Rabies Virus

Presented By: Maliha Rashid

malomir360@gmail.com
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Table of Contents
9. Laboratory Diagnosis
1. Rabies Virus
2. Antigenic types and Classification  Direct Microscopy: Histological Identification of
Characteristic Cell Lesions
3. Replication
 Fluorescent Antibody Technique (FAT)
4. Epidemiology  Rapid Rabies Enzyme Immunodiagnosis
(RREID)
5. Pathogenesis of Virus
 Viral isolation
6. Life Cycle of Rabies Virus  Demonstration of Antibodies
 Direct Rapid Immunohistochemical Test (dRIT)
7. Clinical Manifestations
 Indirect Rapid Immunohistochemistry Test (IRIT)
8. Host Defense Mechanisms  Immunochromatographic techniques

10. Rabies Vaccines

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Rabies
• Rabies is a preventable viral illness spread by the
bite of a rabid animal.

• The rabies virus infects the central nervous system

of mammals, resulting in brain illness and death.

• Raccoons, bats, foxes, and skunks account for the

majority of rabies cases reported to the Centers for
Disease Control and Prevention (CDC) each year.

• Rabies is a disease that may infect any mammal.

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Structure of Rabies Virus

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Shape of Rabies Virus

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Symbol Name Function

L Large structural protein RNA replicase of the Mononegavirales type.

G Glycoprotein Spike. Uses muscular nAChR, NCAM, and p75NTR as

receptors.

M Matrix Keeps nucleoprotein condensed. Important for

assembly; has roles in regulation.

P Phosphoprotein L cofactor and various regulatory functions. Has many

isoforms from multiple initiation

N Nucleoprotein Coats the RNA.

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Properties

The rabies virus genome is made out of RNA, which is

1. roughly 12 kb in size
2. antisense.
3. non-segmented
4. single-stranded
5. It has a 50-nucleotide leader sequence, followed by the five genes N, P, M, G,
and L

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Classification
• Rabies viruses are divided into two types:

1. street, also known as wild type

2. fixed, which has been altered by transit in cell culture and animals.

• The use of monoclonal antibodies and genetic sequencing to

distinguish and identify street rabies viruses has aided in the discovery
of viral variations from key host reservoirs across the world.

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 Replication Process- steps
1. Adsorption (receptors and virion interaction)

2.Penetration (virus entry)

3.Uncoating (envelope removal)

4.Transcription (synthesis of mRNAs)

5.Translation (Synthesis of structural proteins)

6.Processing (G-protein glycosylation)

7.Replication (production of genomic RNA from intermediate strand

8.Assembly

9.Budding (complete virions) 9

Replication

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Epidemiology

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Pathogenesis

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Clinical Manifestations

•Following are the stages of rabies observed in humans:

1. Incubation
2. Prodrome
3. acute neurologic period
4. coma
5. death (or, very rarely, recovery)

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Host Defense Mechanisms

• To evade the immune system, RABV employs a two-pronged technique.

• First, by making it exceedingly difficult for lymphocytes to execute their job,

RABV takes advantage of the host's innate protection of the NS against
overwhelming immunological reaction.

• Second, RABV can stimulate neurite development while also preventing cell
death. This permits virions to use the neurological system of the host as a mode of
transit to the brain.

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Laboratory Techniques
1. Direct Microscopy: Histological i. Direct Rapid Immunohistochemical
Identification of Characteristic Cell Test (dRIT)
Lesions ii. Indirect Rapid Immunohistochemistry
2. Fluorescent Antibody Technique (FAT) Test (IRIT)
3. Rapid Rabies Enzyme iii. Immunochromatographic techniques
Immunodiagnosis (RREID) 7. Nucleic Acid Detection Techniques
4. Viral isolation
5. Demonstration of Antibodies
6. Newer Diagnostic Tests for Rabies
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Conventional Diagnostic Tests for Rabies: Advantages and
Limitations
• Laboratory techniques in rabies were Luzzani in 1913 paved the way to
started as early as 1800 BC when for laboratory confirmation of rabies.
the first time Zinke demonstrated
that the infection could be • A definitive diagnosis of rabies can be
transmitted to a normal animal after made only with the appropriate
inoculating with saliva from a laboratory methods. The basic
rabid animal. techniques are described in the WHO
publication Laboratory Techniques
• The landmark discovery of Negri in Rabies and the OIE Manual of
bodies by Adelchi Negri in 1903 and Diagnostic Tests and Vaccines for
demonstration of their diagnostic Terrestrial Animals
significance by his wife Lina Negri-
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 Direct Microscopy:
Histological Identification of Characteristic
Cell Lesions
• Infected neuronal cells reveal aggregates the form of a rosette, within the
of viral particles “Negri bodies” which eosinophilic matrix.
are intracytoplasmic inclusion bodies
specific to rabies encephalitis,
demonstrated by histological tests Limitations
(Seller's Technique) on smears taken
from various areas of the brain. 1. Seller's method on unfixed tissue
smears has a very low sensitivity and
• Negri bodies vary in size from as small is only suitable for fresh specimens
as 3 μm to as large as 30 μm and are
generally circular or oval and deeply 2. time consuming, less sensitive, and
eosinophilic with characteristic more expensive
basophilic granules, often arranged in
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Demonstration of Viral Agents
1. Fluorescent Antibody Technique (FAT)
The most widely used test for postmortem rabies diagnosis is WHO
and OIE.

