MICROBIOLOGY

4.4C Flaviviridae Dr. Madrid

February 12, 2014
PamantasanngLungsodngMaynila – College of Medicine

Italicized text lifted from the book. FLAVIVIRUS

FLAVIVIRIDAE  3 groups according to symptoms they cause:
 Genus Flavivirus o Meningoencephalitides
o Once classified in the Togaviridae, now constitutes one of three o Fever-arthralgia-rash syndrome
genera in the family Flaviviridae o Hemorrhagic fever syndrome
 Genus Pestivirus
o Includes animal pathogens (bovine viral diarrhea and hog VIRUS DISEASE
cholera viruses) that are of considerable economic importance, Dengue 1,2,3,4  Rash
but contains no known human pathogens  Arthralgia
 Genus Hepacivirus  Fever
o No arthropod vector  Myalgia
o Not an arbovirus Japanese encephalitis  Encephalitis
 All flaviviruses that cause disease in humans are arthropod-borne St. Louis encephalitis  Encephalitis
viruses (arboviruses)
o Formerly included in the togavirus family as "group B West Nile  Rash
arboviruses"  Arthralgia
o Moved to a separate family because of differences in viral  Fever
genome organization  Myalgia
o Transmitted between vertebrates by
 Mosquitoes Yellow Fever  Fever
 Ticks  Hemorrhage
o Transmitted among rodents or bats without any known insect  Jaundice
vectors Omsk hemorrhagic fever  Fever
 All flaviviruses are antigenically related  Hemorrhage
Tick-borne encephalitis  Encephalitis
FLAVIVIRUS STRUCTURE Louping Ill  Encephalitis
Virion  Spherical Powassan  Encephalitis
 40-60 nm in diameter Kyansanur Forest  Encephalitis
Nucleocapsid  Contains capsid protein (C) Disease  Fever
Composition  DNA (13%)  Hemorrhage
 Protein (87%)
Genome  (+)ssRNA Murray Valley  Encephalitis
 Capped at the 5’ end, but unlike alphaviruses, encephalitis/
has no polyA segment at the 3’ end Kunjin Rocio
 Genome RNA infectious
Envelope  Lipid bilayer GENOME
o Envelope protein (E) [51,000-59,000 daltons]
o Small nonglycosylated protein (M) [8,500
daltons]
o Only E (glycosylated in most flaviviruses) is
clearly demonstrable on the virion surface
Outstanding  Virions mature at intracytoplasmic membranes
characteristics  Most members are transmitted by bloodsucking
arthropods
 Upon translation, several enzymes cut the single polypeptide into
functional protein units
Primary Vertebrate Geographic STRUCTURAL PROTEINS
Virus
Vector Reservoir Distribution C Highly basic component of the nucleocapsid
Dengue  Humans
Aedes aegypti  Tropics prM Precursor of M protein
1,2,3,4  Monkeys Membrane-associated and serve a matrix function, linking
Japanese M
Culex  Birds  Asia capsid and envelope
enceph Major envelope protein
St. Louis  Birds E
Culex  Americas virion assembly, receptor binding & membrane fusion
enceph  Pigs
 Africa NON-STRUCTURAL PROTEINS
West Nile Culex  Birds  Tropical Asia NS1 Membrane soluble hemagglutinin
 Mediterranean NS3 Protease/ polymerase
 Humans  Tropical Africa NS5 Polymerase
Yellow Fever Aedes aegypti
Monkeys  America NS2a Works in tandem with NS3 as a protease
Omsk
hemorrhagic Dermacentor  Rodents  Central Russia REPLICATION
fever
 Rodents
 Russia Eastern
Tick-borne  Birds
Ixodes  Europe
encephalitis  Domesticated
 Scandinavia
Animals
 Sheep
Louping Ill Ixodes  British Isles
 Birds
 Canada
 Small
Powassan Ixodes  US
Mammals
 Russia
Kyansanur
Forest Haemaphysalis  Rodents  Southwest India
Disease
Murray Valley
 Australia
encephalitis/ Culex  Birds
Kunjin Rocio  New Guinea

