Virology Lecture 1
VIROLOGY: DEFINITIONS, CLASSIFICATION,
MORPHOLOGY AND CHEMISTRY
AND MUMPS VIRUSES
VIROLOGY - LECTURE TWENTY
Dr Richard Hunt
MBIM lecture 76
Rabies virus belongs to the family:
Rhabdoviridae. (Greek: Rhabdos: rod). They can infect a variety of animals
1 - Rhabdoviruses
stomatitis virus (VSV)
mammals including humans
fish, birds, arthropods
Structure of rhabdoviruses (figure
General structure of a rhabdovirus
Negative stain electron micrograph of rabies virus
Wadsworth Center, NY Dept of Health
1 - Rhabdovirus structure
Rhabdoviruses are negative strand
RNA viruses; that is they have a single strand of RNA that is anti-sense
to the messenger RNA needed to code for viral proteins. This means that
the RNA cannot code directly for protein synthesis and must be copied to
positive strand mRNA. As a result, the virus must carry its own
RNA-dependent RNA polymerase.
As their name suggests these
viruses are rod shaped. They have one end that is rounded and are often
referred to as bullet-shaped. Each virus particle is up to 100nm diameter
and 400 nm long but this is very variable. They have an envelope derived
from the host cell plasma membrane. The virus has only five proteins.
G (Surface) Protein.
This is the surface glycoprotein spike and exists as trimers. There are
about 1200 G proteins (400 trimers) per virus particle. It is a
transmembrane protein with an N-terminal signal sequence. The G protein
binds to cellular receptors and is the target of neutralizing antibodies.
There are three sugar chains that are N-glycosidically attached.
Penetration of the virus into the cytoplasm takes place in the endocytic
pathway and not at the plasma membrane. This is because the G protein
trimer undergoes a change in conformation at pH 6.1 which stabilizes the
trimer and probably allows a hydrophobic region of the molecule to become
exposed and to embed in the membrane of the cell to be infected.
M (matrix) protein.
This is a peripheral membrane protein (originally M stood for membrane)
that appears to line the inner surface of the viral membrane, though this
remains somewhat controversial. It may act as a bridge between the
membrane or G protein and the nucleocapsid.
This is the infectious ribonucleoprotein core of the virus. It is a
helical structure that lies within the membrane. In negative stain
electron micrographs, such as seen in figure 1, the nucleocapsid has a
N (Nucleoprotein) protein.
This is the major structural protein and covers the RNA genome. It
protects the genome from nucleases and holds it in a conformation that
L (Large) protein and NS
(nonstructural, otherwise known as P (phospho)) protein together
form the RNA-dependent RNA polymerase or transcriptase. The L protein
has a molecular weight of 240 kiloDaltons and its gene takes up 60% of
the genome (figure 3).
The cycle of rabies infection and replication CDC
2 - Replication of rabies virus
The receptors for rhabdoviruses have yet to be definitively identified
but some experiments point to phospholipids, particularly phosphatidyl
serine, as the cell surface receptor molecule.
After endocytosis, pH-dependent fusion with the membrane of the
endocytic vesicle occurs. The nucleocapsid enters the cytoplasm. All
subsequent stages take place here with no involvement of the nucleus of
First, the polymerase, which is carried in the entering virus, makes
five individual mRNAs, one for each viral protein. Note, the RNA must be
made before any viral protein synthesis and so the infecting virus must
supply the polymerase enzyme. (As might be expected, this primary
transcription process takes place in the presence of protein synthesis
inhibitors). The mRNAs are capped, methylated and polyadenylated. The
sequence of transcription is N, NS(P), M, G and L with synthesis of the
mRNAs being attenuated at each gene junction (figure 3). This means that
less of the L mRNA is made than any of the others.
The rhabdovirus genome CDC
3 - Rhabdovirus genome
In addition, the polymerase transcribes the negative-sense genomic RNA
into a positive sense strand. This serves as a template for the
transcriptase to transcribe new negative sense genomic RNA molecules. This
replicative phase does require protein synthesis and the same polymerase
is involved. In the replicative phase, this enzyme must ignore signals
that define the individual mRNA species and make one single RNA molecule.
