Morphology pathogenicity,laboratory diagnosis

2. Rhabdoviruses are –
 Rod or bullet shaped, enveloped viruses.
 Have nucleocapsid with helical symmetry.
 Genome is single stranded, unsegmented, negative
sense RNA.
 ( Rhabdos = rod).
Included under family Rhabdoviridae & contain
viruses infecting mammals, reptiles,fish, insects &
plants.
2

3.  Rhabdoviruses infecting mammals grouped in
family Rhabdoviridae belong to TWO Genera.
1. Vesiculovirus - containing vesicular stomatitis virus &
related viruses like Chandipura virus (Arbovirus).
2. Lyssavirus ( lyssa = madness, rage, a synonym for
rabies) containing Rabies virus & related viruses.
3

4. 4

5. Bullet shaped.
180 x 75 nm in size with
one end rounded or conical
& the other end planar or
concave.
Outer lipoprotein envelop
carrying knob like spikes
composed of glycoprotein
G
5

6. Spikes do not cover the
planar end of virion.
Beneath envelop is the
membrane or matrix (M)
protein layer which may be
invaginated at the planar
end.
Membrane may project
outwards from the planar
end.
6

7. Core of virion contains
helically arranged
nucleoprotein.
Genome – unsegmented,
linear, negative sense,
single stranded RNA.
Present in the nucleocapsid
is RNA dependent RNA
polymerase ( transcriptase)
& some structural proteins.
7

8. 8

9.  Sensitive to ethanol, iodine preparations, quaternary
ammonium compounds, soap, detergents & lipid
solvents such as ether, chloroform & acetone.
 Is inactivated by phenol, formalin, beta
propiolactone, ultraviolet irradiation & sunlight.
 Thermal inactivation in 1 hr at 500C & 5 min at 600C.
9

10.  Dies at room temp, can survive for weeks when
stabilised by 50% glycerol.
 Survive at 40C. Can be preserved at - 700C by
lyophilisation.
 For storage in dry ice, the virus has to be sealed in
vials as it is inactivated on exposure to CO2
10

11. 11
 GLYCOPROTEIN – Surface spikes – composed of glycoprotein –
imp. In pathogenesis, virulence & immunity. Imp. Properties as
follows –
 Mediates binding of virus to acetylcholine receptors in neural tissue
 Induces haemagglutination inhibiting (HI) antibodies. Rabies virus
has haemagglutinating activity, optimally seen at 0 - 40C & pH 6.2. It
is inactivated by heat 560C for 30 – 60 min, ether, trypsin etc.
 Induces neutralising antibodies ( protective).
 Stimulates cytotoxic T cell immunity.
 Purified glycoprotein may act as safe, effective subunit vaccine.

12. 12
 NUCLEOPROTEIN – Is a nucleocapsid protein Imp. Having
following properties –
 Induces complement fixing antibodies.
 Antibodies are not protective.
 Antigen is group specific & cross reactions do occur with rabies –
related viruses.
 Antiserum prepared against the nucleocapsid antigen is used in
diagnostic immunofluorescence tests.

13. 13
 OTHER ANTIGENS – Include -
 Two membrane proteins.
 Glycolipid.
 RNA dependent RNA polymerase.

14. 14
 ANIMALS –
• All mammals are susceptible to Rabies
• Cattle, cats & foxes – Highly susceptible.
• Skunks, opossums & fowl – relatively resistent.
• Humans & dogs occupy an intermediate position.
• Pups more susceptible than adults.
• Experimental infection can be produced in any lab.
Animal but mice is choice.

15. Definition: the virus
recovered from naturally
occurring cases of rabies
is called “street virus”
Sources: it is naturally
occurring virus. It is
found in saliva of
infected animal.
(continue)
FIXED VIRUS
Definition: the virus which
has a short, fixed and
reproducible incubation
period is called “fixed
virus
Sources: it is prepared
by repeated culture in
brain of rabbit such that
its I.P. is reduced &
fixed
15
TYPES OF RABIES VIRUS -

16. 1. It produces Negri bodies
2. Can be demo. In the brain
of dying animal.
3. Incubation period is long
i.e. 20 to 60 days
4. It is pathogenic for all
mammals
5. Cannot be used for
preparation of vaccine
FIXED VIRUS
1. It does not form Negri
bodies
2. Incubation period is
constant between 4-6
days
3. It can be pathogenic
for humans under
certain conditions
4. Is used for preparation
of antirabies vaccine
16
TYPES OF RABIES VIRUS -

17. • Rabies virus can be grown in
chick embryo.
• Mode of inoculation – Yolk
sac.
• Serial propagation in chick
embryo – development of
attenuated vaccine strains like
Flury & Kelev.
• Strains adapted to duck egg –
give high yields – used for
preparation of inactivated
vaccine.
17
• Rabies virus can grow in
several primary & continuous
cell cultures – chick embryo
fibroblast, porcine or hamster
kidney
• Fixed virus strains adapted for
growth in human diploid cell,
chick embryo & Vero cell
cultures – used for the
production of vaccine.

