Microbiology (Dra Madrid/Reyes)

Flaviviridae
13 December 07

FLAVIVIRIDAE PATHOGENIC FLAVIVIRUSES

 Flaviviridae family  3 groups according to symptoms they cause
o Genus Flavivirus o Meningoencephalitides
 SLE, JE, MVE and TBE
 Once classified in the Togaviridae, now
o Fever-arthralgia-rash syndrome
constitutes one of three genera in the family
Flaviviridae  DEN, WN, KUN
o Hemorrhagic fever syndrome
o Genus Pestivirus
 Omsk hemorrhagic fever, Kyasanur Forest dse
 Includes animal pathogens (bovine viral
virus, YF, DEN
diarrhea and hog cholera viruses) that are of
considerable economic importance, but contains
FLAVIVIRUS STRUCTURE
no known human pathogens
 Virions: spherical, 40-50 nm in diameter
o Genus Hepacivirus  Nucleocapsid contains capsid protein (C)
o All flaviviruses that cause disease in humans are  RNA: single 405 (10.9 kilobases) positive-sense strand
arthropod-borne viruses (arboviruses) o Capped at the 5’ end, but, unlike alphaviruses, has
no poly A segment at the 3’ end
VIRUS PRIMARY VERTEBRA GEOGRAPHIC  Envelope: lipid bilayer
VECTOR TE DISTRIBUTIO o 1) envelope protein (E) [51,000-59,000 daltons]
RESERVOI N o 2) small nonglycosylated protein (M) [8,500
R daltons]
Dengue Aedes aegypti Humans, Tropics o Only E, which is glycosylated in most flaviviruses, is
1,2,3,4 monkeys clearly demonstrable on the virion surface
Japanese Culex Birds Asia  Virions mature at intracytoplasmic membranes
enceph  Most members are transmitted by bloodsucking
St. Louis Culex Birds, pigs Americas arthropods
enceph
West Nile Culex Birds Africa, tropical FLAVIVIRUS GENOME
Asia,  Upon translation, several enzymes cut single polypeptide
Mediterranean into functional protein units.
Yellow Fever Aedes aegypti Humans, Tropical Africa
 Structural p rotein functions :
monkeys and the
Americas  C: highly basic component of the nucleocapsid
Omsk Dermacentor Rodents Central Russia  prM: precursor of M protein
hemmorhagic  M protein: membrane-associated and serve a
fever matrix function, linking capsid and envelope
Tick-borne Ixodes Rodents, Russia, Eastern  E: major envelope protein; virion assembly, receptor
encephalitis birds, Europe, binding and membrane fusion
domesticate Scandinavia
d animals MULTIPLICATION
Louping III Ixodes Sheep, British Isles  Genomic RNA is capped (not polyadenylated) and serves
birds as mRNA for all proteinds
Powassan Ixodes Small Canada, US,  Structural proteins are encoded at the 5’ end of the
mammals Russia genome; nonstructural proteins (e.g., RNA-dependent
RNA polymerase) are encoded in the 3’ two-thirds
Kyansanur Haemaphysalis Rodents Southwest
Forest India  Complementary (antisense) RNA, made from genomic
Disease RNA, serves as a template for progeny genomic RNA
Murray Valley Culex Birds Austrialia, New
encephalitis/ Guniea (+) Strand parental RNA Proteins encoded in
Kunjin Rocio the entire genome

