FOUR CORNERS DISEASEDr.R.Jayaprada

OVERVIEW

History

Organism

Epidemiology

Transmission

Disease in Humans

Prevention and Control

INTRODUCTION

Two major outbreaks of disease led to the discovery of

hantaviruses in the Old and New Worlds.

The first outbreak occurred during the Korean War (1950 to

1953), where in more than 3,200 United Nations troops fell ill

with Korean hemorrhagic fever, which is commonly referred

to as hemorrhagic fever with renal syndrome (HFRS).

The second outbreak of disease occurred in the Four Corners

region of the United States in 1993 and was initially referred

to as Four Corners disease, which is now called hantavirus

pulmonary syndrome (HPS) or hantavirus cardiopulmonary

syndrome (HCPS).

FOUR CORNERS OUTBREAK The converging point of Colorado,

Utah, Arizona, and New Mexico’s state borders became the location of the 1993 Four Corners Outbreak of Hantavirus Pulmonary Syndrome (HPS).

The Navajo tribal belief is such that mice were responsible for bringing seeds to the earth, allowing humans to survive.

Mice are thought of as the “landlords of the world” and are highly respected in Navajo culture.

HISTORY

In May 1993, an outbreak of an unexplained pulmonary

illness occurred in the southwestern United States, in an

area shared by Arizona, New Mexico, Colorado and

Utah known as "The Four Corners".

A young, physically fit Navajo man suffering from shortness

of breath was rushed to a hospital in New Mexico and died

very rapidly.

The mysterious illness A native Indian Health physician began to notice an outbreak of

an unexplained illness that caused death among normal

healthy young adults

After calling his colleagues, he discovered that 10 people had

already died of a similar respiratory disease

Autopsies did not reveal any sign of viral pneumonia, influenza

or any other common disease that attacked the lungs

Although this disease was not specific to the Navajo people, to

the media, the disease became known as the “Navajo disease”

When the number of cases doubled to about 20 victims, the

CDC was called in

CDC In May 1993, the CDC was called in to investigate the case.

The CDC, using immunofluorescent techniques and their virus

library, was able to positively identify this new virus as a

relative of the hantavirus strains that were found on the

Eurasian continent

However, scientists were skeptical for 3 reasons

The only hantaviruses known were on the Eurasian continent

The diseases caused by the Eurasian hantavirus strain did

not cause respiratory failure

The new virus in the “four corners” appeared to be 5 times

as lethal as the strain in Europe!

HISTORY

HPS was first recognized as a HANTAVIRUS DISEASE.

32 of the 53 people were infected & died.

A warm winter allowed for an increase in the host population.

Outbreak was caused by the SIN NOMBRE STRAIN(SNV,

in Spanish, "Virus sin Nombre", for "nameless virus").

30% of the mice in that area carried this strain.

FOUR CORNERS OUTBREAK

Winter and spring 1993

Drought for several years followed by snow and rain

Vegetation blossomed and rodent population grew

tenfold

Virus was isolated 1 month after the first report of cases

and named as Muerto Canyon virus, then Four Corners

virus, and finally Sin Nombre Virus (SNV –“virus

without a name”).

FOUR CORNERS OUTBREAK

Newly emerging virus has been present since 1959.

38 year old Utah man that had died from an illness

compatible with hanta in 1959.

Researchers located his lung tissue and utilizing current

technology, were able to isolate SNV in 1994.

 The earliest case of HPS to be confirmed by IHC with

direct visualization of hanta viral antigens in postmortem

tissue involved a patient who died in 1978.

HISTORY Hantavirus disease outbreaks have occurred as far back as

American Civil War times.

There are records of a hemorrhagic fever syndrome (HFRS)

during World War I and II.

First outbreaks of hantavirus causing HFRS were reported

in Russia in 1913 and 1932.

Japanese troops in Manchuria reportedly had cases in

1932 and cases referred to as Nephropathia Epidemica

(NE) in Sweden appeared in 1934.