18. • Primarily a Zoonotic disease of
warm blooded animal
• such as :-
18

19. 19
• Mad Dog biting Humans lead to Rabies.
• Latin Rabhas means Frenzy.
• Hydrophobia Fear of Water, Saliva of Rabid
dogs
• Pasture’s success – Vaccination
Fixed virus from Rabbit injected into Joseph
Meister
Injected 13 injection of the cord vaccine.

20. 20
1) URBAN
RABIES:
From Dogs and
cats.

21. 21
2) WILD LIFE
RABIES:
From jackals and
foxes.

22. 22
3) BAT RABIES:
Vampire bats
which live on the
blood of animals
and men. These
are one of the main
causes of the
death of bovine,
around 0.5 to 1
million per year.

23. 23
Saliva of Rabid animal

24. HOST FACTORS
• All warm blooded
animals including man.
• Rabies in man is a
dead-end infection.
MODE OF TRANSMISSION
24
1. ANIMAL BITES
2. LICKS
3. AEROSOL
4. PERSON TO PERSON

25. • Normally it is 3 - 8 wks .
• May be short that is 4 days.
• Or may be prolonged for years.
25

26. 26
• Bite by Rabid dog or other animals
• Virus are carried in saliva virus deposited on
the wound site.
• If untreated 50% will Develop rabies.
• Rabies can be produced by licks and corneal
transplantation.
• Virus multiply in the muscle ,connective
tissue, nerves after 48 – 72 hours.
• Penetrated nerve endings.

27. 27
Live virus Epidermis, Mucus membrane
Peripheral nerve
Centripetally
CNS ( gray matter )
Centrifugally
Other tissue (salivary glands,…)

28. 28

29. 29

30. 30
• From Brain virus
spread to
Salivary glands,
Conjunctival cell
released into tears
Kidney
Lactating glands
and Milk after
pregnancy

31. 31

32. 32
• Virus travels through
axoplasam toward the
spinal cord, at the rate of 3
mm/hour,
• Towards the brain
• Spread from brain
centrifugally to various
parts of the body.
• Multiplies in the salivary
glands and shed in the
saliva.
• Cornea, facial tissues skin.
• Incubation 1 – 3 months.
• May be average from 7
days to 3 years.
• Stages of the disease.
Prodrome
Acute encephalitis.
Coma / Death.

33. 33
• Furious Rabies
• Dumb ( Rage tranquille )
• (Landry/Guillain-Barre
Syndrome

34. CLINICAL FINDINGS SYMPTOMS
34
• Bizarre behavior.
• Agitation
• Seizures.
• Difficulty in drinking.
• Patients will be able to eat
solids
• Afraid of water - Hydrophobia.
• Even sight or sound of water
disturbs the patient.
• But suffer with intense thirst.
• Spasms of Pharynx produces
choking
• Death in 1 -6 days.
• Respiratory arrest / Death /
Some may survive.
• Headache, fever, sore throat
• Nervousness, confusion
• Pain or tingling at the site of the
bite
• Hallucinations
– Seeing things that are not
really there
• Hydrophobia
– “Fear of water" due to
spasms in the throat
• Paralysis
– Unable to move parts of the
body
• Coma and death

35.  1 – Non specific prodrome
 2 – Acute neurologic
encephalitis
 Acute encephalitis
 Profound dysfunction of
brainstem
 3 – Coma
 4 - Death ( Rare cases 
recovery )
 Non specific prodrome
 1 - 2 days  1 week
 Fever, headache, sore
throat
 Anorexia, nausea, vomiting,
 Agitation, depression
 Parenthesis or
fasciculation's at or Around
the site of inoculation of
virus.
35

36. 36
• 1 – 2 days to < 1 week
• Excessive motor activity,
Excitation, Agitation
• Confusion, Hallucinations,
Delirium,
• Bizarre aberrations of
thought, Seizures,
• Muscle spasms,
Meningismus,
• Opisthotonic posturing
• Mental aberration ( Lucid
period  coma )
• Hypersalivation, Aphasia,
Pharyngeal spasms
• Incordination, Hyperactivity
• Fever T > 40.6
• Dilated irregular pupils
• Lacrimation, Salivation &
Perspiration
• Upper motor neuron
paralysis
• Deep tendon reflexes
• Extensor plantar responses (
as a rule )
• Hydrophobia or Aerophobia
(50 -70% )

37. Demo. Of Rabies virus antigens by
immunofluorescence. Direct immunofluorescence is
done using monoclonal antibodies tagged with
fluorescein isothiocynate. Immunoperoxidase staining
used for Ag in tissues.
Negri bodies
37

38. Negri bodies
38
• Negri bodies round or
oval inclusion bodies
seen in the cytoplasm and
sometimes in the
processes of neurons of
rabid animals after death.
• Negri bodies are
Eosinophilic, sharply
outlined, pathognomonic
inclusion bodies (2-10 µm
in diameter) found in the
cytoplasm of certain
nerve ..