 Genomic RNA is capped (not polyadenylated) and serves as

mRNA for all proteins
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o Structural proteins are encoded at the 5' end of the genome
o Nonstructural proteins (e.g., RNA-dependent RNA polymerase)
are encoded in the 3' two-thirds
 Complementary (antisense) RNA, made from genomic RNA,
serves as a template for progeny genomic RNA
 Replication occurs in the cytoplasm (20-30 hours)
o Genome-length mRNA  large precursor protein  cleavage
by viral and host proteases  all viral proteins (structural and
nonstructural)
 Entire virus genome is translated as a single polyprotein which is
then cleaved into the mature proteins
 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)
o Proliferation of intracellular membranes is characteristic of flavi-
virus infected cells
 Release occurs when cell lyses
REACTION TO PHYSICAL AND CHEMICAL AGENTS
 Inactivated by:
o Acid pH
o Heat
o Lipid solvents
o Detergents
o Bleach
o Phenol
o 70% alcohol
o Formaldehyde
 Exhibit hemagglutinating ability
CLINICAL FINDINGS
 Incubation for encephalitides: 4-21 days
 Inapparent infection common
 Some may develop mild flu-like illness, others encephalitis
o Sudden onset of severe headache, chills and fever, nausea and
vomiting, generalized pains and malaise
o In 24-48h: marked drowsiness, may become stuporous
o Severe cases: mental confusion, tremors, convulsions, coma
 Mortality rate varies, JBE as high as 80% in older age groups
 Fever 4-10 days
 Sequelae:
o Mental deterioration
o Personality changes
o Paralysis
o Aphasia
o Cerebellar signs
 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 encephalitis (SLE) & Japan
encephalitis (JE) viruses have either no apparent disease or a
nonspecific febrile illness with headache
 Infection resolves, and lasting immunity is produced
 CNS invasion may develop: aseptic meningitis or encephalitis
 In the great majority of flavivirus infections, virus is cleared by the
immune system
o Persistence in neurological tissue noted in tick-borne
encephalitis viruses
o Recurrent encephalitic bouts in children associated with JE virus
recovered from peripheral blood mononuclear cells
PATHOGENESIS AND PATHOLOGY
flaviviridae
 Primary viral multiplication (susceptible vertebrate host) in
o Myeloid and lymphoid cells or
o Vascular endothelium
 In natural infection of birds and mammals, an inapparent infection is
usual
 For several days there is viremia, and arthropod vectors acquire the
virus by sucking blood during this period—the first step in its
dissemination to other hosts
 In experimental mice
o Subcutaneous inoculation  local viral replication  bloodstream
 dissemination
o Different tissues support further viral replication depending on
specific agent: monocyte-macrophages, endothelial cells, lung,
liver, muscles
o Cross blood-brain barrier
o Widespread neuronal degeneration in all arbovirus-induced
encephalitides
 Majority of infections control of virus occurs before neuroinvasion
 Humans show an age-dependent susceptibility to central nervous
system infections, with infants and the elderly being most
susceptible
 Equine encephalitides – diphasic
o 1st phase (minor illness) – multiplication in nonneural tissue
o 2nd phase (major illness) – multiplication in the brain with cells
injured and destroyed  apparent encephalitis
LABORATORY DIAGNOSIS
 Virus occurs in the blood only early in the infection, usually before
the onset of symptoms
 Virus can also be found in CSF and tissue specimens, depending on
the agent
 Able to grow in common cell lines, such as Vero, BHK, Hela, and
MRC-5
o Mosquito cell lines are useful
 Antigen detection
 Polymerase chain reaction assays
 Virus-specific monoclonal antibodies in immunofluorescence assays
 Neutralizing and hemagglutination-inhibiting antibodies are
detectable within a few days after the onset of illness
 HI test – Simplest, Identifies the group rather than the specific
causative virus
 Specific IgM (ELISA): most sensitive serologic assays
 cross-reactivity within the alphavirus or other flavivirus group must
be considered in making the diagnosis
IMMUNITY
 Permanent after a single infection
 Lasting protection is generally restricted to the same flavivirus, and
is associated with neutralizing antibodies
 Both humoral antibody and cellular immune responses are
thought to be important in protection and recovery from infection
EPIDEMIOLOGY
 Most infections asymptomatic, occurring in summer months in the
northern hemisphere when arthropods are more active
 St. Louis Encephalitis – most important cause of epidemic
encephalitis of humans in North America
 West Nile Fever –leading cause of arboviral encephalitis in the US
 JBE – leading cause of viral encephalitis in Asia
ARTHROPOD-BORNE VIRUSES (ARBOVIRUSES)
 Viruses maintained in nature principally through biological
transmission between susceptible vertebrate hosts by
hematophagus arthropods
 Belong to 3 families
o Togaviruses – EEE, WEE, VEE
o Bunyaviruses – Sandfly Fever, Rift Valley Fever, Crimean-Congo
Hemorrhagic Fever
o Flaviviruses – Yellow Fever, Dengue, Japanese Encephalitis
TRANSMISSION CYCLES
MAN-ARTHROPOD-MAN
 Dengue, Urban Yellow Fever
 Reservoir may either be man or arthropod vector
 Transovarian transmission may take place in arthropod
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ANIMAL-ARHTROPOD VECTOR-MAN
 JE, Jungle YF
 Reservoir is an animal
