Togaviruses and

Flaviviruses

Features
• Enveloped ss(+) RNA
• Replicate in the cytoplasm
• Toga - bud at the plasma membrane,
• Flavi – bud at the internal membrane
• Inactivated by drying, soap, detergents
• Spread by specific arthropods (except
Rubella for Toga and Hepatitis C for Flavi)
• Can be asymptomatic, or nonspecific
symptoms (fever, chills), but could also
lead to encephalitis, arthritis and
hemorrhagic fever
• Live attenuated vaccine for yellow fever
and Japanese encephalitis
• Mosquitoes acquire virus
by blood meal from a
viremic host.
• The virus then infects the
epithelial cells of the
mosquito’s gut
• Goes to circulation and
infects the salivary glands.

• It then persists in the salivary

glands which release the virus in
to the saliva.
• Only those species that can
get infection of the salivary
glands, will transmit the virus to
humans. Only female arthropods can
support viral replication.

• When the mosquito bites its host

and regurgitates virus-containing
saliva into the host’s
bloodstream.
 Name of Disease Vector Host Disease
• Most are arboviruses have a
broad host range including
vertebrates and invertebrates.

• Most viral infections do not

progress beyond the initial
viremia in the blood.
Arboviruses

• If sufficient virus is produced

during initial replication in the
WBC of the blood (usually in
monocytes) then other organs
such as brain, liver, skin and
vasculature can be effected.

• Although these viruses exit by

exocytosis, they usually lyse the
cell (even being enveloped)
because they change the
permeability of the cell.

• Both cell mediated and

humoral immunity are
important for to control
infections.
Togavirus and Flavivirus Diseases
Encephalitis (Arbovirus encephalitis is similar for Toga and Flavi)

• Acute onset of fever, headache, and vomiting; progress to signs of meningeal

involvement (stiff neck and back)

• After acute symptoms, evidence of neuronal damage (drowsiness, coma,

paralysis, convulsions, ataxia, organic psychoses).

• Cerebrospinal fluid (CSF) pleocytosis is usual, with up to 1,000

leukocytes/mm3. Mononuclear cells usually predominate, although early in
fulminant encephalitis, polymorphonuclear leukocytes predominate, glucose
concentration in the CSF is normal, and the protein is increased.

• The peripheral blood shows a moderate polymorphonuclear leukocytosis.

• With recovery from acute viral encephalitis, evidence of neuronal injury and
death becomes apparent as residual neurologic defects, impairment of
intelligence, and psychiatric disturbances.

• The severity of these sequelae vary according to the causative virus.

Togaviruses arbovirus disease examples:

VEE, WEE, EEE

Often asymptomatic but can also cause flu-

like symptoms (fever, chills, rash, aches) that
can progress to encephalitis, with a
possibility of paralysis, mental disability,
tremors and seizures.
Flavivirus arbovirus disease example:
West Nile encephalitis causes mild symptoms in 20% of the
cases that last a few days and will cause encephalitis in 1%
of WNE-infected people.
Typical transmission of arboviruses that cause meningitis/ encephalitis

Many mammals
bitted by infected
WNV mosquitos will test
transmitted positive for WNV
when without getting ill.
mosquito
bites
infected
bird.

Infected mosquitos
“Dead-end” hosts
transmit the virus to birds.
such as mammals do
Infected birds of some
not produce enough
species will get ill and die,
virus for it to go back
other bird species become
to mosquito.
infected but do not show
signs of disease.
Before WNE After WNE
 Hemorrhagic fevers

• Symptoms: Lab findings:

– Flu-like Leukopenia (often neutropenia)
• Chills Thrombocytopenia
• High fever  hematocrit
• Malaise Proteinuria
• Myalgias +/-  AST/ALT
• +/- headache
• Nausia and vomiting

– Followed by:
• Hemorrhagic diathesis
• Hypotension
• Shock
 Flavivirus: Dengue fever
• Endemic to North and Central Africa, S.E. Asia and some areas in
Caribbean

