Transcript Hantavirus Stephanie Bagley Eugene Khandros Nicholas Bevins

Hantavirus
Stephanie Bagley
Eugene Khandros
Nicholas Bevins
History
• Hantaviruses are rodent-borne viruses which
may be transmitted to humans in aerosolized
urine, feces, or saliva, and occasionally by bite.
• Hantaviruses often cause either hemorrhagic
fever with renal syndrome (HFRS) or hantavirus
pulmonary syndrome (HPS)
• Other hantaviruses are not known to be human
pathogens.
Hantavirus Outbreak in the US
• HPS was first described in the United States in
May 1993 during the investigation of a cluster of
cases of acute adult respiratory distress in the
Four Corners region.
• HPS was found to be caused by a previously
unknown hantavirus, Sin Nombre, detected in
deer mice.
• Sin Nombre caused approximately 200
confirmed cases of HPS during the outbreak,
that led to a 50% mortality rate.
Hantavirus in the US
• Prior to the HPS outbreak, the only known
hantaviruses were those that caused
HFRS
• At least three other hantaviruses, New
York Virus, Bayou Virus, and Black Creek
Canal virus have since been confirmed to
cause HPS in the U.S.
HPS in South America
• 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.
History of HFRS
• While HPS has only been identified since
1993, HFRS has a much longer and
complex history.
• HFRS may have been recognized in China
as early as 1000 years ago.
• HFRS described in 1913 Russian records
History of HFRS
Outbreaks of HFRS in the 1930’s:
• Russia (1932)
• Japanese troops in Manchuria (1934)
• Sweden (1934)
Characterization of HFRS
• 1934 -Japanese outbreak led to studies by
Japanese physicians
• 1940- Japanese physicians compiled a clinical
and pathological description of what was then
called “epidemic hemorrhagic fever.”
• First to implicate mice in disease transmission.
• Physicians injected filtrates of tissue from
Apodemus agrarius carrying the hantavirus
Hantaan into human subjects.
History of HFRS
• 1939-Russian studies also implicated mice
in disease transmission.
• 1961- Moscow outbreak affecting 113 of
186 workers linked to rodent shipment
Characterization of HFRS
1967-Russian scientists Yankovski and
Povalishina provide more insight into
disease.
• Incubation period up to 6 weeks
• Cycles of virus activity parallel population
cycles of field mice
• Transmission of disease occurred via
inhalation of dust contaminated by rodent
excretions.
Recent HFRS Cases
• 1951- outbreak in Korea during the war affected
3,200 soldiers and resulted in a 7-15% mortality
rate.
• Korean hemorrhagic fever (KHF) drew
widespread international attention and was
reclassified as HFRS in 1983 by the WHO.
• 1983-Several past outbreaks including KHF,
“epidemic hemorrhagic fever” , Russian
outbreaks all reclassified as HFRS by the WHO
Family Bunyaviridae
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
Hantavirus 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.
Epidemiology and Rodent Hosts
• Each strain of hantavirus has a specific
rodent host
• Hantavirus species appear to have coevolved with host rodent species
• Rodents carrying hantavirus are
asymptomatic
Transmission of Hantaviruses
Chronically infected
rodent
Horizontal transmission of infection by
intraspecific aggressive behavior
Virus is present in
aerosolized excreta,
particularly urine
Virus also present in
throat swab and feces
Secondary aerosols, mucous
membrane contact, and skin
breaches are also sources of
infection
Courtesy of CDC
Rodent Hosts
Virus Strain
Rodent Host
Murinae
Hantaan
Dobrav
aSeoul
Thailand
Apodemus agrarius
Prospect Hill
Microtus pennsylvanicus
Puumala
Clethrionomys glareolus
Sin Nombre
New York
Peromyscus maniculatus
Peromyscus leucopus
Apodemus flavicollis
Rattus norvegicus
Bandicota indicus
Arvicolinae
Oryzomys palustris
Bayou
Black Creek Canal Sigmodon hispidus
Sigmodontinae
New World Hantaviruses
New York
Sin Nombre
Peromyscus leucopus
Prospect Hill
Peromyscus maniculatus
Muleshoe
Microtus pennsylvanicus
Bloodland Lake
Sigmodon hispidus
Microtus ochrogaster
Isla Vista
Bayou
Oryzomys palustris
Black Creek
Canal
Microtus californicus
El Moro Canyon
Reithrodontomys megalotis
Calabazo
Zygodontomys
brevicauda
Choclo
Sigmodon hispidus
Rio Segundo
Reithrodontomys mexicanus
Caño Delgadito
Sigmodon alstoni
Oligoryzomys
Rio Mamore
fulvescens
Oligoryzomys microtis
Orán
Oligoryzomys longicaudatus
Bermejo
Oligoryzomys chacoensis
Andes
Oligoryzomys longicaudatus
Juquitiba
Unknown Host Laguna Negra
Calomys laucha
Maciel
Necromys benefactus
Hu39694
Unknown Host
Lechiguanas
Oligoryzomys flavescens
Pergamino
Akodon azarae
Courtesy of CDC
Hantavirus Pulmonary Syndrome
Countries
with reported
cases of
HPS
(no of cases)
Canada (36)
Countries with
no reported
cases of HPS
United
States (335)
Panama (31)
Bolivia (20)
Chile (273)
Brazil
(168)
Paraguay (74)
Uruguay (23)
Argentina (404)
Rodent Hosts in the United States
Deer mouse (Peromyscus maniculatus)
Carrier of Sin Nombre strain, primary agent of HPS in
the US. 250-300 cases since discovery.