Virus Disease
Dengue 1,2.3.4 Fever, rash, arthralgia, myalgia
Japanese enceph Encephalitis
St. Louis enceph Encephalitis Nonstructural proteins
(-) Strand
West Nile Fever, rash, arthralgia, myalgia encoded in the 3’ end of
RNA
Yellow Fever Fever, hemorrhage, jaundice genome RNA
Omsk hemorrhagic fever Fever, hemorrhage
Tick-borne encephalitis Encephalitis (+) Strand progeny RNA
Louping III Encephalitis
Powassan Encephalitis Structural protein
Kyansanur Forest Disease Fever, hemorrhage, encephalitis encoded in the 5’
Murray Valley Encephalitis
encephalitis/Kunjin Rocio
Progeny virus
Leu, virns, brim, ate candz 1 of 6
 Microbiology – Flaviviridae by Dra Madrid/Reyes
FLAVIVIRUS REPLICATION
 Replication occurs in the cytoplasm (20-30 hrs)
 Entire virus genome is translated as a single polyprotein
which is then cleaved into the mature proteins
o Structural proteins: encoded at the 5’ end
o Nonstructural proteins (e.g., NS-1 and RNA-
dependent RNA polymerase) are encoded in the 3’
two-thirds
 Complementary negative strand RNA is synthesized by
NS proteins and then is used as a template for genomic
progeny RNA synthesis
 Assembly occurs characteristically into cytoplasmic
vacuoles (in association with Golgi or smooth
membranes)
 Release occurs when cell lyses
FLAVIVIRUS PATHOGENESIS AND CLINICAL
MANIFESTATIONS
 Flaviviruses vary widely in their pathogenic potential and
mechanisms for producing human disease
 It is useful to consider them in three major categories
o Those associated primarily with the encephalitis
Syndrome (prototype: St. Louis encephalitis)
o Those associated with fever-arthralgia-rash
(prototype: dengue fever), or
o with hemorrhagic fever (prototype: yellow fever)
 Human infection initiated by deposition of virus through
the skin via the saliva of an infected arthropod 
Replicates locally & in regional lymph nodes  viremia
 Most human infections with St. Louis enceph (SLE) and
Japanese enceph (JE) viruses, there is either no apparent
disease or a nonspecific febrile illness with headache
 Infection resolves, and lasting immunity is produced
 CNS lesions may develop: aseptic meningitis or
encephalitis
 In the great majority of flavivirus infections, virus is
cleared by the immune system
o However, persistence in neurological tissue has
been noted with tick-borne encephalitis viruses, and
recent reports of recurrent encephalitic bouts in
children have been associated with JE virus
recovery from peripheral blood mononuclear cells
HOST DEFENSES
 Lasting protection is generally restricted to the same
flavivirus, and is associated with neutralizing antibodies
ANTHROPOD-BORNE VIRUSES
 Anthropod-borne viruses (arboviruses) WHO definition:
“Viruses maintained in nature principally through
biological transmission between susceptible vertebrate
hosts by haematophagus arthropods.”
 Arboviruses belong to three families
1. Togaviruses
- e.g. EEE, WEE, and VEE
2. Bunyaviruses
- e.g. Sandfly Fever, Rift Valley Fever, Crimean-
Congo Haemorrhagic Fever
3. Flaviviruses
- e.g. Yellow Fever, Dengue, Japaneses
Encephalitis
TRANSMISSION CYCLES
 Man-arthropod-man
o e.g. Dengue, Urban yellow fever
o Reservoir may be in either man or arthropod vector
o In the latter transovarial transmission may take
place
 Animal-arthropod vector-man
o e.g. Japanese encephalitis, Jungle yellow fever
Page 2 of 6
o Reservoir is in an animal
o Virus is maintained in nature in a transmission cycle
involving the arthropod vector and animal. Man
becomes infected incidentally.
 Both cycles may be seen with some arboviruses such as
yellow fever
ARTHROPOD VECTORS
 Mosquitoes
o Japanese encephalitis, dengue, yellow fever, St.
Louis encephalitis
 Ticks
o Various tick-borne encephalitides etc.
ANIMAL RESERVOIRS
 In many cases, the actual reservoir is not known. The
following animals are implicated as reservoirs
o Birds: Japanese encephalitis, St. Louis encephalitis
o Pigs: Japanese encephalitis
o Monkeys: Yellow Fever
o Rodents: Russian Spring-Summer encephalitis
DISEASES CAUSED
 Fever and rash
o This is usually a non-specific illness resembling a
number of other viral illnesses such as influenza,
rubella, and enterovirus infections. The patients
may go on to develop encephalitis or haemorrhagic
fever
 Encephalitis
o St. Louis encephalitis, Japanese encephalitis
 Haemorrhagic fever
o e.g, Yellow fever, dengue
DIAGNOSIS
 Serology – usually used to make a diagnosis of arbovirus
infections
 Culture – a number of cell lines may be used, including
mosquito cell lines. However, it is rarely carried out since
many of the pathogens are group 3 or 4 pathogens
 Direct detection tests – e.g. detection of antigen and
nucleic acids are available but again there are safety
issues
PREVENTION
 Surveillance – of disease and vector
 Control of vector – pesticides, elimination of breeding
grounds
 Personal protection – screening of houses, bed nets,
insect repellants
 Vaccination – available for a number of arboreal
infections e.g. Yellow fever, Japanese encephalitis,
Russian tick-borne encephalitis
JAPANESE ENCEPHALITIS
 First discovered and originally restricted to Japan. Now
large scale epidemics occur in China, India and other
parts of Asia.
 Flavivirus, transmitted by culex mosquitoes
 The virus is maintained in nature in a transmission cycle
involving mosquitoes, birds and pigs.
 Most human infections are subclinical, the unapparent to
clinical cases is 300/1.
 In clinical cases, a life-threatening encephalitis occurs.
 The disease is usually diagnosed by serology. No specific
therapy is available.
 Since culex has a flight range of 20km, all local control
measures will fail. An effective vaccine is available.
 Microbiology – Flaviviridae by Dra Madrid/Reyes Page 3 of 6