2. Rapid Rabies Enzyme Immunodiagnosis (RREID)

The rabies N antigen can also be detected by applying
immunohistochemical techniques as well as enzyme immunoassays.
An ELISA-based technique was developed by Perrin et al. in 1986
which is known as rapid rabies enzyme immunodiagnosis (RREID)

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FAT
• involves demonstration of the rabies virus nucleoprotein antigen (N) in
fresh brain smears of a suspected rabies case by using
immunofluorescence technique.
• It can also be used to confirm the presence of rabies antigen in cell
culture or in brain tissue of mice that have been inoculated for diagnosis.
• specificity and sensitivity = 99%
• Reliable results are obtained only when fresh brain tissue is used.
• Obtaining a postmortem brain biopsy/autopsy - challenge due to
religious, cultural values.
• FAT on corneal smears and nuchal skin biopsy

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 Rapid Rabies Enzyme Immunodiagnosis (RREID)

• based on capturing rabies N protein in a brain homogenate by a

polyclonal or monoclonal anti-N antibody coated on the solid

phase.

• the captured antigen is detected by adding peroxidase

conjugated monoclonal or polyclonal antibody raised in a

different species or even better by the addition of biotinylated

N antibody followed by streptavidin peroxidase and colour

development with OPD and hydrogen peroxide.

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Continued…..

• In various studies, the test is found to be as sensitive and specific as

FAT.

• An added advantage is that partial decomposition of the brain will

not affect the test result.

• A limitation of the test is requirement of brain tissue, which

precludes its use in antemortem diagnosis.

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Virus Isolation
• Used as a confirmatory test when FAT gives uncertain results
• Molecular characterization in area that was rabies-free before.
• Two kinds of tests are included:
1. Mouse Inoculation Test
2. Rapid Tissue Culture Infection Test (RTCT)

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Demonstration of Antibodies

• As neutralizing antibodies are considered a key component of the

adaptive immune response against rabies virus, virus neutralization (VN)
assays in cell cultures are prescribed tests for checking vaccination
responses.

• MNT, RFFIT, and FAVN have been described for this purpose

• The RFFIT is considered the gold standard assay and has been used to
estimate the titre of rabies virus neutralizing antibodies for several years.
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Newer Diagnostic Tests for Rabies

Demonstration of Viral Antigen

1. Direct Rapid Immunohistochemical Test
(dRIT)
2. Indirect Rapid Immunohistochemistry Test
(IRIT)
3. Immunochromatographic Techniques
4. Other Antigen Detection Assays

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Rabies Vaccine
 In 1885, the first rabies vaccine was released, followed by a
better one in 1908. Vaccination against the virus has been
given to millions of individuals throughout the world. It is
listed as an essential medicine by the WHO

 disease-prevention vaccine

 Availibility of safe and effective rabies vaccinations on the

market

 used to prevent rabies before and after exposure to the

rabies virus

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 
• Rabies immunizations are safe for people of all
ages. A brief period of redness and soreness at the
injection site occurs in 35 to 45 percent of
persons, and 5 to 15% of people report fever,
headaches, or nausea.

• There are no contraindications to using it after

being exposed to rabies because the virus is
almost always lethal if left untreated.

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 
o The vaccination is given in three doses over
the course of a month, on days zero, seven,
and either twenty-one or twenty-eight
o One research indicated that after 10 years,
97 percent of immunocompetent patients
had protective levels of neutralising

o Four doses over two weeks are suggested antibodies after receiving a booster dosage.

for people who have been exposed to the

virus, as well as an injection of rabies
immunoglobulin with the first dosage. Post-
exposure vaccination is the term for this.

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References
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THANKS

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