• There are 100 mil. cases per year of Dengue. 300,000 cases per yr. are
of a more serious infection-Dengue hemorrhagic fever (DHF)

• Dengue fever is also known as break-bone fever, the symptoms include

• Headache
• Fever
• Rash
• Platelet loss (clotting dysregulated)
• Bleeding from mucous membranes
(esp. gums)
• Severe bone pain
• Severe disease is marked by:
• dysfunction of endothelium
• disordered blood clotting.
If a person with Dengue fever is re-
challenged with another strain of Dengue
fever, he will develop Dengue Shock
Syndrome (DSS)/DHF which results in:

• weakening and rupture of vasculature

• internal bleeding
Antibody dependent enhancement in Dengue fever

Immunity to Flaviruses is problematic as non-

neutralizing Ab can enhance the uptake of the virus
by cells that express Fc receptors.
 Dengue hemorrhagic fever/
Dengue shock syndrome
Classical Dengue fever DHF/DSS
Dengue world wide
2013
Diagnosis:

Antigen genome/protein testing:

• Patient blood can be directly examined by RT-PCR and monoclonal
antibodies that are directed against the individual strains of viruses.

Serology:
• Serological methods including ELISA can be used to diagnose a recent
infection, but serological cross-reactivity among viruses limits the distinction
of actual viral species.

Other:
• In the cases of encephalitis, the measurement of IgM in a patient’s
CSF can be used for diagnosis.

In many cases specimens collected from people infected with Flavi or

Toga are send to CDC for further analysis.

Both Toga and Flavi viruses are difficult to grow and analyze in tissue
culture.
• In a statement, Sanofi said it had
recently examined six years of
patient data.
• Scientists concluded that while the
vaccine protects people against
further infection if they've already
been infected with dengue, that's not
the case for people who haven't
previously been sickened by the
disease.
• "For those not previously infected
by dengue virus...the analysis
found that in the longer term,
more cases of severe disease
could occur
following vaccination," Sanofi
said.
• "These findings highlight the
complex nature of dengue infection."

19
 Yellow fever
• Incubation is 3 to 6 days, after which the patient may present with high
fevers associated with bradycardia, leukopenia, and transaminase
elevations

• The major organ target is the liver, and massive necrosis of

hepatocytes leads to a decrease in the rate of formation of prothrombin
as well as to jaundice.

• This is followed by a period of remission,

but within 24-48 hours up to 30% of
patients progress to the intoxication
stage, with high fevers, gastrointestinal
hemorrhages (black vomit), jaundice,
hypotension, dehydration, proteinuria,
kidney failure, CNS dysfunction, and
shock.

• Infection of the skin, bone marrow and blood

vessels is noted
• Mortality rate can be as high as 50%.
Yellow fever
Yellow fever world wide
 In 1889, during Spanish-American
war, more American soldiers were
killed (in Cuba) by yellow fever than
by the war
 Walter Reed and
The Yellow Fever Commission
1900
Spanish – American War
Experiment
  A large room divided into two
 A single room with as much sections by a floor-to-ceiling wire
foul air as possible. mesh screen.

 A small stove kept the  Configured to keep infected

temperature and humidity at mosquitoes inside one section
tropical levels only.