VIROLOGY DIFFERENT PATTERNS OF AGE DISTRIBUTION CASES

 Japanese encephalitis (JE) serocomplex
o 10 viruses
o 6 human pathogens (JE, West Nile, Kunjin,
Usutu, St. Louis Encephalitis, Murray Valley
Ecephalitis viruses)
o Most are amplified bird – mosquito – bird
 5 genotypes in Asia (most isolates in genotype 1)

HISTORY OF DISCOVERY
 1871: “Summer encephalitis” epidemic in Japan
 1924: Agent from human brain tissue isolated in rabbits
 1934: Isolate of this virus produced experimental
encephalitis in monkeys
 1938: First isolate from Culex tritaeniorhynchus
 1930s: First mouse brain-derived vaccines developed
 1954: “Refined” mouse brain vaccine developed

WHY IS JE A PROBLEM?
 JE is the leading cause of viral encephalitis in Asia,
now that poliomyelitis has nearly been eradicated.
 More than 3 billion people live in areas where JE are
reported to WHO each year.
TWO PATTERNS OF TRANSMISSION OF JE
 10,000 to 15,000 deaths are reported each year. 1. Seasonal, called an epidemic pattern (e.g., southern
China)
CASES ARE UNDER-REPORTED
 Cases are under-reported due to
o Lack of good surveillance
o Lack of diagnostics
 Actual number of cases is probable more than 175,000
per year

CLINICAL SPECTRUM OF JE DISEASE

Die
Sever

Moderat 2. Year-round, called an endemic pattern (e.g., Bali,

Indonesia)
Mild

Asymptomati
c

DEATH AND DISABILITY FROM JE

 Up to 30% of all patients with JE die
 For those that survive the illness, 30% to 75% cases are
left with disability
 Disability is with both physical and cognitive

AGE GROUPS AFFECTED BY JE

 Children 1 to 15 years of age are mainly affected in
endemic areas.
 But people of any age can be affected. Adult infection
most often occurs in areas where the disease is newly
introduced.
TRANSMISSION OF JE
 JE is spread by mosquitoes
 Culex tritaeniorhynchus is the main vector in most of
Asia, but tohe species that breed in rice paddies, ditches,
and ground pools are also important.
 Microbiology – Flaviviridae by Dra Madrid/Reyes Page 4 of 6

 Dengue haemorrhagic fever and shock syndrome appear

JE TRANSMISSION CYCLE most often in patients previously infected by a different
serotype of dengue, thus suggesting an
immunopathological mechanism.
 Diagnosis is made by serology.
 No specific antiviral therapy is available.
 Prevention of dengue in endemic areas depends on
mosquito eradication. The population should remove all
containers from their premises which may serve as
vessels for egg deposition.
 A live attenuated vaccine is being tried in Thailand
with encouraging results.

