Everything you always wanted to know about

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Everything you always wanted to know
about but were afraid to ask!
by Srilatha Bodla
SARS
Evaluation of modified vaccinia virus
Ankara based recombinant SARS vaccine in ferrets
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Introduction
Severe Acute Respiratory Syndrome (SARS) is an
acute respiratory illness caused by Corona virus
infection .
Fever followed by a respiratory compromise are
the signs and symptoms, which also include
chills, muscular aches, headache and loss of
appetite.
The first world-wide SARS epidemic occurred
between late 2002 and the first half of 2003
caused severe stress in global society.
Much has been learned about SARS, including its
causation by a new coronavirus (SARS-CoV)
however, our knowledge about the ecology of SARS
coronavirus infection remains limited.
At present, the most efficacious treatment
regimen for SARS is still subject to debate.
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Introduction (cont.)
The coronaviruses ( family Coronaviridae) are
members of a family of large, enveloped,
positive-sense single-stranded RNA viruses .
The genomes of coronaviruses range in length from
27 to 32 kb and about 100 and 140 nanometers in
diameter.
Human coronaviruses (HCoVs) were previously only
associated with mild diseases.
Several coronaviruses can cause fatal systemic
diseases in animals, including feline infectious
peritonitis virus (FIPV), hemagglutinating
encephalomyelitis virus (HEV) of swine, etc.
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Transmission

• Coronaviruses may be transmitted from
  person-to-person by droplets, hand contamination,
  and small particle aerosols .

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Organization

• The SARS-CoV genome contains five major open
  reading frames (ORFs) that encode the replicase
  polyprotein the spike (S), envelope (E), and
  membrane (M) glycoproteins and the nucleocapsid
  protein (N).

The main function of the S protein is to bind to
species-specific host cell receptors and to
trigger a fusion event between the viral envelope
and a cellular membrane.
The spike protein has been shown to be a
virulence factor in many different coronaviruses.

The S protein is the principal viral antigen that
elicits neutralizing antibody on behalf of the
host.
The M protein is the major component of the
virion envelope.
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Detection

• SARS Co-V has been detected from extracts of lung
  and kidney ,sputum or upper respiratory tract
  swab, by virus isolation, electron microscopy and
  PCR.

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Antiviral Drugs Vaccine
Efforts are underway to assess potential
anti-SARS-CoV agents in vitro.

• The availability of vaccines against animal
  coronaviruses, (avian infectious bronchitis
  virus, feline infectious peritonitis virus) are
  encouraging.

