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