MICRO-BIOLOGY OF

RABIES VIRUS
CLASSIFICATION.
MORPHOLOGY.
RESISTANCE.
REPLICATION.
ANTIGENICITY.
LAB.DIAGNOSIS.
Classification
Order :Mononegavirales

Family :Rhabdoviridae

Genus :Lyssavirus
MORPHOLOGY
Bullet shaped
180nm long &75 nm wide
It consists of a helically arranged
ribonucleoprotein core and a surrounding
envelope.


ENVELOPE
Formed when newly formed virus buds out
through the plasma membrane.
The envelope is lipoprotein in nature.
Carries knob like glycoprotein spikes.
Beneath the envelope is the matrix protein
layer or membrane.
CORE
50nm in diameter.

Tightly coiled RNP with helical symmetry.

RNA with RNA polymerase,
phosphoprotein & nucleoprotein.

Genome
The rabies virus genome consists of
Leader sequence,
N gene-nucleoprotein,
P gene-phospho protein,
M gene-matrix protein,
G gene-glycoprotein,
L gene-RNA polymerase,
Length of genome 11kb.




RESISTANCE
Sensitive to ethanol, trypsin, detergents
and lipid solvent.
Inactivated by phenol, formalin, uv rays
and sunlight.
Thermal inactivation occurs in 1 hour at
50C and 5min at 60C
Preserved at -70Cor by lyophilisation.

REPLICATION
BINDING:
Virus attaches to susceptible cell
membrane mediated by glycoprotein.
PENETRATION:
Enters the cell by endocytosis.
pH-dependent fusion with the membrane
of the endocytic vesicle occurs. The
nucleocapsid enters the cytoplasm.
Transcription:
Gives rise to 5 individual m RNA one
for each viral protein.
Replication:
polymerase transcribes the negative-
sense genomic RNA into a positive sense
strand.
this is in turn replicated into progeny
virions negative strand.


EXIT FROM THE CELL:
Negative strand RNA associates with
N, L, & P protein forming nucleocapsid
core.
This in turn associates with G&M
protein and buds out through the
membrane.

Antigenic properties
Glycoprotein G spikes are important in
virulence and immunity.
Induce binding of virus to Ach receptors of
neural tissue and induce HI & cytotoxic T
cell immunity.
Nucleocapsid protein stimulate CF Ab.
Other Ag are two membrane protein,
glycolipid, RNA-polymerase.
LAB DIAGNOSIS
IMMUNOFLUORESCENCE.
DEMONSTRATION OF NEGRI BODIES.
ISOLATION OF VIRUS.
REVERSE TRANSCRIPTION PCR.
SEROLOGY.

Immunofluorescence test
The ideal tissue to test for rabies antigen
is brain.
The flouresecently-labelled anti-rabies
antibody directed against nucleoprotein
of virus is incubated with suspected brain
tissue.
Unbound Ab are washed away& areas
where Ag is present can be visualized by
fluorescent apple green areas.

DEMONSTRATION OF NEGRI
BODIES
Round or oval inclusions within the
cytoplasm of nerve cells of animals
infected with rabies.
Negri bodies may vary in size from3 to 27
m.
On Staining with Mann's, giemsa, or
Sellers stains. Negri bodies appear as
purplish pink in color and have small dark-
blue interior basophilic granules.

Negri body
Isolation of rabies virus
MICE ISOLATION:
Infected tissue inoculated intra
cerebrally into suckling mice.
infection results in encephalitis
and death.
CNS is examined for Negri
bodies& rabies Ag.


Cell culture lines:
Mouse neuroblastoma cells (MNA) and
baby hamster kidney (BHK) cells provide
an excellent environment for amplification
of rabies virus without the use of animals.
isolated virus identified fluorescent Ab
test with specific serum.
Virus isolation in cell cultures increases
the virus concentration because the virus
replicates in cell cultures.
RT-PCR
Rabies virus RNA can be enzymatically
amplified as DNA copies.
Rabies RNA can be copied into a DNA
molecule using reverse transcriptase (RT).
The DNA copy of rabies can then be
amplified using polymerase chain reaction
(PCR). This technique can confirm dFA
results and can detect rabies virus in
saliva and skin biopsy samples

The figure below shows PCR test results
for rabies virus. The arrows indicate
positions of positive bands.