39. Isolation of virus by intracerebral
inoculation in mice attempted from brain, CSF, saliva
& urine
Isolation of virus in tissue culture cell
lines ( WI 38, BHK 21, CER ). CPE minimal so identified
by immunofluorescence.
Demo. Of antibodies by ELISA.
Detection of rabies virus RNA by
reverse transcriptase PCR is a sensitive method.
39

40. • Demonstration of Negri bodies in brain postmortem
• Impression smears stained by Seller’s technique
(basic fuchsin + methylene blue in methanol).
• Demonstration of rabies virus antigen by
immunofluorescence in salivary glands.
• Isolation of rabies virus by animal inoculation.
40

41. 41
1. General consideration:-
Aim is to neutralize virus
before entering CNS
2. LOCAL WOUND
TREATMENT
a, Cleansing of wound(soap
& water)
b, Chemical treatment:
• Either Alcohol 400-700 ml
/liter
• Tincture Iodine
• No more treatment with
Ammonium
compound
• No Carbolic acid and Nitric
acid as it leave very bad
scar
c, Suturing avoided.
d, Anti Rabies Serum
e, Antibiotic and ATS
f, Observe the animal for 10
days
• 3, Immunization
• 1,NERVOUS TISSUE
VACCINE (NTV 2, Human
diploid cell vaccine (HDCV)

42. 42
Definition:
it is fluid or dried preparation of Rabies “Fixed” virus
grown in the Neural tissue of
Rabbits,
Sheep,
Goats,
Mice or Rats
OR in embryonated duck eggs
OR in cell culture

43. 43
Nervous Tissue
vaccine
Duck embryo
vaccine
Cell culture
vaccine
preparation From fixed virus grown in
brain of sheep or other
animals
potency Low or variable Eliminate Neuroparalytic
factors
More potent
more safer
Doses Large nos: are required Fewer doses of small
volume
Side effects Severe & fatal reactions Allergic risks Fewer
Uses Exposed subjects Used in UK,USA in past 1, (HDC) safe, potent
Pre & post
expos:Immunization
Suckling mouse brain V
Devoid of Neuroparalytic
effect
Used in Latin America
Improvement over adult
animal nervous tissue V
Now purified DEV
developed
Improvement over adult
animal nervous tissue V
Not available in India &
Pakistan
2Tissue culture 2nd G
(Non-human)
Potent, low cost
WHO recommendatio

44. 44

45. 45

46. 46
• TISSUE CULTURE VACCINES –
• Human diploid cell vaccine (HDCV)
Developed by Koprowsky,Wiktor,and
Plotkin
• Purified chick embryo cell vaccine
(PCEC)
• Purified Vero cell vaccine (PVRV)
• Purified duck embryo vaccine
(PDEV)

47. 47
• The vaccination is
given on
0, 3, 7, 14, 30, and
90th day
Immunity lasts for
5 years
Injected on deltoid
region IM/SC
Not to be given in
the gluteal region

48. 48
• Dosage
• Pre- exposure prophylaxis
0 – 7 – 21 – or 28 – 56 days
A booster after 1 year,
Repeat once in 5 days,
Post exposure Prophylaxis
Sex doses
0 -3 -7-14 – 30 - 90 days
Given IM or SC in the Deltoid region
Don't inject in Gluteal region.

49. 49
• Given on the
following days
0, 7, 21,or 28 and
56th day
Generally given to Vet
nary personal

50. 50
• Human Rabies
Immunoglobulin HRIG
• High Risk bitten on face
and neck
• Given a dose of 20 IU /Kg
wt
• Half at the site of bite and
rest IM route.
• Active immunization
should be initiated with
passive immunization.

51. 51
• A number of experimental vaccines are under
development that may provide alternative safe
and potent but less expensive vaccine options.
These include DNA vaccines, recombinant viral
vaccines, and recombinant protein vaccines.
Further testing is needed to determine if and
which one of these novel vaccines will make
their way into mass production and application
in the future.

52. 52
• A viral immunizing agent that
has been treated to remove
traces of viral nucleic acid so
that only protein subunits
remain. The subunits have less
risk of causing adverse
reactions.
• Several trails in progress

53. 53
1. Class I (slight Risk) 07 injection
a. Licks on healthy unbroken skin.
b. Scratches without oozing of blood.
2. Class II (Moderate Risk) according to the Schedule plus one
booster dose after 3 week
a. Licks on fresh cuts.
b. Scratches with oozing of blood.
c. All bites except those on head, neck, face, palms and fingers.
d. Minor wounds less than 5 in number.
3. Class III (Severe Risk) according to the Schedule plus Two
booster dose one after one week and another 2 week
a. all bites or scratches with oozing of blood on neck, head, face,
palms and fingers.
b. Lacerated wounds on any part of the body.
c. Multiple wounds 5 or more in number.
d. Bites from wild animals.

54. 54