Rodent Hosts in the United States
White-footed Mouse (Peromyscus leucopus)
Carrier of New York strain.
Molecular Biology of Hantavirus
• Physical Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
Molecular Biology of Hantavirus
• Physical Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
Virion Properties
• Spherical or oval-shaped.
• 80-120 nm diameter
• Unique grid-like surface pattern,
with 7-8 nm projections
• Lipid bilayer envelope
• Granulofilamentous interior
• Survive 12 hours at 4C, high salt
concentration and nonphysiological pH.
• Survives 1-3 days after drying.
• Exposure to lipid solvents and
nonionic detergents destroys viral
envelope
Molecular Biology of Hantavirus
• Virion Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
Structure
Structural Proteins
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 (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
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
Molecular Biology of Hantavirus
• Physical Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
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
Coding Strategy
Panhandle Structure
• Conserved repeated complementary
sequences at 5’ and 3’ ends form
panhandle structures
Transcription
• Viral polymerase transcribes negative-strand vRNA
to mRNA
• Polymerase acts as a endonuclease and cleaves
host mRNAs 7-18 nt from the 5’ cap
• The capped oligonucleotides act as primers
required to initiate transcription
• After transcription is primed and the first repeat of
the terminal sequence is transcribed, polymerase
slips and realigns the nascent RNA, then continues
transcription
Replication
• Viral polymerase transcribes negativestrand 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
Prime and Realign
Transcription and Replication
Molecular Biology of Hantavirus
• Physical Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
Attachment
Entry
Uncoating
Release
Transcription
Replication
Assembly
Translation
Attachment
• Viral G1 and G2 glycoproteins interact with
cell surface receptors
• Pathogenic hantaviruses bind 3 integrins
• Non-pathogenic hantaviruses bind 1
receptors
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 and G2
• Viral and cell membrane fuse
• Genomic material and polymerase are
released into cytoplasm
Entry
Primary Transcription
• Transcription of negative sense vRNA to
mRNA
• Viral polymerase (RdRp) transcribes
nucleoprotein-coated vRNA
• Capped oligonucleotides from cell’s own
mRNA are used to prime transcription
• Follows “prime and realign” model
Translation
• L and S segment mRNA is translated on free
ribosomes in cytoplasm
• M segment mRNA translated on ER-bound
ribosomes
• G1 and G2 peptides produced from M mRNA
are cleaved cotranslationally
• Separate signal sequences for G1 and G2 cause
ER attachment and embed the peptides in ER
membrane (Signal Hypothesis)
Translation
Genome Replication
• vRNA is used as a template by viral
polymerase to make sense strand cRNA
• cRNA is used as a template to make more
negative strand vRNA
• More genetic material means more virions
produced
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
Virion Assembly
• Membrane-bound G1 and G2 peptides are
transported to Golgi and carbohydrates
are attached by N-linked glycosylation
• vRNA complexes with N nucleopcapsid
protein, forms looped panhandle structure,
and complexes with L polymerase
Virion Assembly
Virion Release – Scenario 1
• Nucleocapsid complexes bud into the
Golgi membrane with G1 and G2
embedded
• Virion particle is formed inside the Golgi
• Virions are transported to cell membrane
by vesicles and released by exocytosis,
just like in secretion
• Viruses may prefer different cell surfaces
for release
Virion Release – Scenario 2
• Sin Nombre and Black Creek Canal
viruses
• G1 and G2 embedded into cell membrane
through Golgi vesicles
• Virions bud from cell membrane, not
through Golgi
Molecular Biology of Hantavirus
• Physical Properties
• Structure
• Genetics
• Replication Cycle
• Pathogenesis
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
• Heterodimeric 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 vasodilation
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
Laboratory Diagnosis of Hantavirus
• 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
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
HFRS