 Screening secured doorways  Cots with bedding were steam

against intrusion by sterilized.
mosquitoes.
 Windows exposed the entire
room to the clean, steady ocean
breezes and to sunlight.
Set up
• Volunteers wore the clothing and
slept on the mattresses used by
yellow fever patients
• Volunteers smeared their bedding
with blood drawn from cases in the
early stages of yellow fever
Set up
• Non-immune men remained in
a mosquito room for about
twenty minutes -- enough time
to suffer several mosquito
bites -- then exited to a
quarantine room outside.
RESULTS
 None of the people became sick
with yellow fever in room #1

Many people became sick with

yellow fever symptoms in room
#2

People in room #1 were not

somehow immune, because some
of them got exposed to mosquitoes
later and became sick
 Zika virus
• Flaviviridae

• Transmission:

• Aedes mosquito bite

• Sexually transmitted
• Mother to child in the womb

• Symptoms:
• Replicates in skin layers, fibroblasts and causes
viremia
• 70% asymptomatic
• Fever
• Red eyes
• Joint pain
• Headache
• Maculopapular rash
• No hemorrhages!
• Microcephaly in neonates!
Stopping Zika:
The GMO mosquito that kills his own offspring
Sandee LaMotte, CNN 3-11, 2016

• OX513A may soon be a household

name in the fight against Zika in Florida.
• OX513A is a mutated version of a
"sterile" mosquito.
• This male is created by injecting very small
amounts of synthetic DNA into thousands of
mosquito eggs until finally one of the eggs accepts
the DNA into its genome, creating two genes.
• One is a lethal gene, synthesized from E. coli and
the herpes simplex virus. It's lethal because it
creates a protein called tTAV which interferes with
a cell's activity, killing the infected mosquito before
it can reach adulthood.
• The second synthetic gene, based on DNA from
coral, implants a fluorescent red marker in
OX513A so researchers can more easily track
him.
 Chikungunya virus

• Togaviridae

• Transmission:

• Aedes mosquito bite

• Symptoms:

• Replicates in epithelial and endothelial cells, primary fibroblasts,

muscle tissue and monocyte-derived macrophages
• 15-20% asymptomatic
• High fever
• Conjunctivitis
• Joint pain
• Headache
• Maculopapular rash
• No hemorrhages!
• No neutropenia, thrombocytopenia
Exception to arthropod-transmitted Time
course
diseases caused by Toga: of
RUBELLA (German measles): rubella
disease

• Strictly human disease

• Not transmitted by vectors, but by

aerosol/respiratory route

• From respiratory route virus travels to

lymphatic and blood

• After viremia, virus multiplies in many

organs, particularly lymph nodes
(lymphadenopathy)

• can also cross the placenta

• The virus is not cytolytic

 RUBELLA (German measles):

• Characteristic pink, continuous

maculopapular rash appears 2 weeks after
infection and lasts 3 days- patient is
infectious before and after rash onset.

• The rash, fever and swollen glands are the

main characteristics in children.

• In adults, in addition to the rash, it may

cause more severe disease including bone
pain and arthralgia (joint pain).

• Rash is may be mediated by immune

complexes rather than direct viral damage.
• Congenital defects -
the most serious
outcome of rubella

• Most risk prior to week

20 post gestation

• Child born with

cataracts, mental
retardation, deafness,
and heart defects.

Effect of rubella virus vaccination on the

incidence of rubella and congenital
MMR – live vaccine against rubella rubella syndrome (CRS).
 Exception to arthropod-transmitted diseases caused by Flavi
HEPATITS C:
• Not transmitted by vectors
• Strictly human disease
• Not cytolytic
• Acquired by transfusion, needle stick injury, rarely sexual intercourse,
rarely breast-feeding

Outcomes of hepatitis C virus infection.

If you're in a long-term,
monogamous relationship
with a partner who has
hepatitis C, your risk of
sexual transmission is low-
0 percent to 0.6 percent a
year.

Steatosis (fatty liver) is common

 Treatment for Hepatitis C
Traditional drugs:

• Interferon alpha –binds to INF alpha receptor and induces apoptosis in infected
cells
• Ribavirin - a guanosine analog used to stop viral RNA synthesis and viral mRNA
capping, thus, it is a nucleoside inhibitor.

