MICR 454L

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MICR 454L

• Emerging and Re-Emerging
• Infectious Diseases
• Lecture 12
• Ebola Virus, Dengue virus
• (ReadingEmerging Viruses)
• Dr. Nancy McQueen Dr. Edith Porter

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Overview

• Ebola Virus
• Dengue Virus

Brief history Morphology Genome Repl
ication cycle Diseases Pathogenesis Diagnosis Trea
tment Prevention Threats
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Ebola Virus
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Ebola Virus - Brief History

• In 1976 two epidemics of hemorrhagic fever
  occurred simultaneously in Zaire and Sudan.
• Over 500 cases were reported with a mortality
  rate of 88 in Zaire and 54 in Sudan
• A new virus that was isolated as the causative
  agent was named after the Ebola River in Zaire.
• Subsequent outbreaks occurred in Sudan in 1977
  and 1979.
• In 1994 the first Ebola case was reported in West
  Africa.
• In 1989 an outbreak occurred in cynomolgus
  monkeys imported from the Philippines to a
  facility in Reston, Virginia .
• Luckily that species does not appear to be
  pathogenic to humans

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Ebola Virus - Brief History

• Sporadic outbreaks, most in equatorial Africa,
  continue to occur
• Endemic in Sudan, Zaire, and the Ivory Coast.
• Very pathogenic for monkeys and apes - argues
  against these animals as the natural host.
• Recent studies - virus can replicate in fruit and
  insect-eating bats without any ill effects to the
  bat.
• Thus, bats may be the natural reservoir for the
  virus.

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Ebola Virus - Taxonomy

• Ebola virus belongs to the family Filoviridae,
  genus Ebolavirus
• There are four identified subtypes, three of
  which infect humans
• Filamentous, helical, enveloped virus
• Linear SS, - RNA genome

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Ebola Virus Replication Cycle
Budding from the plasma membrane
Penetration via receptor mediated endocytosis
Fusion with endosomal membrane (uncoating)
mRNA synthesis and genome replication in the
cytoplasm
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Ebola Virus Transmission to Humans

• How the virus first appears in a human at the
  start of an outbreak has not been determined.
• ? contact with an infected animal.
• After the first case-patient in an outbreak
  setting is infected, virus can be transmitted
  via
• Direct contact with the blood and/or secretions
  of an infected person.
• Contact with objects, such as needles, that have
  been contaminated with infected secretions.
• Sexual contact
• Aerosol transmission has been documented in
  non-human primates, but not in humans

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Ebola Virus - Diseases/Pathogenesis

• Incubation - 2 to 21 days
• Symptoms
• abrupt - characterized by fever, headache, joint
  and muscle aches, sore throat, and weakness
• followed by diarrhea, vomiting, and stomach pain
• rash, red eyes and hiccups may also occur
• dendritic cells and macrophages initially
  infected followed by hepatocytes, and, in latter
  stages, endothelial cells.
• virus evades host defenses by producing proteins
  (vp24, vp35) that interfere with interferon
  signaling pathway.

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Ebola Virus Pathophysiology of the Disease

• Clinical infection in human and nonhuman primates
  is associated with rapid and extensive viral
  replication in all tissues.
• Viral replication is accompanied by widespread
  and severe focal necrosis.
• The most severe necrosis occurs in the liver.
• A viral glycoprotein, sGP binds to a
  neutrophil-specific receptor and inhibits early
  neutrophil activation.
• sGP also may be responsible for the profound
  lymphopenia that characterizes Ebola infection
• A second viral glycoprotein binds to endothelial
  cells but not to neutrophils, allowing Ebola
  virus to invade, replicate in, and destroy
  endothelial cells.