The switch between transcription of mRNAs and replication of genomic RNAs
seems to be controlled by the level of N protein
The G protein mRNA is translated in association with the endoplasmic
reticulum and transported via the Golgi body to the cell surface. Here, it
forms patches with which the M protein associates. The genomic length
negative strand RNA molecules associate with N, L and NS (P) proteins
forming the core nucleocapsids. This, in turn, associates with the M
protein at the inner surface of the plasma membrane or perhaps in the
cytoplasm. The interaction between nucleocapsid and M protein causes the
former to change configuration so that it appears more condensed. The
nucleocapsid then buds through the membrane.
Vesicular Stomatitis Virus (VSV)
VSV infects cattle in Carribean and
occasionally in US. It is also found in horses and pigs but rarely humans
Rabid animals become aggressive and
harbor the virus in saliva and thus transmission is frequently via animal
bites, although there is some evidence for spread via aerosols such as in
bat urine in caves. The virus has also been transmitted by transplantation
of corneas from infected individuals.
The virus binds to nerve or muscle
cells at the site of the inoculation via nicotinic acetylcholine
receptors. Here the virus can remain for a prolonged period of time (up to
several months). The virus can replicate in muscle cells at the site of
the bite with no obvious symptoms. This is the incubation phase.
The virus then moves along the
nerve axons to the central nervous system using retrograde transport. The
virus arrives at the dorsal root ganglia and the spinal cord. From here,
spread to the brain occurs. A variety of cells in the brain can be
infected including in the cerebellum, the Purkinje=s cells and also cells
of the hippocampus and pontine nuclei. This is the prodromal phase.
Infection of the brain leads to encephalitis and neural degeneration
although elsewhere the virus seems to cause little in the way of a
cytopathic effect. Involvement of the brain leads to coma and death. This
is the neurological phase and during this period, the virus can spread
from the central nervous system, via neurons, to the skin, eye and various
other sites (adrenals, kidneys, pancreatic acinar cells) and the salivary
glands (figure 4).
There are various factors that
determine the timing of the onset of symptomatic rabies but most important
are the number of virus particles in the infection and how close the bite
is to the brain. The immunological status of the patient is also
important. It should be noted that the immune response to naturally
acquired virus is slow and a good neutralizing response is not seen until
the virus has reached the brain which is too late for survival.
Cell-mediated immunity plays little role in a rabies infection. Rabies is
almost always fatal and only three survivors of symptomatic rabies have
been documented. Nevertheless, a good immune response that eliminates the
infection, can be achieved using a vaccine even after infection because of
the long incubation phase.
© Richard Hunt
1. Raccoon is bitten by a rabid animal
2. Virus enters wound via saliva
3. Virus spreads through nerves to spinal cord and brain
4. Incubation period of 3-12 weeks with no symptoms
5. In brain the virus replicates and spreads to other tissues
including the salivary glands. Signs of disease occur
6. The animal dies within a week
4 - Rabies pathogenesis
Rabies is spread, usually by bites
from animals, to other animals and to man. It is thus a zoonotic
infection. The most important reservoir as far as humans are concerned is
the dog but other animal reservoirs of importance include, in the United
States, racoons, bats, skunks and foxes. Rabies is found in most
continental countries of the world but not in some island nations such as
the United Kingdom and Australia. The incidence of rabies cases in various
animals is shown in figure 5.
2 - Major animal reservoirs of rabies
raccoons, bats, foxes
dogs, vampire bats
In many western countries where
rabies is endemic, vaccination of animals has reduced the rate of human
disease and in the United States there is approximately one case of human
rabies per year. In countries such as the United Kingdom, where there is
no rabies in the wild animal population, vaccination is not used. In some
other countries, rabies is much more of a problem. For example, India
records about 25,000 cases of human rabies per year, mainly from dog
bites. In South America, rabies transmission by vampire bats is a major
problem for the cattle industry (table 2).