Newer drugs:

Harvoni (sofobuvir + ledipasvir) - $1,125 per pill (12 week treatment)

• Sofosbuvir (Sovaldi) - nucleotide analog, inhibitor of RdRpol

• Ledipasvir - NS5a phosphoprotein inhibitor

Newest drug (6-2016)

Epclusa, first pill to treat all major forms of hepatitis C

• $74,760 for a 12-week course of treatment, or roughly $890 per pill.

• Combines sofosbuvir and velaptasvir
Pegylated interferon (Peg-IFN)
Interferon is similar to a protein your body makes to fight off infection. Pegylated
interferon is a long-acting form of interferon that’s administered as an injection.
Ribavirin (RBV)
RdR pol inhibitor (nucleoside analogue)

May 2011
The FDA approved two new medications that are part of a drug group called
protease inhibitors:
VICTRELIS (boceprevir) and INCIVEK (telaprevir)
The protease inhibitors were approved to be used in people with HCV genotype 1
in combination with Peg-IFN/RBV therapy. They could not be used alone; using
Peg-IFN/RBV plus one of the protease inhibitors was called triple therapy. These
medications were no longer used after interferon-free regimens became widely
available in 2014.

November 2013
The FDA approved the first once-daily protease inhibitor:
OLYSIO (simeprevir)
Broad use of simeprevir occurred when it was used in combination with a newer
drug, sofosbuvir (SOVALDI). This all-oral regimen was initially prescribed off-label
(meaning that it wasn’t part of the FDA-approved labeling) for genotype 1 patients
and was offered with and without ribavirin. FDA approval for the simeprevir and
sofosbuvir combination was ultimately received in November 2014.
December 2013
Polymerase inhibitors.
SOVALDI (sofosbuvir)
Sofosbuvir, a once-daily pill, was approved to treat HCV genotypes 1, 2, 3 and 4. This
was the first drug that allowed genotype 2 and 3 patients to be treated with pills only,
offering an interferon-free regimen with ribavirin. The first line therapy for genotype 1
and 4 patients became a 12-week combination regimen with peginterferon and
ribavirin.
October 2014
HARVONI (ledipasvir/sofosbuvir)
This once-daily pill combined sofosbuvir (Sovaldi) and a new drug called ledipasvir.
Ledipasvir inhibits an important viral phosphoprotein, NS5A, which is involved in viral
replication, assembly, and secretion.
Harvoni was approved to treat adults with HCV genotype 1, the most common form of
HCV in the U.S. This was the first drug that allowed people with genotype 1 to be
treated with only one pill, eliminating the need for weekly injections of interferon or a
second antiviral, ribavirin, both of which are challenging to take and tolerate.
June 2016
The FDA approved a new combination medicine, the first therapy approved to treat
all HCV genotypes (1, 2, 3, 4, 5, or 6). It is also the first single tablet regimen
approved for the treatment of patients with HCV genotype 2 and 3, without the need
for ribavirin.
EPCLUSA (sofosbuvir /velpatasvir (NS5A inhibitor)
Epclusa is a once-daily, fixed-dose combination tablet approved for the treatment of
adults with chronic HCV genotype 1-6. Epclusa for 12 weeks was approved in
patients without cirrhosis or with compensated cirrhosis, and in combination with
ribavirin for patients with decompensated (advanced) cirrhosis.
 Diagnosis
HCV RNA
Ag test Ab test
(Qualitative
RT-
HCV (EIA) Anti-HCV (RIBA) PCR or TMA) Interpretation

Non-reactive Non-reactive Undetectable No present or past infection

False positive EIA; no present or past
Reactive Non-reactive Undetectable infection
In the absence of risk factors, probable
Reactive Undeterminate Undetectable false positive EIA
Probable past exposure with clearance
of infection. Qualitative RNA testing
should be repeated to exclude
Reactive Positive Undetectable fluctuating low levels of viremia

Reactive Positive Detectable Ongoing infection

Acute HCV infection or chronic HCV
infection in an immunocompromised
person unable to make adequate
Non-reactive Non-reactive Detectable antibodies