Western medicine diagnosed Korean Hemorrhagic Fever

(KHF) during the Korean War in the 1950’s.

HISTORY

1951-1954: Korean War

3,200 U.N. troops develop disease

Hantaan River separated N. & S. Korea

1977

Hantaan agent was isolated and characterized

1990: 94% of serum samples from soldiers in 1950’s

had antibodies

1979

Seoul virus found in Japan and Europe

HISTORY Its original name was "Four Corners virus" or "Navajo

Flu", but the name was changed after local residents

raised objections.

Its rodent host, the Deer mouse (Peromyscus

maniculatus), was first identified by Terry Yates, a

professor at the University of New Mexico.

HISTORY 1951-1954: HEMORAGIC FEVER WITH RENAL FAILURE FIRST

RECOGNIZED AS A PATHOGEN AFTER AN OUTBREAK IN HANTAAN

KOREA.

1977: DISEASE ISOLATED AND NAMED AFTER HANTAAN RIVER.

1978: IT WAS CONFIRMED THAT THE VIRUS IS CARRIED BY

RODENTS.

1981: FIRST SUCCESSFUL PROPAGATION OF VIRUS IN CELL

CULTURE.

1993: OUTBREAK OF HPS IN FOUR CORNER REGION OF

COLORADO, NEW MEXICO, ARIZONA AND UTAH.

RECENT CASES

May 2003: Montana

Three cases

Two deaths

Contracted virus from rodents in home

First cases since fall of 2001

Overall cases in Montana

Virus first appeared in state in 1993

20 cases

5 deaths

ORGANISM

BUNYAVIRIDAE

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

HANTA VIRUS GENUS

Hantavirus Similarities

RNA viruses

Lipid membrane

Tri-segmented genome

Hantavirus Differences

Hantavirus transmitted through aerosolized rodent urine,

feces and saliva.

Others genera transmitted through arthropod vectors.

IMPORTANT SPECIES OF HANTA

The genus Hanta contains at least four species—

1.Hantaan virus causing the severe HFRS in the Far

East, North Asia and Russia,

2.Seoul virus causing a milder type of disease and

probably present worldwide,

3.Puumala virus responsible for Nephropathia

epidemica in Northern and Eastern Europe, and

4.Prospect Hill virus isolated from voles in the USA,

which has not been associated with human illness.

INTRODUCTION

Hantaviruses belong to the bunyaviridae family of viruses.

Hanta viruses are rodent borne diseases transmitted from

humans to humans in aerosolized urine/ saliva &

occasionally by bite.

International committee on taxonomy of viruses recognized

30 species in the genus Hanta virus (of which 21 are

pathogenic to humans).

TAXONOMY Rodent borne Hantaviruses are divided into 3 groups

based on the taxonomic assignment of their principal hosts belonging to families muridae & cricetidae.

FAMILY SUBFAMILY PRINICIPAL HOST

MURIDAE Murinae old world rats & mice

Cricetidae Arvicolinae Voles & Lemmings

Cricetidae Sigmodontinae New world rats & Mice

Cricetidae Neotominae

TAXONOMY

ASSOCIATION WITH DISEASE Murine rodents are the principal hosts of Hanta virus

associated with severe HFRS.

Voles are the principal hosts of Puumula virus, which is

the cause for relative mild form of HFRS called

Nephropathia epidemica.

Sigmodontinae & Neotominae rodents the principal hosts

of Hanta virus Known to cause HPS.

Physical PropertiesStructureGenetics

Replication CyclePathogenesis

Molecular Biology of Hantavirus

DESCRIPTION OF THE AGENT

Spherical

Enveloped, ss negative sense

RNA virus.

Virions 80-120nm in diameter

with a characteristic square

grid-like structure.

Helical nucleocapsid.

Genome consists of three RNA

segments: L, M, and S.

NO matrix protein.

Lipid bilayer.

DESCRIPTION OF THE AGENT

Spikes protrude from the lipid bilayer envelope.