• A group of clinically similar diseases that
occur throughout Europe and Asia
• Includes several diseases that formerly
had other names, including Korean
hemorrhagic fever, epidemic hemorrhagic
fever and nephropathia epidemica
• ~15% fatality
Stages of Hemorrhagic Fever with
Renal Syndrome (HFRS)
1)Incubation (4-40 days)
2)Febrile Phase (3-5 days)
3)Hypotensive Phase (hours to days)
4)Oliguric Phase (3-7 days)
Recovery:
5)Diuretic Phase (2-21 days)
6)Convalescent Phase (2-3 months)
Febrile Phase
• 3-5 days
• Characterized by fever, chills
• Headache, severe myalgia
(muscle pain), nausea
• Blurred vision, photophobia,
eye pain caused 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
Hypotensive phase
• Hours to days
• Blood pressure decrease, hypovolemia (decreased
blood volume), shock
• Worsening of bleeding manifestations: petechiae,
epistaxis (nosebleed), gastrointestinal and
intracranial bleeding
• Levels of urea and creatinine in blood rise,
proteinuria (excessive protein in urine)
• Leukocytosis, thrombocytopenia (decreased # of
platelets)
Oliguric Phase
• 3-7 days
• Marked by decreased urine production due to
renal (kidney) dysfunction
• Hypervolemia (high blood volume) leading to
hypertension
• Blood electrolyte imbalance
• Continuation of hemorrhagic symptoms
• Severe complications: cardiac failure
pulmonary edema (swelling of lungs), and
cerebral bleeding
Diuretic Phase
• Several days to several weeks
• Beginning of recovery
• 3-6 liters of urine/ day; return to normal
renal activity
• Anorexia, fatigue due to dehydration
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
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 of the Americas
• ~50% fatality
Stages of Hantavirus Pulmonary
Syndrome (HPS)
1)
2)
3)
4)
5)
Incubation (4-30 days)
Febrile phase
Cardiopulmonary phase
Diuretic phase
Convalescent phase
Febrile Phase
• 3-5 days
• Fever, myalgia, malaise
• Other symptoms: headache, dizziness,
anorexia, nausea, vomiting, and
diarrhea.
Cardiopulmonary Phase
• 4-24 hours
• Presentation and rapid progression of shock and
pulmonary edema (4-24h non-productive cough
and tachypnea (shortness of breath)
• Hypovolemia due to progressive leakage of high
protein fluid from blood to lung interstitium and
alveoli
• Hypotension and oliguria
• Thrombocytopenia (often present in febrile
phase as well)
• Death within 24-48 hours due to hypoxia (lack of
oxygen) and/or myocardial failure
Diuretic Phase
•
•
•
•
•
Several days to several weeks
Beginning of recovery
Rapid clearance of pulmonary edema
Resolution of fever and shock
Anorexia, fatigue due to dehydration
Convalescent Phase
• Up to 2 months
• Results in chronic decreased small-airway
volume and diminished alveolar diffusing
capacity
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
Problems Diagnosing HPS
• Symptoms often confused with influenza
• Common signs of upper respiratory
disease such as sore throat, sinusitis, and
ear pain not usually present
• Abdominal pain often misinterpreted as
appendicitis
• Many doctors outside endemic regions fail
to recognize or have sufficient testing
Treatment of Hantavirus
Infection
• General care, alleviation of symptoms
• Ribavirin (HFRS)
• ECMO (HPS)
General Care
HFRS
• General treatment for
renal failure
• Hydration
• Dialysis
HPS
• General treatment for
pulmonary pathology
• Administration of
oxygen
Extra Corporeal Membrane
Oxygenation
• Removes blood from the body and
artificially removes CO2 and adds O2
• Costly
• Difficult
ECMO
Ribavirin
• Administered intravenously
• Shown to be effective against HFRS
causing strains
• Not shown to be effective against HPS
causing strains
Terrorism
Terrorism
A policy intended to strike with terror
those against whom it is adopted; the
employment of methods of intimidation;
the fact of terrorizing or condition of being
terrorized.
One man’s terrorist is another
man’s freedom fighter.
Considerations of Pathogens for
Use in Bioterrorism
•
•
•
•
•
Health effects
Epidemiology
Cost effectiveness
Psychological effects
Economic impact
CDC classifications
Category A
• Highest priority organisms that can be
easily disseminated or transmitted form
person to person, results in high mortality
rates and have the potential for major
public health impact, might cause public
panic and social disruption, and require
special action for public health
preparedness.