 Virus maintained in nature by transmission cycle between arthropod
vector and animal
 Incidental infection of man
ARBOVIRUS HOST-VECTOR
 Infection of humans by mosquito-borne encephalitis viruses occurs
when a mosquito or another arthropod bites first an infected animal
and later a human
 The equine encephalitides—eastern, western, and Venezuelan—
are transmitted by culicine mosquitoes to horses or humans from a
mosquito-bird-mosquito cycle
 Swine are an important host of Japanese B encephalitis
 Mosquitoes remain infected for life (several weeks to months)
o Only the female feeds on blood and can feed and transmit the
virus more than once
o Cells of the mosquito's midgut (primary viral multiplication) 
viremia and invasion of organs, chiefly salivary glands and nerve
tissue (secondary viral multiplication)
 Tick-borne flavivirus encephalitides, such as Russian spring-
summer encephalitis (RSSE)
o Occurs in humans exposed to the ticks Ixodes persulcatus and
Ixodes ricinus in uncleared forest areas
o Virus is secreted in the milk of infected goats for long periods
o Infection may be transmitted to those who drink unpasteurized
milk
OVERWINTERING OF ARBOVIRUS
 Viruses have been isolated from mosquitoes and ticks, which
serve as reservoirs of infection
 In ticks, the viruses may pass from generation to generation by the
transovarian route, and in such instances the tick acts as a true
reservoir of the virus as well as its vector
 Virus has been found in the blood of wild snakes
 Mosquitoes can be infected by feeding on emerged snakes and
then transmit the virus
ANIMAL RESERVOIR
 In many cases, not known
 Birds – JE, SLE
 Pigs, monkeys – JE, YF
 Rodents – RSSE
PREVENTION
 Surveillance of disease and vector populations
 Control of vector: pesticides, elimination of breeding grounds
 Personal protection: screening of houses, bed nets, insect
repellants
 Vaccination: available for a number of arboviral infections e.g.
Yellow fever, Japanese encephalitis, Russian tick-borne encephalitis
TREAMENT AND CONTROL
 There is no specific treatment
 The most effective method is arthropod control, such as spraying
of insecticides to kill mosquitoes
 The vaccines are not for human use
 A killed-virus vaccine for Japanese B encephalitis is used in some
Asian countries
WEST NILE FEVER VIRUS
 Caused by a member of JBE complex
 Fatal encephalitis more common in older people
 Person-to-person transmission through organ transplantation, blood
transfusion, in utero, breastfeeding
 Produce viremia and an acute mild febrile disease with
lymphadenopathy and rash
o Transitory meningeal involvement may occur during acute stage
 Only 1 antigenic type, immunity presumably permanent
 No human vaccine
JAPANESE ENCEPHALITIS VIRUS
 Japanese encephalitis (JE) serocomplex
o 10 viruses
o 6 human pathogens (JE, West Nile, Kunjin, Usutu, St. Louis
encephalitis, Murray Valley encephalitis viruses)
o Most are amplified bird – mosquito – bird
 5 genotypes in Asia (most isolates in genotype 1)
 Leading cause of viral encephalitis in Asia
o Up to 30% die
o 30-75% of survivors left with neurologic and psychiatric sequelae
 Infections during the 1st and 2nd trimesters of pregnancy have
reportedly lead to fetal death
 People in rural areas (with rice paddies and pools of water) have
highest risk of disease (breeding of vector mosquito)
 Patterns of transmission
o Epidemic – seasonal (eg Southern China)
o Endemic – year-round (eg Bali, Indonesia)
 Culex tritaeniorhynchus – main vector in most of Asia
YELLOW FEVER
 Mainly found in West Africa and S. America
 Yellow fever occurs in 2 major forms:
o Jungle (sylvatic) Yellow Fever – natural reservoir of the disease
 Cycle involving nonhuman primates and forest mosquitoes
 Man may become incidentally infected on venturing into jungle
areas
o Urban Yellow Fever – transmitted between humans by the
Aedes aegypti mosquito
 Classically presents with chills, fever, 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 hemorrhagic manifestations
 50% of patients with frank YF will develop fatal disease
characterized by severe hemorrhagic manifestations, oliguria and
hypotension
 Diagnosis is usually made by serology
 There is no specific antiviral treatment
 Neutralizing antibodies endure for life and provide complete
protection
 An effective live attenuated vaccine is available against yellow fever
and is used for persons living in or traveling to endemic areas
PATHOGENESIS AND PATHOLOGY
 The virus is introduced by a mosquito through the skin where it
multiplies
 It spreads to the local lymph nodes, liver, spleen, kidney, bone
marrow, and myocardium, where it may persist for days
 It is present in the blood early during infection
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 The lesions of yellow fever are due to the localization and o Only effective way to avoid infection in endemic areas is through
propagation of the virus in a particular organ prevention of being bitten (use of insect repellants, or other insect
 Infections may result in necrotic lesions in the liver and kidney barriers)
 A live attenuated vaccine is being tried in Thailand with encouraging
CLINICAL FINDINGS results
 IP: 3–6 days
 At the abrupt onset, the patient has fever, chills, headache, REPLICATION AND TRANSMISSION
dizziness, myalgia, and backache—followed by nausea, vomiting,  Virus in mosquito saliva transmitted to human
and bradycardia  Replication in target organs