DENGUE PREVENTION
 Currently, the only effective way to avoid dengue virus
infection in areas where the disease is endemic or
epidemic is to avoid being bitten by infected mosquitoes
throught he use of personal insect repellant and other
PEOPLE AT RISK insect barriers.
 People living at rural areas have the highest risk of
disease because the mosquitoes that spread JE breed in REPLICATION AND TRANSMISSION OF DENGUE VIRUS
rice paddies and pool water. 1. Virus transmitted to human in mosquito saliva
 Cases in urban areas also occur. 2. Virus replicates in target organs
3. Virus infects white blood cells and lymphatic tissues
YELLOW FEVER 4. Virus released and circulates in blood
 Flavivirus, mainly found in West Africa and S America 5. Second mosquito ingests virus with blood
6. Virus replicates in mosquito midgut and other
 Yellow fever occurs in 2 major forms: urban and jungle organs, infects salivary glands
(sylvatic) yellow fever. 7. Virus replicates in salivary glands
 Jungle YF is the natural reservoir of the disease in a
cycle involving nonhuman primates and forest
mosquitoes. Man may become incidentally infected on
venturing into jungle areas.
 The urban form is transmitted between humans by the
Aedes aegypti mosquito
 Classically Yellow Fever presents with chills, fever, and
headache. Generalized myalgias and GI complaints
(N+V).
 Some patients may experience an asymptomatic
infection or a mild undifferentiated febrile illness.
 After a period of 3 to 4 days, the more severely ill
patients with a classical YF course will develop
bradycardia (Faget's sign), jaundice, and haemorrhagic
manifestations.
 50% of patients with frank YF will develop fatal disease
characterized by severe haemorrhagic manifestations,
oliguria and hypotension.
 Diagnosis is usually made by serology
 There is no specific antiviral treatment
 An effective live attenuated vaccine is available
against yellow fever and is used for persons living in or
traveling to endemic areas.

DENGUE
Aedes aegypti Mosquito
 Dengue is the biggest arbovirus problem in the
world today with over 2 million cases per year. Dengue
is found in SE Asia, Africa and the Caribbean and S
America.
 Flavivirus, 4 serotypes, transmitted by Aedes
mosquitoes which reside in water-filled containers.
 Human infections arise from a human-mosquito-
human cycle  Dengue transmitted by infected female mosquito
 Classically, dengue presents with a high fever,  Primarily a daytime feeder
lymphadenopathy, myalgia, bone and joint pains,  Lives around human habitation
headache, and a maculopapular rash.  Lays eggs and produces larvae preferentially in artificial
 Severe cases may present with haemorrhagic fever and containers
shock with a mortality of 5-10%. (Dengue
haemorrhagic fever or Dengue shock syndrome.) DENGUE CLINICAL SYNDROMES
 Microbiology – Flaviviridae by Dra Madrid/Reyes Page 5 of 6

 Undifferentiated fever o Grade 1 manifestations + spontaneous bleeding

 Classic dengue fever  Grade 3
 Dengue hemorrhagic fever o Signs of circulatory failure (rapid/weak pulse, narrow
 Dengue shock syndrome pulse pressure, hypotension, cold/clammy skin)
 Grade 4
o Profound shock (undetectable pulse and BP)

UNDIFFERENTIATED FEVER DANGER SIGNS IN DENGUE HEMORRHAGIC FEVER

 May be the most common manifestation of dengue  Abdominal pain - intense and sustained
 Prospective study found that 87% of students infected  Persistent vomiting
were either asymptomatic or only mildly symptomatic  Abrupt change from fever to hypothermia, with sweating
 Other prospective studies including all age-groups also and prostration
demonstrate silent transmission  Restlessness or somnolence

CLINICAL CHARACTERISTICS OF DENGUE FEVER WARNING SIGNS FOR DENGUE SHOCK

 Fever
 Headache
 Muscle and joint pain
 Nausea/vomiting
 Rash
 Hemorrhagic manifestations

SIGNS AND SYMPTOMS OF

ENCEPHALITIS/ENCEPHALOPATHY ASSOCIATED WITH
ACUTE DENGUE INFECTION
 Decreased level of consciousness: lethargy, confusion,
coma
 Seizures
 Nuchal rigidity
 Paresis

HEMORRHAGIC MANIFESTATIONS OF DENGUE

 Skin hemorrhages: petechiae, purpura, ecchymoses
 Gingival bleeding HYPOTHESIS ON PATHOGENESIS OF DHF (PART 1)
 Nasal bleeding  Persons who have experienced a dengue infection
 Gastrointestinal bleeding: hematemesis, melena, develop serum antibodies that can neutralize the dengue
hematochezia virus of that same (homologous) serotype
 Hematuria
 Increased menstrual flow Homologous Antibodies Form Non-Infectious
Complexes
CLINICAL CASE DEFINITION FOR DENGUE
HEMORRHAGIC FEVER
 4 Necessary Criteria:
o Fever, or recent history of acute fever
o Hemorrhagic manifestations
o Low platelet count (100,000/mm3 or less)
o Objective evidence of “leaky capillaries:”
 elevated hematocrit (20% or more over
baseline)
 low albumin
 pleural or other effusions