Vaccination, even after exposure,
is extremely effective at preventing disease. Without such treatment,
rabies is almost invariably fatal. During the incubation/prodromal period,
symptoms include: pain or itching at the site of the wound, fever,
headache and gastrointestinal problems. After this period (usually of up
to two weeks), CNS infection is apparent. In up to half of patients,
hydrophobia is seen. This fear of water is the result of the pain
associated with drinking. There are also seizures and hallucinations. In
some patients paralysis is the only symptom and this may lead to
respiratory failure. Following the neurological phase, the patient becomes
comatose. Because of the neurological problems including respiratory
paralysis, death ensues.
Overt symptoms clearly define
symptomatic rabies in people who suffer animal bites but by this time,
therapeutic intervention is too late. After a bite, laboratory tests can
determine whether an animal is indeed rabid. The presence of rabies virus
in an animal or an infected person can be determined by serology and
immunofluorescence antigen determination using biopsy skin, brain or
corneal specimens (figure 8). Histologically very characteristic is the
presence of Negri bodies. These are intracytoplasmic inclusions formed by
aggregates of nucleocapsids in neurons of about 90% of infected humans
(table 3 and figure 7). Other tests include the growing of virus in the
brains of mice or in culture, after which antigen tests are used to
determine the presence of virus. Also anti-rabies antibodies can be
detected BUT only very late in the disease. Polymerase chain reaction (PCR)
can also be used to detect virus (figure 6).
evidence of rabies encephalomyelitis (inflammation) in brain
tissue and meninges
Perivascular cuffing of lymphocytes or polymorphonuclear cells
or inflammation around a blood vessel CDC
Babes nodules consisting of glial cells Image: CDC
bodies (see below)
PCR test results for the presence of rabies virus. The arrows
indicate positions of positive bands CDC
Direct fluorescent antibody test (dFA)
The dFA test is based on the principle that an animal infected
by rabies virus will have rabies virus protein (antigen) present
in its tissue. Because rabies is present in nervous tissue (and
not blood like many other viruses), the ideal tissue to test for
the presence of rabies antigen is brain. The most important part
of a dFA test is fluoresecently-labeled anti-rabies antibody.
When labeled antibody is added to rabies-suspect brain tissue,
it will bind to rabies antigen if it is present. Unbound
antibody can be washed away and the areas where the antigen has
bound antibody will appear as a bright fluorescent green color
when viewed with a fluorescence microscope. If rabies virus is
absent there will be no staining. The rabies antibody in the dFA
test is primarily directed against the nucleoprotein of
the virus. Rabies virus replicates in the cytoplasm of cells,
and infected cells may contain large round or oval inclusions
containing collections of nucleoprotein (N) or smaller
collections of antigen that appear as dust-like fluorescent
particles if stained by the dFA procedure CDC
Post-exposure prophylaxis is the
major form of treatment when it is suspected that the patient may have
come in contact with a rabid animal. Such contact is most often a bite but
could also include contact with tissue from a rabid animal. In the United
States up to 20,000 rabies inoculations are given to humans every year.
After a bite, it is recommended to wash the wound and to instill
anti-rabies serum into the wound. After this, the vaccine is given,
accompanied by either horse anti-rabies serum or human rabies immune
globulin. This suffices until the patient develops an immune response to
the virus. Four more vaccinations are then given over a period of a month
(days 3, 7, 14 and 28 after exposure to the virus). Pre-exposure
vaccination is recommended for persons who may come in contact with tissue
of rabid animals and seems effective for up to two years.
Vaccination against rabies has a
long history, the first vaccine having been developed by Louis Pasteur.
His vaccine was an attenuated form of the virus produced by inoculation of
rabbit spinal cord. The present (HDCV) rabies vaccine is a chemically
inactivated one that is produced in human diploid tissue culture cells.
Previous vaccines were produced in the brains of animals but these gave
more side effects.