o The patient is viremic and a source of infection for mosquitoes  Infection of WBCs and lymphatic tissues
o There may be a brief abatement of fever and symptoms; some  Release and circulation in the blood
patients recover at this point
 The disease progresses to a more severe form, with fever, jaundice,  Aedes aegypti, female – vector
renal failure, and hemorrhagic manifestations o Primarily a daytime feeder
o Vomitus may be black with altered blood o Lives around human habitation
o Mortality rate is high especially among young children and the o Lays eggs preferentially in artificial containers
elderly
o Death occurs on day 7–10 of illness CLINICAL SYNDROMES
 Regardless of severity, there are no sequelae; patients either die or UNDIFFERENTIATED FEVER
recover completely.  May be the most common manifestation of dengue
 Prospective study found that 87% of students infected were either
LABORATORY DIAGNOSIS asymptomatic or only mildly symptomatic
 Virus Detection or Isolation  Other prospective studies including all age- groups also
o May be recovered from the blood the first 4 days after onset, or demonstrate silent transmission
by use of cell lines
o Virus antigen or nucleic acid can be identified in tissue specimens CLASSIC DENGUE FEVER
using immunohistochemistry, ELISA antigen capture, or  Fever
polymerase chain reaction tests  Headache
 Muscle and joint pain
 Serology
 Nausea and vomiting
o Presumptive diagnosis: The detection of IgM antibody by ELISA
 Rash
capture in a single sample
o Confirmation: fourfold or greater rise in titer of neutralizing  Hemorrhagic manifestations
antibody between acute phase and convalescent phase serum
samples Encephalitis/encephalopathy Hemorrhage
o Specific hemagglutination-inhibiting antibodies appear first, ↓ level of consciousness Skin: petechiae, purpura,
followed rapidly by antibodies to other flaviviruses ecchymoses
Seizures Gingival bleeding
TREATMENT, PREVENTION, CONTROL Nuchal rigidity Nasal bleeding
 No antiviral drug therapy Paresis GI bleeding: hematemesis,
 Proper mosquito control on airplanes and vaccination of all melena, hematochezia
persons at least 10 days before arrival in or from an endemic zone Hematuria
↑ menstrual flow
 Avirulent strain: The 17D strain of yellow fever virus is an excellent
attenuated live-virus vaccine  4–7 days (range of 3–14 days) after an infective mosquito bite:
 Virulent strain: Asibi strain of yellow fever virus clinical disease begins
 Vaccine is prepared in eggs, dispensed as a dried powder and is a o There is sudden onset of fever
live virus o Prodromal symptoms: malaise, chills, and headache
 Vaccination is contraindicated for infants under 9 months of age, o Pains soon develop, especially in the back, joints, muscles, and
during pregnancy, and in persons with egg allergies or altered eyeballs
immune systems (eg, human immunodeficiency virus infection, o Viremia is present at the onset of fever and may persist for 3–5
malignancy, organ transplantation) days
o After 5–6 days: The temperature returns to normal or may
 Yellow fever vaccine-associated viscerotropic disease: cases of
subside on about the third day and rise again about 5–8 days
multiple organ system failure in vaccine recipients have been
after onset ("saddleback" form)
reported worldwide
 Myalgia and deep bone pain (breakbone fever) are characteristic
 Vaccination is the most effective preventive measure against yellow
fever  On the 3rd or 4th day: A rash may appear and last for 1–5 days.
 Lymph nodes are frequently enlarged
DENGUE VIRUS  Classic dengue fever is a self-limited disease
 Dengue is the biggest arbovirus problem in the world today with  Convalescence may take weeks, although complications and death
over 2 million cases per year are rare
o Found in SE Asia, Africa and the Caribbean and S America  Especially in young children, dengue may be a mild febrile illness
o Breakbone fever principally affecting children lasting a short time
 5 serotypes (DEN-1, 2, 3, 4, 5)
o Each serotype provides specific lifetime immunity, and short-term DENGUE HEMORRHAGIC FEVER
cross-immunity  Passively acquired (as maternal antibody) or preexisting
o All serotypes can cause severe and fatal disease nonneutralizing heterologous dengue antibody
o Reinfection with a virus of a different serotype after the primary  Initial symptoms simulate normal dengue, but the patient's condition
attack is more apt to result in severe disease (DHF) abruptly worsens
 Human infections arise from a human-mosquito-human cycle
 Classically, dengue presents with a high fever, lymphadenopathy,  Clinical case definition: 4 Necessary Criteria
myalgia, bone and joint pains, headache, and a maculopapular rash o Fever or recurrent history of acute fever
 Severe cases may present with hemorrhagic fever and shock with a o Hemorrhagic manifestations
mortality of 5-10% (Dengue hemorrhagic fever or Dengue shock o Low platelet count (< 100,000/mm3)
syndrome) o Objective evidence of “leaky capillaries”
 Dengue hemorrhagic fever and shock syndrome appear most often  ↑ haematocrit (20% or more over baseline)
in patients previously infected by a different serotype of dengue,  ↓ albumin
thus suggesting an immunopathological mechanism.  Pleural or other effusions
 Diagnosis is made by serology
 No specific antiviral therapy is available
 Prevention of dengue in endemic areas depends on mosquito
eradication