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Ebola Virus - Pathophysiology of the Diseases

• Destruction of endothelial surfaces causes the
  vessels to leak and bleed.
• Destruction of the endothelial surfaces can lead
  to disseminated intravascular coagulation (DIC),
  and this, combined with the liver destruction may
  contribute to the hemorrhagic manifestations that
  characterize Ebola infections.
• The two major factors in Ebola virus pathogenesis
  are the impairment of the immune response and
  vascular dysfunction.
• Death results from liver damage and dysfunction,
  shock, and the DIC which leads to internal and
  external bleeding
• mortality rate ranges from 30-90

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Ebola Hemorrhagic Fever
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Ebola Virus- Diagnosis

• Serology
• ELISA
• Molecular tests
• RT-PCR
• Virus isolation - Must use biocontainment level
  IV facility!

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Ebola Virus - Treatment

• There is no standard treatment for Ebola
  hemorrhagic fever (HF).
• Patients receive supportive therapy - balancing
  the patients fluids and electrolytes,
  maintaining their oxygen status and blood
  pressure, and treating them for any complicating
  infections.
• Ribavirin treatment has been tried with little
  success.

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Ebola Virus - Prevention

• No vaccine is currently available, but many are
  in development
• Practical viral hemorrhagic fever isolation
  precautions, or barrier nursing techniques must
  be used. These techniques include
• the wearing of protective clothing, such as
  masks, gloves, gowns, and goggles
• the use of infection-control measures, including
  complete equipment sterilization
• the isolation of Ebola HF patients from contact
  with unprotected persons.
• direct contact with the body of a deceased
  patient should be prevented.

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Ebola Virus - Threats

• Sporadic epidemics continue to occur
• challenge of developing additional diagnostic
  tools to assist in early diagnosis of Ebola HF
• challenge of conducting ecological investigations
  of Ebola virus and its possible reservoir (bats).
• challenge to determine how the virus is
  transmitted to humans must be acquired to prevent
  future outbreaks effectively.
• Use as bioterrorism weapon
• Kills too quickly?
• Aerosol spread?

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Take Home Message

• Ebola virus belongs to the family Filoviridae
• Enveloped with linear SS, - RNA genome
• Reservoir appears to be bats
• Virus targets dendritic cells, macrophages,
  hepatocytes, endothelial cells
• Virus impairs immune function in several ways
• Liver and endothelial cell destruction? DIC ?
  internal and external bleeding
• High mortality rate
• Diagnosis via serology, molecular tests, virus
  isolation (level IV biocontainment)
• Treatment - supportive care
• Vaccines in development

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Dengue Virus
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Brief History

• The first cases of Dengue Fever (DF) were
  recorded in 1779 in Batavia, Indonesia, and Cairo
  
• In 1780, there was an epidemic reported in
  Philadelphia, PA.
• For the past 200 years, pandemics have been
  recorded in tropical and subtropical climates at
  10 to 30 year intervals.
• Although DF is not a new disease, it can be
  classified as an emerging disease.
• Since 1945, the number of reported cases of DF
  surged because of increased urbanization and
  travel

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Dengue Virus- Classification

• Is in the family Flaviviridae, genus flavivirus
• Belong to Group B arboviruses (arthropod borne
  animal viruses)
• Transmitted by female Aedes aegypti mosquitoes
• Has 4 serotypes (DEN-1, 2, 3, 4)
• Enveloped, single-stranded RNA genome

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Dengue Virus Replication Cycle
mRNA synthesis and genome replication in the
cytoplasm
Budding from ER
exocytosis
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Transmission of Dengue Virusby Aedes aegypti
Mosquito refeeds /
Mosquito feeds /
transmits virus
acquires virus
Intrinsic incubation period
Viremia
Viremia
0
5
8
12
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DAYS
Human 1
Human 2
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Replication and Transmissionof Dengue Virus
1. Virus transmitted to human in mosquito
saliva
2. Virus replicates in target organs
3. Virus infects white blood cells and
lymphatic tissues
4. Virus released and circulates in blood
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Replication and Transmissionof Dengue Virus
5. Second mosquito ingests virus with blood
6. Virus replicates in mosquito midgut
and other organs, infects salivary glands
7. Virus replicates in salivary glands
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Diseases