CLINICAL CASE DEFINITION FOR DENGUE SHOCK

SYNDROME This slide presents a graphic depiction of the development of
homologous antibodies. When an individual is infected with
 4 criteria for DHF
any dengue serotype—here called dengue type A, to indicate
 Evidence of circulatory failure manifested indirectly by all that the infection may be with DEN-1, 2, 3 or 4—the immune
of the following: system responds by producing anti-A antibodies. These anti-A
o Rapid and weak pulse
antibodies form homologous complexes with the type A virus,
o Narrow pulse pressure (< 20 mm Hg) OR which results in the neutralization of the virus.
hypotension for age
o Cold, clammy skin and altered mental status
HYPOTHESIS ON PATHOGENESIS OF DHF (PART 2)
 Frank shock is direct evidence of circulatory failure  In a subsequent infection, the pre-existing heterologous
antibodies form complexes with the new infecting virus
FOUR GRADES OF DHF serotype, but do not neutralize the new virus
 Grade 1
o Fever and nonspecific constitutional symptoms Heterologous Antibodies Form Infectious
o Positive tourniquet test is only hemorrhagic Complexes
manifestation
 Grade 2
 Microbiology – Flaviviridae by Dra Madrid/Reyes
In this depiction of the second step in antibody-dependent
enhancement, dengue type B represents the new serotype.
The anti-A antibodies produced in response to the original
infection can form complexes with the new serotype, as we
see here. However, the new virus serotype is not neutralized
by these heterologous antibodies. The virus retains its
capacity to become active and replicate.
HYPOTHESIS ON PATHOGENESIS OF DHF (PART 3)
 Antibody-dependent enhancement is the process in which
certain strains of dengue virus, complexed with non-
neutralizing antibodies, can enter a greater proportion of
cells of the mononuclear lineage, thus increasing virus
production
Heterologous Complexes Enter More Monocytes, Where
Virus Replicates
This slide shows the same information graphically. We can
see that the heterologous antibody-virus complexes can enter
a greater proportion of monocytes than the virus alone. The
greater proportion of infected cells results in greater virus
production.
HYPOTHESIS ON PATHOGENESIS OF DHF (PART 4)
 Infected monocytes release vasoactive mediators,
resulting in increased vascular permeability and
hemorrhagic manifestations that characterize DHF and
DSS
PETECHIAE
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)
Positive Tourniquet Test
Page 6 of 6
LABORATORY TESTS IN DENGUE FEVER
 Clinical laboratory tests
 CBC—WBC, platelets, hematocrit
 Albumin
 Liver function tests
 Urine—check for microscopic hematuria
* CBC. In patients with dengue, the leukocyte counts are
often low, and the patient may even be neutropenic. Platelet
levels should also be checked, and you should do serial
hematocrits for hemoconcentration.
 Dengue-specific tests
 Virus isolation
 Serology
LABORATORY METHODS FOR DENGUE DIAGNOSIS, CDC
DENGUE BRANCH
 Virus isolation to determine serotype of the infecting
virus
 This can be performed either by using mosquito
cell cultures or by mosquito inoculation.
o IgM ELISA test for serologic diagnosis
DENGUE: MANAGEMENT
 No hemorrhagic manifestations and patient is well-
hydrated: home treatment
 Hemorrhagic manifestations or hydration borderline:
outpatient observation center or hospitalization
 Warning signs (even without profound shock) or DSS:
hospitalize
 Fluids
 Patients should be encouraged to take small,
frequent sips of fluids. If the patient cannot be
rehydrated by mouth, fluids should be
administered intravenously. At times large
amounts of intravenous fluids are needed.
 Rest
 Antipyretics (avoid aspirin and non-steroidal anti-
inflammatory drugs so that platelet function will not be
impaired)
 Monitor blood pressure, hematocrit, platelet count, level
of consciousness
DENGUE VACCINE?
 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