CDC Classifications
Category A
• Anthrax (bacillus anthracis)
• Botulism (clostridium botulinum)
• Plague (Yersinia pestis)
• Smallpox (variola major)
• Tularemia (Francisella tularensis)
• Viral hemorrhagic fevers
CDC Classifications
Category B
• Second highest priority organisms that are
moderately easy to disseminate, results in
moderate mortality rates, and require
specific enhancements of CDC's
diagnostic capacity and enhanced disease
surveillance.
CDC Classifications
Category B
• Typhus fever
• Viral encephalitis
• Ricin toxin
• Food and water safety threats
CDC Classifications
Category C
• Third highest priority organisms that
include emerging pathogens that could be
engineered for mass dissemination in the
future because of availability, ease of
production and dissemination, and
potential for high morbidity and mortality
rates and major health impact.
CDC Classifications
Category C
• Nipah Virus
• Crimean-Congo Hemorrhagic Fever virus
• Yellow fever
• Multi-drug resistant TB
• Influenza
• Rabies
CDC Classifications
• HFRS causing strains are Category A
because of high infectivity and morbidity
• HPS causing strains are Category C
because of low infectivity
Considerations of Pathogens for
Use in Bioterrorism
• Health effects
• Epidemiology
• Cost effectiveness
• Psychological effects
• Economic impact
Health Effects
• High lethality
• No or ineffective treatment
Health Effects
Hemorrhagic Fever with
Renal Syndrome
• Medium lethality
• Dramatic visual
change in patients
(psychological)
• Some success with
antiviral treatments
Hantavirus Pulmonary
Syndrome
• High Lethality
• No effective treatment
Considerations of Pathogens for
Use in Bioterrorism
• Health effects
• Epidemiology
• Cost effectiveness
• Psychological effects
• Economic impact
Epidemiology
• Medium incubation time in order to cause
secondary infections
• Rodent vector
• Spread through aerosol
• HFRS causing strains known to transmit
human to human
• Suspected human to human transmission
of HPS causing strain in Argentina (Andes
Virus)
Andes Virus
• 1996 outbreak in rural
Argentina
• Spread to people
whose only contact
was a car ride
• Spread to several
doctors caring for
HPS patients
• Low rodent
population in
Argentina at the time
(early spring)
Aerosolization
• Virus only infectious for 1-3 days outside
of a host because of a weak lipid envelope
• The number of particles needed to cause
a human infection is not known
Considerations of Pathogens for
Use in Bioterrorism
• Health effects
• Epidemiology
• Cost effectiveness
• Psychological effects
• Economic impact
Cost Effectiveness
• What resources would be required to
successfully disseminate Hantavirus into a
large population?
Cost Effectiveness
• Preparation of virus using cell culture
(difficult, need training and equipment)
• Aerosolization?
• Contaminated water?
• Creation of infected rodent population?
Considerations of Pathogens for
Use in Bioterrorism
• Health effects
• Epidemiology
• Cost effectiveness
• Psychological effects
• Economic impact
Psychological Effects
• HFRS causes dramatic visual effects in
patients
• HPS would be especially difficult to
diagnose outside of its normal range
• Media coverage ‘super flu’ ‘hemorrhagic
fever’
Mouse Virus in City!
Everyone will Die!
“Nothing to Fear” say Liberal ‘Doctors’
Considerations of Pathogens for
Use in Bioterrorism
• Health effects
• Epidemiology
• Cost effectiveness
• Psychological effects
• Economic impact
Economic Impact
What effect would an outbreak of
hantavirus have on the economy?
• Treatment
• Disruption of work
• Hysteria
• Even a small outbreak could cause a large
disruption
Why Hantavirus Would Be a
Good Terrorist Weapon
• There is no cure for HPS infection
• Fairly long incubation period between
infection and onset of symptoms
• Difficult diagnosis of HPS
• High lethality of HPS
Why Hantavirus Would Not Be
a Good Terrorist Weapon
• Low infectivity of HPS
• Difficult production
• Not stable
Prevention
• Vaccines
• Hygiene
Vaccines
• E. Coli expressed truncated nucleocapsid
as an immunogen
• Naked DNA
• Recombinant non-pathogenic virus
• Rodent brain-derived
• Cell culture derived
• Inactivated virus
Hygiene
• Prevent aerosolization of virus from rodent
excrement
• Dampen surfaces with detergent before
cleaning
• General hygiene
“Weapons of mass
destruction, including evil
chemistry and evil biology, are
all matters of great concern,
not only to the United States
but also to the world
community”
~ John Ashcroft