Rhabdoviruses

MBCHB II, PHA II, BNS II, PHS II

Kennedy Kassaza, Dept for Microbiology – 15-07-2021

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 Introduction
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.

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 Classification
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.
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 Rabies virus
STRUCTURE
MORPHOLOGY

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

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 Morphology….
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.

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Morphology
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.

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 Rabies virus proteins

Proteins L-RNA dependent RNA

polymerase
Protein G surface antigen
Protein N RNA binding protein
Protein NS – phosphoprotein
Protein M – membrane/matrix protein

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

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 Antigenic properties
• 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.
NUCLEOPROTEIN – IS A NUCLEOCAPSID
PROTEIN IMP.
➢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.
OTHER ANTIGENS – Include -
➢ Two membrane proteins.
➢ Glycolipid.
➢ RNA dependent RNA polymerase.
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Host range and cultivation
ANIMALS –
• All mammals are susceptible to Rabies
• Cattle, cats & foxes – Highly susceptible.
• Skunks, opossums & fowl – relatively resistant.
• Humans & dogs occupy an intermediate position.
• Pups more susceptible than adults.
• Experimental infection can be produced in any lab animal but mice is
choice.

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 TYPES OF RABIES VIRUS
STREET VIRUS
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.
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
Definition: the virus which has a short, fixed and
reproducible incubation period is called “fixed virus
Sources: it is prepared in the lab by repeated culture in
brain of rabbit such that its I.P. is reduced & fixed
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 anti-rabies vaccine
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 Host range and cultivation cont.’
CHICKEN EMBRYO
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.
TISSUE CULTURE
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.
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Rabies
Primarily a Zoonotic disease of warm blooded animal such as :-
Dogs
Wild cata
Jakals
Wolves etc.

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Rabies common facts
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
13 injection of the cord vaccine.

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

2) WILD LIFE RABIES:

From jackals and
foxes.

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Source of infection
Saliva of rabid animal

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Rabies transmission and associated factors
MODE OF TRANSMISSION
HOST FACTORS
All warm blooded animals including 1. animal bites
man.
2. licks
Rabies in man is a dead-end infection.
3. aerosol
4. person to person
Incubation period

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

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Pathogenesis
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.

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Pathogenesis
Live virus Epidermis, Mucus membrane

Peripheral nerve

Centripetally
CNS ( gray matter )

Centrifugally
Other tissue (salivary glands,…)

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Pathogenesis

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Pathogenesis

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Spread of virus
From Brain virus spread to
- Salivary glands,
- Conjunctival cell - released into tears
- Kidney
- Lactating glands and Milk after pregnancy

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

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Bad category presentation
• Furious Rabies
• Dumb ( Rage tranquille )
• (Landry/Guillain-Barre Syndrome

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

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 Acute neurologic encephalitis: Presentation
1 – 2 days to < 1 week
• Excessive motor activity, Excitation, Agitation
Confusion, Hallucinations, Delirium, Bizarre
aberrations of thought, Seizures, Muscle spasms,
Meningismus,
• Opisthotonic posturing
• Hypersalivation, Aphasia, Pharyngeal spasms
• Incordination, Hyperactivity
• Mental aberration ( Lucid period → coma
– death
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% )
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 Laboratory diagnosis
1. Specimens: specimens tested are corneal smears, skin
biopsies (from face and neck) or saliva antemortem, and brain
postmortem
2. Direct microscopy
Antemortem: Demonstration of rabies virus antigens by
immunofluorescence. Direct immunofluorescence is done by
mono clonal antibodies tagged with fluorescein
isothiocyanate. Immunoperosidase staining used for Ag
tissues.
Postmortem: Diagnosis on postmortem is by demonstration of
Negri bodies in the brain. May be absent in 20%
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 ..

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Isolation
Animal inoculation: isolation of the virus by intracerebral inoculation in mice attempted from
the brain, CSF, saliva and urine.
Tissue culture: isolation of virus in tissue culture cell lines (WI 38, BHK21, CER)
Antibody Demonstration: demonstration of antibodies by ELISA
Molecular methods: detection of rabies RNA virus by reverse transcriptase PCR is a sensitive
method
In dogs and other animals:
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.

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

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Antirabic vaccine
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

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Antirabic vaccine

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Antirabic vaccine

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Antirabic vaccine

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Human diploid cell vaccine
Dosage
• Pre- exposure prophylaxis
0 – 7 – 21 – or 28 – 56 days
Generally given to Veterinary personal

A booster after 1 year,

Repeat once in 5 days,

Post exposure Prophylaxis doses

0 -3 -7-14 – 30 - 90 days
Given IM or SC in the Deltoid region

Don't inject in Gluteal region

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Passive immunization
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.

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