Spikes consists of glycoproteins Gn & Gc (formerly G1

&G2 respectively).

Virion contains ribonucleocapsid--SS RNA complexed with

nucleocapsid protein- L protein.

Genomes of Hanta virus consists of trisegmented

negative sense Linear ss RNA.

Three segments:

Large (L) codes for viral polymerase

Medium (M) codes for G1 and G2 glycoproteins

Small (S) codes for nucleocapsid

STRUCTURAL PROTEINS

Membrane Glycoproteins

Nucleocapsid Protein

Viral Polymerase

Membrane glycoproteins (G1 and G2)

Nucleocapsid proteins (N)

Polymerase (L)

Membrane Glycoproteins

G1: 64-67kDa

G2: 54 kDa, highly conserved

Integral membrane proteins

G1-G2 heterodimers form 8 nm projections on

virion surface

Cysteine-rich

Contain asparagine-linked sugar groups

Important in cell entry and pathogenesis

Nucleocapsid Protein

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

Viral Polymerase

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

Genomic Organization

Tripartite negative sense genome

Small (S) segment, 1.7-2.1kb, codes for N

nucleocapsid protein

Medium (M) segment, 3.6-3.7kb, codes for G1 and

G2 glycoproteins

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

protein

Sin Nombre Virus

• Circular single stranded; RNA

• Total genome 10500-22700 nucleotides long

• 100 (80-120) nm in diameter

• Surface projections of envelope distinct; spikes

(of about 10 nm).

• Reservoir for Sin Nombre virus is deer mice

Viral Replication

Receptor mediated endocytosis

Occurs in cytoplasm

Budding at Golgi apparatus or cell membrane

Hantavirus Replication Cycle

Attachment

Entry and Uncoating

Primary Transcription

Translation

Genome Replication

Secondary Transcription

Virion Assembly

Virion Release

Attachment

Viral G1 and G2 glycoproteins interact with cell

surface receptors

Pathogenic hantavirus bind β3 integrins

Non-pathogenic hantaviruses bind β1 receptors

Virion Assembly

Membrane-bound G1 and

G2 peptides are

transported to Golgi

apparatus and

carbohydrates are

attached by N-linked

glycosylation

vRNA complexes with N

nucleocapsid protein,

forms looped panhandle

structure, and complexes

with polymerase

Virion ReleaseTwo Mechanisms

Nucleocapsid complexes

bud into the Golgi

membrane with G1 and G2

embedded

Virion particle is formed

inside Golgi apparatus

Virions are transported to

cell membrane by vesicles

and released by exocytosis

G1 and G2 embed

into cell

membrane through

Golgi vesicles

Virions bud from

cell membrane,

not through Golgi

apparatus

Old World & New World Hantavirus

Hantaviruses are commonly referred to as Old World

and New World hantaviruses due to the geographic

distribution of their rodent reservoirs and the type of

illness (HFRS or HPS) that manifests upon transmission

to humans.

Old World Hantavirus vs New World Hantavirus

Found mostly in Europe

and Asia

Carried by rodents

Causes HFRS

Targets the kidney

Mortality rate is <10-15%

Vaccine exists for these

strains

Discovered in the “four

corners in America

Carried by rodents

Causes HPS

Targets the lungs

Mortality rate is 50-60%

Vaccine does not exist for

these strains

Serotype Host Location

Hantaan Apodemus agrarius(striped field mouse)

Asia, Far East Russia

Dobrava A. agrarius, A. flavicollis (yellow neck mouse)

Europe Balkans

Seoul Rattus norvegicus, R. rattus(Norway brown rat, roof rat)

Worldwide

Puumala Clethrionomys glareolus (red bank vole)

Europe

HANTA VIRUSES IN THE OLD WORLD

Serotype Host LocationSin Nombre Peromyscus

maniculatus (deer mouse)

Central & West U.S., Canada

Monongahela Peromyscus maniculatus (deer mouse)

Eastern U.S., Canada

New York Peromyscus leucopus (white-footed mouse)

Eastern U.S., Canada

Bayou Oryzomys palustris(rice rat)

SE U.S.