The S protein is generally thought to be a good
target for vaccines because it will elicit
neutralizing antibody.
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Present study
Highly attenuated vaccinia virus ,modified
vaccinia Ankara (MVA) used as a vector.
Construction of rMVA expressing the S(rMVA-S) and
N(rMVA-N)
2 major antigenic proteins responsible for
inducing protective immune responses against
coronavirus.
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Animal model
Ferrets (male castrated).
They are susceptible to SARS-CoV infection
To evaluate the efficacy and safety of rMVA based
SARS vaccines.
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Results and Discussion
Expression of SARS-CoV S and N proteins by rMVA
N and S recombinant viruses
Confirmed by Western Blot.
S-specific mouse monoclonal antibody (detection
of SARS-CoV S protein)
SARS patient serum (detection of SARS-CoV N
protein)
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Immunization of ferrets with rMVA-S N
12 ferrets divided to 4 groups of 3 animals.
Immunized with PBS ,Parental MVA, rMVA-S, or
rMVA-N.
ELISA and Micro plaque reduction neutralizing
assay
Antibody was detected in ferrets immunized with
rMVA-S.
Ferrets immunized with rMVA-S showed peek
antibody titre between 7 and 9 days.
Other ferrets showed comparable levels of
antibodies between 19 and 21 days.
Rapid memory immune response occurred in ferrets
immunized with rMVA-S.
The presence of anribody did not lead to the
prevention of SARS-CoV dissemination.
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Challenge of Immunized Ferrets
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SARS-CoV Challenge of Immunized Ferrets
Challenged the vaccinated and control animals
with SARS-CoV
No clinical signs were found up to 29 days .
Detected viral RNA in feces, pharyngeal swabs and
blood samples by RTPCR.
Viral RNA detected in pharyngeal and feces within
7 days, but not in the blood.
The viral RNA persisted in blood longer than in
pharyngeal excretion and feces .SARS-CoV
replicates in ferrets.
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No viral RNA was found in any tissue collected
from the post-mortem examination.
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Blood Chemistry Histopathology
Performed to investigate any pathological effects
as consequence of rMVA vaccination and SARS-CoV
challenge.
Vet Test dry chemistry analyzer was used.
Blood samples taken were examined for levels of
alkaline phosphatase, alanine amino transferase,
albumin, creatinine, total bilirubin, total
keratin, and urea.
Ferrets vaccinated with rMVA-N or rMVA-S showed
higher levels of ALT.
Elevated level of ALT was evidenced on 5th dpi
and lasted until day 21.
All other parameters were almost normal.
All the animals infected with SARS-CoV developed
periportal and pan-lobular hepatitis.
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ALT level following rMVA immunization and
SARS-CoV challenge.
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Ferrets immunized with rMVA-S developed more
severe lesions than other animals.
Ferret 9,(immunized with rMVA-S) which
developed rapid antibody response had most severe
hepatitis.
Mild hepatitis was observed in control animals.
Ferrets immunized with rMVA-N also showed
elevated level of ALT, only ferret 4 developed
severe hepatitis.
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Pictures of Livers From Ferrets
Mild Hepatitis
MVA
PBS
Severe Hepatitis with Focal Necrosis
rMVA-SARS-N
rMVA-SARS-S
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Perivascular mononuclear infiltrates were present
in all livers.
The tissue samples for pathological sectioning
for collected postmortem. (ie 27-29days after
challenge) ALT level had already declines to the
normal range (27-29 days after the challenge)
The liver inflammation may not truly reflect the
severity of the hepatitis associated with rMVA-S
or N.
Other organs were mildly affected.
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Antibody-Dependant Enhancement of Virus
Infectivity
ADE is invitro serological phenomemon, in which
viral infection of susceptible cells is modified
by the addition of virus reactive abs.
Neutralizing antibody induced by the spike
protein of feline infectious peritonitis virus
failed to protect cats from the virus challenge
Antibodies acquired either through a passive
transfer of immune serum against the spike
protein or
Immunization with a recombinant vaccinia virus
expressing the spike protein often lead to
accelerated infection.
SARSCoV infect hepatocytes and cause hepatitis
in human.
Immunization with rMVA-S induced hepatitis in
ferrets after SARS-CoV challenge.is in line with
reports of ADE of FIPV infection.
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FUTURE STUDIES Detailed pathological examination
must be perform when the ALT level is high. To
improve the immune responses by the use of
different immunization regimen. More ferrets
which would allow post-mortem examination at
various time points after vaccination and
challenge. More detailed analysis of the immune
responses and immuno histological studies should
be done. Other vaccination strategies should be
examined.
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CONCLUSION
rMVA-S can induce rapid and vigorous neutralizing
antibody response in ferrets.
Neutralizing antibody did not prevent virus
infection and spreading.
Vaccination with SARS-CoV S/N protein may lead to
enhanced pathology during infection and may cause
damage of the liver.
SARS-CoV does not cause clinical disease in
ferrets.
Ferrets are useful model in evaluating the safety
of the vaccination strategy.
Extra caution must be taken in future human
trials of SARS vaccination.
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SARS Panic Humor
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Unanswered Questions
What is the origin of SARS-CoV?
What is the animal reservoir, if any?
If SARS-CoV was present in an unknown animal
species, did it jump to humans because of a
unique combination of random mutations?
Can SARS-CoV now infect both its original host
and humans?
Why are children less likely to develop SARS ? Do
they have a lower clinical manifestation index,
or do they possess a (relative) (cross-?)
immunity against SARS-CoV?
Are there environmental sources of SARS-CoV
infection, such as foodstuff, water, sewage?
How important is genetic diversity among SARS-CoV
strains?
Which factors determine the period of time
between infection and the onset of
infectiousness?
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THE END Thank you!