• Undifferentiated fever
• Classic dengue fever
• Dengue hemorrhagic fever
• Dengue shock syndrome

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Undifferentiated Fever

• most common manifestation of dengue
• 87 of students infected are either asymptomatic
  or only mildly symptomatic

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Classic Dengue Fever

• Fever
• Headache
• Muscle and joint pain
• Nausea/vomiting
• Rash (petechiae)
• Hemorrhagic manifestations
• Encephalitis
• Decreased level of consciousness lethargy,
  confusion, coma
• Seizures
• Nuchal rigidity
• Paresis (slight paralysis)

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Petechiae
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Dengue Hemorrhagic Fever (DHF)

• Skin hemorrhages
  petechiae, purpura, ecchymoses
• Gingival bleeding
• Nasal bleeding
• Gastro-intestinal bleeding
  hematemesis, melena (dark stools),
  hematochezia (bloody stools)
• Hematuria
• Increased menstrual flow

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Clinical Case Definition forDengue Hemorrhagic
Fever
4 Necessary Criteria

• Fever, or recent history of acute fever
• Hemorrhagic manifestations
• Low platelet count (100,000/mm3 or less)
• Objective evidence of leaky capillaries
• elevated hematocrit (20 or more over baseline)
• low albumin
• pleural or other effusions

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Pleural Effusion Index
Vaughn DW, Green S, Kalayanarooj S, et al. Dengue
in the early febrile phase viremia and antibody
responses. J Infect Dis 1997 176322-30.
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Clinical Case Definition for Dengue Shock Syndrome

• 4 criteria for DHF
• Evidence of circulatory failure manifested
  indirectly by all of the following
• Rapid and weak pulse
• Narrow pulse pressure (? 20 mm Hg) OR hypotension
  for age
• Cold, clammy skin and altered mental status
• Shock is direct evidence of circulatory failure

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Risk Factors For Dengue Hemorrhagic Fever (DHF)

• Second infection with a different serotype
• Due to pre-existing, non-neutralizing anti-dengue
  antibody
• Presence of maternal antibody to a different
  serotype
• Virus strain
• Virus serotype
• DHF risk is greatest for DEN-2, followed by
  DEN-3, DEN-4 and DEN-1
• Host genetics
• Age
• Higher risk in locations with two or more
  serotypes circulating simultaneously at high
  levels (hyperendemic transmission)

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Hypothesis on Pathophysiologyof DHF

• Persons who have experienced a dengue infection
  develop serum antibodies that can neutralize the
  dengue virus of that same (homologous) serotype.

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1
Dengue 1 virus
Neutralizing antibody to Dengue 1 virus
Non-neutralizing antibody
Complex formed by neutralizing antibody and virus
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Hypothesis on Pathophysiologyof DHF

• In a subsequent infection, the pre-existing
  heterologous non-neutralizing antibodies form
  complexes with the new infecting virus serotype,
  but do not neutralize the new virus.

Dengue 2 virus
Non-neutralizing antibody to Dengue 1 virus
Complex formed by non-neutralizing antibody and
virus
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Hypothesis on Pathophysiologyof DHF

• Antibody-dependent enhancement of virus uptake -
  Dengue virus, complexed with non-neutralizing
  antibodies binds to Fc receptor and enters
  monocytes/macrophages (Note - This entry into
  monocytes or macrophages is not dependent on the
  cell having the receptor to which the virus must
  normally bind for entry.)

Fc receptor
Fc region
Dengue 2 virus
Non-neutralizing antibody
Complex formed by non-neutralizing antibody and
Dengue 2 virus
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Hypothesis on Pathophysiologyof DHF

• Hemorrhagic manifestations that characterize DHF
  and DSS due to
• Infected monocytes/macrophages release of
  vasoactive mediators, resulting in increased
  vascular permeability.
• circulating dengue antigen-antibody complexes
  that activate complement, resulting in the
  release of vasoactive mediaters.
• the process of immune elimination of infected
  cells, that releases proteases and lymphokines
  that activate complement, coagulation cascades
  (leads to DIC) and vascular permeability factors.

