only known hantaviruses were those that caused HFRS

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Hantavirus

Clinical characteristics - HPS

Clinical stages:

1 - Incubation (4-30 d)2 - Febrile Phase 3 - Cardiopulmonary phase 4 - Diuretic phase5 - Convalescent phase

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Hantavirus


Clinical stages:


3-5 days

Fever, myalgia, malaise

Other symptoms: headache,

dizziness, anorexia, nausea,

vomiting, and diarrhea.

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Hantavirus


Clinical stages:


Lasts between 4 and 24 hrs

Non-productive cough and tachypnea

Rapid progression of shock

Worsenning of pulmonary edema

Hypovolemia (progressive leakage syndrome)

Hypotension and oliguria due to hypovolemia

Thrombocytopenia (especially during febrile phase)

Death within 24-48 hrs due to hypoxia and/or

myocardial failure

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Hantavirus


Clinical stages:


Bilateral interstitial infiltrates

Moderate to rapid progression

Bilateral alveolar infiltrates

Pleural effusion

Normal heart size (not cardiogenic edema)

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Hantavirus


Clinical stages:


Several days to several weeks

Beginning of recovery

Rapid clearance of pulmonary edema

Resolution of fever and shock

Anorexia, fatigue due to dehydration

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Hantavirus


Clinical stages:


Up to 2 months

Results in chronic decreased small-airway volume Diminished alveolar diffusing capacity

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Hantavirus

Clinical characteristics - HFRS

Occurs throughout Europe and Asia

Formerly had other names, including Korean hemorrhagic fever, epidemic hemorrhagic fever & nephropathia epidemica

~15% fatality

Worldwide 150,000–200,000 cases per year

Outbreaks linked to contact with field rodents during planting and harvesting of crops

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Hantavirus


Clinical stages:

1 - Incubation (4-40 d)2 - Febrile Phase 3 - Hypotensive Phase 4 - Oliguric Phase5 – Recovery:

- Diuretic Phase- Convalescent Phase

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Hantavirus


•3-5 days

•Characterized by fever, chills

•Headache, severe myalgia, nausea

•Blurred vision, photophobia, eye pain by movement

•Flushing of face, V-area of the neck and back

•Petechiae (small red spots on skin)

•Abdominal pain and back pain.

•Thirst, edema, hemoconcentration, postural hypotension

Clinical stages:



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Hantavirus


•Hours to days

•Blood pressure decrease, hypovolemia, shock

•Worsening of bleeding manifestations: petechiae,

epistaxis, gastrointestinal & intracranial bleeding

•↑Urea & ↑Creatinine in blood.

•Proteinuria

•Leukocytosis, thrombocytopenia.

Clinical stages:



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Hantavirus


•3-7 days

•Oliguria due to renal dysfunction

•Hypervolemia leading to hypertension

•Blood electrolyte imbalance

•Hemorrhagic symptoms continue

•Severe complications: cardiac failure pulmonary edema,

& cerebral bleeding

Clinical stages:



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Hantavirus


•Several days to several weeks

•Beginning of recovery

•3-6 liters of urine/ day; return to normal renal activity

•Anorexia, fatigue due to dehydration

•2-3 months

•Progressive improvement in:

•glomerular filtration,

•renal blood flow, and

•urine concentrating ability

Clinical stages:



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Hantavirus

Clinical characteristics - NE

• Puumala hantavirus strain

• Common mild form of HFRS in Europe

• Similar sequence of symptoms as HFRS, but much milder

• Only 6% of serologically confirmed cases require hospitalization

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Hantavirus

Diagnosis

• Early symptoms resemble influenza, though common signs of upper respiratory disease such as sore throat, sinusitis, and ear pain not usually present

• More serious symptoms of hypotensive phase of HFRS or cardiopulmonary phase of HPS have acute onset

• Hantavirus is difficult to culture, so morphological identification is difficult.

• RT-PCR using primers for conserved genome regions allows confirmation of infection.

• PCR product can be sequenced and compared to known viral sequence database for species identification

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Hantavirus

Diagnosis - HPS

• Abdominal pain often misinterpreted as appendicitis

• Many doctors outside endemic regions fail to recognize or have sufficient testing

• Many lab tests and radiographs appear normal

• Suggestive labs

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Hantavirus

Diagnosis - HPS

• Serological tests more effective

• ELISA IgM capture assay, using either SNV, Laguna Negra, or Andes antigens are used in all countries that have previously detected cases

• Immunofluorescent test for the presence of antibodies

• Blood analysis also may find thrombocytopenia with platelet count less than 150,000 mm in 98% of cases

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Hantavirus

Treatment

• No FDA approved antiviral drugs available for HFRS.

• Prompt diagnosis and good management of illness improves patient’s survival.

• No vaccine

• HPS is more serious and rapidly progressive illness.

• IV ribavirin effective for HFRS, not for HPS.

• Aggressive supportive care.

• Extra Corporeal Membrane Oxygenation (ECMO) = $

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Hantavirus

Control and prevention

• Avoid contact with rodents and their habitats.

• Do not keep wild rodents as pets.

• Keep all food in sealed containers.

• Virions may be stable for 2 days on a dry surface so disinfect areas contaminated by rodents using a 10% solution of household bleach or detergent.

• Eradication unlikely due to ubiquitous rodent populations

• Prevent aerosolization of virus from rodent excrement

• General hygiene

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Hantavirus

HPS in Mexico

hantavirus christian a. garcía sepúlveda md phd laboratorio de biología molecular facultad de...

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