Black Creek Canal

Sigmodon hispidus(cotton rat)

SE U.S.

HANTA VIRUSES IN THE NEW WORLD

Serotype Host Location

Andes Oligoryzomys longicaudatus (long-tailed pygmy rice rat)

Argentina/Chile

Oran O. longicaudatus NW Argentina

Lechiguanas O. flavescens Central Argentina

Hu39694 Unknown Central Argentina

Laguna Negra Calomys laucha Paraguay/ Bolivia

Juquitiba Unknown Brazil

HANTA VIRUSES IN THE NEW WORLD

DISTRIBUTION OF NEW AND OLD WORLD HANTAVIRUSES

Epidemiology and Rodent Hosts

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

EPIDEMIOLOGY

Hantan virus ,prototypical member of genus Hanta virus is the

cause of a severe form HFRS endemic in Korea, China and

Eastern Russia.

Dobrava- Belgrade virus is an agent of Severe form of HFRS in

the Balkans Greece and Russia

Seoul Virus is endemic in Asia, Europe and America

Puumala viurs is endemic in Europe and Scandinivea

Saaremaa virus – in Europe .

Amur virus – Eastern Russia.

SinNOmbre virus is the major cause of HPS in North America .

Andes virus is the major cause of HPS inSouth America

Geographical representation of approximate hanta viral disease incidence by country per year.

Jonsson C B et al. Clin. Microbiol. Rev. 2010;23:412-441

TransmissionVectors

Transmitted via aerosolized rodent urine, feces, and saliva

Deer mouse (Peromyscus maniculatus)

Cotton rat (Sigmodon hispidus)

White-footed mouse (Peromyscus leucopus)

Striped field mouse (Apodemus agrarius)

Bank vole (Clethrionomys glareolus)

Rat (Rattus)

Deer Mouse Cotton Rat House Rat (Mus musculus)

Rice Rat   White Footed Mouse

Hantavirus and Host Cells

Virus replication typically halts host macromolecule

synthesis

Hantavirus replication does not affect host cell’s natural

functions

Hantavirus release does not require host cell lysis

Hantavirus is able to establish a persistent infection in

rodent host cells

Integrins

Hetero dimeric receptors composed of α and β subunits

Present on endothelial cells, macrophages, and platelets –

cells affected by Hantavirus infection

Normally involved in regulation of endothelial cell

adhesion, platelet aggregation, Ca++ channel activation,

and extracellular matrix interactions, including cell

migration

β3-Integrins

Required for infection by pathogenic

Hantaviruses

β1 integrins are used by non-pathogenic strains

Attachment of G1/G2 proteins of viroid to

integrin initiates endocytosis, but also activates

the receptor

Variation in virus G1/G2 protein may account for

severity of disease

Hantavirus Infection Pathogenesis

Binding of Hantavirus glycoproteins to β3 integrin causes

disruption of vascular integrity

Capillaries become more permeable

Arteriole vasoconstriction and vasodilatation are disrupted

Binding to platelet receptors affects clotting and platelet

function

Immune Reaction

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

Clinical Presentation of Hantavirus Infection

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

MAJOR DIFFERENCE B/N HFRS & HPS

HFRS HPS Retroperitoneum is the Lungs & thoracic cavity are

the major site of the vascular major sites of vascular

leaks in leak in HFRS. HPS.