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Diagnosis

• ELISA
• Virus isolation
• Cell culture
• Mosquito inoculation
• Fluorescent antibody test
• RT-PCR

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Diagnosis -Tourniquet Test

• Inflate blood pressure cuff to a point midway
  between systolic and diastolic pressure for 5
  minutes
• Positive test 20 or more petechiae per 1 inch2
  (6.25 cm2)

Pan American Health Organization Dengue and
Dengue Hemorrhagic Fever Guidelines for
Prevention and Control. PAHO Washington, D.C.,
1994 12.
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Positive Tourniquet Test
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Treatment

• Fluids
• Rest
• Antipyretics (avoid aspirin and non-steroidal
  anti-inflammatory drugs)
• Monitor blood pressure, hematocrit, platelet
  count, level of consciousness
• Avoid invasive procedures when possible
• Unknown if the use of steroids, intravenous
  immune globulin, or platelet transfusions to
  shorten the duration or decrease the severity of
  thrombocytopenia is effective
• Patients in shock may require treatment in an
  intensive care unit

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Mosquito Barriers

• Only needed until fever subsides, to prevent
  Aedes aegypti mosquitoes from biting patients and
  acquiring virus
• Keep patient in screened sickroom or under a
  mosquito net

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Prevention

• No licensed vaccine at present
• Effective vaccine must be tetravalent
• Field testing of an attenuated tetravalent
  vaccine currently underway
• Effective, safe and affordable vaccine will not
  be available in the immediate future

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Threats

• Dengue virus causes about 100 million cases of
  acute febrile disease annually, including more
  than 500,000 reported cases of DHF/DSS and up to
  50,000 deaths.
• Currently, dengue is endemic in 112 countries.
• From 1977 to 2004, a total of 3,806 suspected
  cases of imported dengue were reported in the
  United States.
• Dengue epidemic in Brazil in April, 2008 killed
  106 but now seems to be abating

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Take Home Message

• Dengue virus is in the family Flaviviridae
• Enveloped, single-stranded RNA genome
• 4 serotypes
• Transmitted by female Aedes aegypti mosquito
• Causes undifferentiated fever, dengue fever,
  dengue hemorrhagic fever (skin hemorrhages, low
  platlet count, leaky capillaries) , dengue shock
  syndrome ( circulatory collapse and shock)
• Risk factors for DHF include secondary infection
  with a different serotype - due to
  non-neutralizing antibodies
• Hemorrhagic manifestations due to increased
  vascular permeability and coagulation activation
• Diagnosis includes tourniquet test
• Treatment is rest, fluids, antipyretics
• Tetravalent vaccine in development

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Resources

• The Microbial Challenge, by Krasner, ASM Press,
  Washington DC, 2002.
• Brock Biology of Microorganisms, by Madigan and
  Martinko, Pearson Prentice Hall, Upper Saddle
  River, NJ, 11th ed, 2006.
• Microbiology An Introduction, by Tortora, Funke
  and Case Pearson Prentice Hall 9th ed, 2007.
• Fundamentals of Molecular Virology, by Nicholas
  Acheson Wiley and Sons 2007
• Human Virology by Collier and Oxford, Oxford
  University Press 2nd edition, 2000.
• www.cdc.gov
• http//www.defenseindustrydaily.com/images/MISC_Eb
  ola_Patient.jpg
• http//www.kcom.edu/faculty/chamberlain/website/le
  ctures/lecture/IMAGE/HEMFEVM.GIF
• http//www.cdc.gov/ncidod/dvbid/dengue/index.htmc
  urrent

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Resources

• www.cdc.gov
• http//www.hepcprimer.com/images/cut-model-with-te
  xt.gif