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 Grades DENGUE SHOCK SYNDROME

Grade 1 Fever and nonspecific constitutional symptoms  A more severe form of the disease characterized by shock and
hemoconcentration, may ensue
(+) tourniquet test  Secondary infection with dengue type 2 following a type 1 infection
Grade 2 Grade 1 + spontaneous bleeding is a particular risk factor
Grade 3 Signs of circulatory failure (rapid weak pulse, narrow  Non-neutralizing enhancing antibodies promote infection of
pulse pressure, hypotension, cold/clammy skin) higher numbers of mononuclear cells, followed by the release of
Grade 4 Profound shock (undetectable pulse and BP) cytokines, vasoactive mediators, and procoagulants, leading to the
disseminated intravascular coagulation (DIC) seen in the
 Danger signs hemorrhagic fever syndrome
o Abdominal pain, intense and sustained
o Persistent vomiting  Clinical case definition
o Abrupt change from fever to hypothermia, with sweating and o 4 criteria of DHF
prostration o Evidence of circulatory failure manifested indirectly by all of the
o Restlessness or somnolence following:
 Rapid and weak pulse
 Hypotheses on pathogenesis  Narrow pulse pressure (< or = 20mm Hg) or hypotension for
1. Persons who have experienced a dengue infection develop age
serum antibodies that can neutralize the virus of that same  Cold, clammy skin and altered mental status
homologous serotype o Frank shock is direct evidence of circulatory failure