Stages of Hemorrhagic Fever with Renal Syndrome (HFRS)

1)Incubation (4-40 days)

2)Febrile Phase (3-5 days):Characterized by fever, chills,

headache, severe myalgia (muscle pain), nausea

3)Hypotensive Phase (hours to days): Decrease in blood

pressure, hypovolemia (decreased blood volume), shock

4)Oliguric Phase (3-7 days):Marked by decreased urine

production due to renal (kidney) dysfunction

Stages of Hemorrhagic Fever with Renal Syndrome (HFRS)

Recovery:

5)Diuretic Phase (2-21 days):Beginning of recovery, 3-6

liters of urine/ day; return to normal renal activity

6)Convalescent Phase (2-3 months):

Progressive improvement in glomerular filtration, renal blood

flow, and urine concentrating ability

Clinical Testing for HFRS

Thrombocytopenia (low platelet count) is a signifier

Urine tests for albuminuria (abnormally high

amounts of the plasma protein albumin in the

urine)

Urine tests for microhematuria (microscopic

amounts of blood in the urine)

Problems Diagnosing HFRS

Early symptoms resemble influenza

More serious symptoms of hypotensive phase

have acute onset

PATHOLOGY

In HFRS, pathology findings are:

1.Effusions in the body cavities,

2.Retroperitoneal edema,

3.Enlarged, congested & hemorrhagic kidneys.

Nephropathia Epidemica (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

HPS

1993 four corners outbreak

Cases found in almost all parts of the Americas

~50% fatality

Stages of Hantavirus Pulmonary Syndrome (HPS)

1)Incubation (4-30 days)

2)Febrile phase (3-5 days): Characterized by fever, myalgia,

malaise, headache, dizziness, anorexia, nausea, vomiting,

and diarrhea.

3)Cardiopulmonary phase ( 4-24 hours): Presentation and

rapid progression of shock and pulmonary edema (4-24h

non-productive cough and tachypnea (shortness of breath)

4)Diuretic phase

5)Convalescent phase: Results in chronic decreased small-

airway volume and diminished alveolar diffusing

capacity

HPS Radiographic Findings

Bilateral interstitial infiltrates

Moderate to rapid progression

Bilateral alveolar infiltrates

Pleural effusion

Normal heart size

Clinical Testing for HPS

Many lab tests and radiographs appear normal

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

PATHOLOGY

In HPS, pathology findings are:

1.Copious amounts of frothy fluid in bronchi & other

airways,

2.Edematous lungs &

3.Pleural effusions.

LABORATORY DIAGNOSIS

COLLECTION,TRANSPORT & STORAGE OF SPECIMENS

SPECIMENS:

Blood, Serum, Urine, CSF, Respiratory secretions.

Specimens are collected during acute phase of illness.

Precautions must be taken while handling the specimens of Hanta

virus.

1)Sera from HFRS/ HPS patients should be handled at BSL-2.

2) Potentially infectious tissue specimens should be handled at BSL-

2 using BSL-3 practices.

Blood, Serum/plasma samples for serology should be strored at 4º

C & sent to lab on ice packs.

Samples for RNA isolation & subsequent testing by RT PCR are

stored continuously at -70º C.

LABORATORY DIAGNOSIS

1.Direct detection—a. Microscopy.

b. Antigen detection.

c. Nucleic acid detection.

2.Virus isolation.

3.Serologic tests.

4.Identification– a. Serologic methods.

b. Genetic methods.

5.Typing Systems.—Sequence analysis.

DIRECT DETECTION

Microscopy : It has limited diagnostic value.

Electron microscopic examination of autopsy tissues from HFRS & HPS

patients showed more number of HANTA viral inclusion bodies containing

mature intact virions.

Antigen detection : Immunohistochemistry: can test formalin fixed

tissues with specific monoclonal (murine) and polyclonal antibodies

retrospective diagnosis.

Nucleic acid detection : By RT PCR, Nested PCR, real time RT

PCR.

Use of genus specific Oligonucleotide primers that anneal to regions of

‘S’& ‘M’ genomic segments confirms genotype of the infecting virus.

Exponential production of product that may be sequenced for viral

characterization .

VIRUS ISOLATION

Virus isolation is not commonly used for diagnosis of Hanta

viral infections in humans.

Vero E6 cell line (ATCC CRL-1586) is used to isolate hanta

virus from blood, urine, tissue samples of HFRS patients

and from serum &urine of patients with Andes virus HPS.