 Warning signs

2. In a subsequent infection, the pre-existing heterologous

antibodies form complexes with the new infecting virus serotype,
but do not neutralize the new virus

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)

LABORATORY TESTS
 Clinical laboratory tests
o CBC--WBC, platelets, hematocrit
o Albumin
o Liver function tests
o Urine--check for microscopic hematuria
 Dengue-specific tests
o Virus isolation
o Serology
 Virus isolation to determine serotype of the infecting virus; IgM
ELISA test for serologic diagnosis
3. Antibody-dependent enhancement – certain strains of dengue
virus complexed with nonneutralizing antibodies can enter a  Reverse transcriptase-polymerase chain reaction-based methods
greater proportion of cells of the mononuclear lineage. Thus  Inoculation of a mosquito cell line with patient serum, coupled with
increasing virus production nucleic acid assays
 Cross-reactivity of IgG antibodies to heterologous flavivirus antigens
 E/M viral protein-specific capture IgM or IgG ELISA and the
hemagglutination inhibition test
 Neutralizing and hemagglutination-inhibiting antibodies
 Analysis of paired acute and convalescent sera to show a significant
rise in antibody titer is the most reliable evidence of an active
dengue infection

MANAGEMENT
 Well-hydrated, with no hemorrhagic manifestations: home treatment
 Hemorrhagic manifestations or hydration borderline: outpatient
observation center or hospitalization
 Warning signs (even without profound shock) or DSS: hospitalize

 Fluids
 Rest
4. Infected monocytes release vasoactive mediators, resulting in  Antipyretics (avoid aspirin and non-steroidal anti-inflammatory
increased vascular permeability and hemorrhagic manifestations drugs)
that characterize DHF and DSS  Monitor blood pressure, hematocrit, platelet count, level of
consciousness

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VACCINE
 No licensed vaccine at present
 Effective vaccine must be tetravalent
 Field testing of an attenuated tetravalent vaccine currently underway

EPIDEMIOLOGY
 Most subtropical and tropical regions around the world where Aedes
vectors exist are endemic areas
 Leading cause of childhood death in several Asian countries
 Often start during the rainy season when the vector mosquito is
abundant
o A aegypti is the primary vector mosquito for dengue in the
western hemisphere
 After a period of 8–14 days, mosquitoes are infective and probably
remain so for life (1–3 months)

2009 WHO Classification of Dengue Fever

Without Warning With Warning Signs Severe Dengue

Signs
Fever with 2 of the Abdominal pain or Severe plasma
ff: tenderness leakage leading to
shock or fluid
Nausea, vomiting accumulation with
Rash respiratory distress
Aches and pains Persistent vomiting Severe bleeding as
Leukopenia evaluated by clinician
Positive tourniquet Clinical fluid Severe organ
test accumulations involvement
(ascites, pleural
effusion) Liver: AST or ALT >
Mucosal bleeding or = 1000
Lethargy, CNS: impaired
restlessness consciousness
Liver enlargement, Failure of heart or
>2cm other organs
↑ Hct + rapid ↓ platelet
count

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