A monolayer cell cultures of Vero E6 cells are inoculated

with a crude / clarified tissue homogenate & maintained

under a fluid overlay for 10-14 days.

VIRUS ISOLATION

Hanta viruses are neither cytopathic in cultured cells nor

pathogenic in laboratory rodents.

Detection of infection in cultured cells & in tissues of

laboratory rodents is by an indirect method i.e

1.Fluorescent antibody testing for viral antigen.

2.RT PCR assay for Hantavirus specific RNA.

SEROLOGIC TESTS

Detection of circulating immunoglobulins.

Usually there is a robust immune response by the time

symptoms are present (24hours –within one week of

infection)

IgM present 3-6 months after infection.

IgG can be detected for years post infection.

IgG & IgM responses are directed first against nucleocapsid

protein, then against the glycoproteins.

Neutralizing antibodies appear during acute phase of HFRS

& HPS—Reactive against glycoproteins Gn & Gc.

IgG against Gn is more specific than anti nucleocapsid

protein IgG.

SEROLOGIC TESTS Methods to detect antibodies against hanta viruses in serum/ plasma

are;

1.High particle density agglutination.

2.Indirect Immunofluorescence assay.

3. Immunoprecipitation.

4.RadioImmunoassay.

5.Haemagglutination inhibition.

6.Plaque & Focus reduction Neutralization -Foci of infected cells(viral

antigen in focus) is revealed by IHC staining/ Chemiluminescence.

7.Western Immunoblotting-*using recombinant antigens and isotype

specific conjugates for IgM/IgG differentiation.

8.µ capture (IgM) ELISA.

9.IgG ELISA.

IgM Capture ELISA Highly sensitive in detecting antiHantavirus IgM, but not

specific for virus.

It uses a Hanta virus infected cell lysate as test antigen.

Uninfected cell lysate as control.

Appropriate positive & negative control.

Treatment of Hantavirus Infection

General care, alleviation of symptoms

Ribavirin (HFRS)

ECMO (HPS).

Hyper immune (Neutralizing ) serum.

GENERAL CARE

HFRS

General treatment for

renal failure

Hydration

Dialysis

HPS

General treatment for

pulmonary pathology

Administration of

oxygen

THERAPY

Aggressive supportive care

Fluid management

Hemodynamic monitoring

Ventilatory support

Peritoneal dialysis

Pressor agents (blood pressure support)

Inotropic agents (cardiac support)

Increases cardiac muscle contractility

Broad spectrum antibiotic therapy until HPS is proven

(to cover for differential diagnoses)

Intravenous ceftriaxone or aminoglycoside

Doxycycline

Ribavirin

Administered intravenously

Shown to be effective

against Hemorrhagic Fever

with Renal Syndrome

Not shown to be effective

against Hantavirus

Pulmonary Syndrome

causing strains

ECMO-Takes over the function of heart and lungs while the patient recovers from initial cause of pulmonary/cardiac failure

Three components: 1)membrane

artificial lung that adds oxygen and removes carbon dioxide

2) roller pump that moves the patient’s deoxygenated blood to the membrane and back into the body

3) heat exchanger that warms the blood back to body temperature

PREVENTION & CONTROL

Vaccines Hygiene

VACCINES

No WHO approved vaccines are available.

Inactivated vaccines have been developed in Asia & used

locally in Korea for protection of humans against HFRS.

Inactivated vaccines are prepared from brains of suckling

rats/ mice or from cell cultures infected with Hantaan virus/

seoul virus.----Hantavax, commercially produced in S.korea.

STRATAGIES for development of new Hanta virus vaccines

include:

1.Recombinant nonpathogenic viruses,

2. Rodent/ cell culture derived inactivated virus.

3. Naked DNA

4.E. Coli expressed truncated nucleocapsid as an immunogen

PREVENTION & CONTROL

Rodent Control - control measures should be

aimed at reducing contact between humans and

rodents.

Prevent aerosolization of virus from rodent

excrement

Dampen surfaces with detergent before cleaning

General hygiene

Thank

You