To know the
different types of rhabdoviruses
about the structure and replication of these negative strand RNA viruses
understand the pathology of rabies
Rabies virus belongs to the family: Rhabdoviridae. (Greek:
They can infect a variety of animals and plants
Worldwide, it is estimated that approximately 55 000 persons die of rabies
stomatitis virus (VSV)
||Acute, self limiting
Figure 1A - Rhabdovirus structure General structure of a rhabdovirus
Figure 1B - Negative stain electron micrograph of rabies virus
Center, NY Dept of Health
- Replication of rabies virus The cycle of rabies infection and replication CDC
|Structure of rhabdoviruses
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
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 striated
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 allows transcription
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).
- Rhabdovirus genome The rhabdovirus genome CDC
Replication (figure 2)
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 the cell.
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
This means that less of the L mRNA is made than any of the others.
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
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 Caribbean 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. In rare cases, rabies has been transmitted by corneal
transplant or transplant of other tissues, or through contact of infected saliva
with mucosal membranes or an open wound in the absence of a bite. The CDC
states: “Inhalation of aerosolized rabies virus is also a potential non-bite
route of exposure, but other than laboratory workers, most people are unlikely
to encounter an aerosol of rabies virus”. It has been suggested that people in
infected bat caves may be exposed to aerosolized virus. Most bats are not
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
Rabies is usually transmitted by an animal bite. Worldwide most cases arise
from a dog bite. Canine rabies is prevalent in Latin America, Asia and Africa.
In recent years (1990 - 2004), in the US the majority of cases (35 out of 47)
have been associated with bat rabies; of the remaining cases, two were acquired
in the US (one dog/coyote like-strain and one raccoon strain) and 10 were
acquired outside the US (all dog/coyote like strains).
Many animals in the US are infected with rabies viruses, including raccoons
(especially along the eastern seaboard states), skunks, coyotes, and foxes.
Small rodents are rarely infected, but there have been cases reported,
especially in woodchucks. Dogs, cats and cattle are potential vectors - in the
US immunization of pets has lessened the risk of pets acquiring rabies from wild
animals. Bats also carry rabies, although most bats are not infected. Bats have
very small, sharp teeth, and people who are bitten may not be aware of the bite,
or do not bother to do anything about it. With most bites from other rabid
animals, the victim normally seeks treatment because the bite is more serious
and also because the animal appeared to behave in a suspicious fashion; the
level of awareness seems to be lower for suspiciously behaving bats.
Immunization of pets and prompt response for bites from most suspicious animals
may explain why bat-transmission of rabies has been the predominant mode of
transmission in recent years.
In many cases of bat-associated rabies, there is no record of a bite. In some
cases, the victim or their family may be aware that they handled a bat or that
an oddly behaving bat was found (e.g. a bat which is active by day, is easily
approached, is unable to fly, is in a room in a house or on a lawn). However, if
the victim is not able to answer questions it may be difficult to obtain a
history of bat contact since they may not have found the incident worth
mentioning to anyone.
Human to human transmission has occurred in a few cases of corneal transplants
(when it was not realized that the encephalitis was due to rabies). This has led
to stricter criteria in screening of potential donors for encephalitis so that
those who might have rabies (or Creutzfeld-Jakob disease) are not accepted.
Recently (2004) an organ donor who died of a brain hemorrhage also had rabies
and it was transmitted to 4 recipients. Apart from transplant cases, no
human-human spread of the disease has ever been documented.
Table 2 -
Major animal reservoirs of rabies
raccoons, bats, foxes
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).
of a rabid raccoon
North Carolina Depatment of Health and Human Services
sound of a rabid cow
North Carolina Depatment of Health and Human Services
4 - Rabies pathogenesis
© 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
and Rabies (CDC)
Question and Answer (CDC)
Vaccination, even after exposure, is extremely effective at preventing
disease. Without such treatment, rabies is almost invariably fatal (although,
see the case report at left). 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.
of a Patient from Clinical Rabies --- Wisconsin, 2004
Investigation of Rabies
Infections in Organ Donor and Transplant Recipients
Reported cases of rabies in the United States, 1999
Reported cases of rabies in raccoons in the United States, 1999
Reported cases of rabies in skunks in the United States, 1999
Reported cases of rabies in bats in the United States, 1999
Reported cases of rabies in coyotes in the United States, 1999
Reported cases of rabies in other wild animals in the United States,
Distribution of major terrestrial reservoirs of rabies in the United
Reported cases of rabies in cats in the United States, 1999
Reported cases of rabies in cattle in the United States, 1999
The Expanding Epizootic of Raccoon Rabies, Eastern United States, 1977-1996
Cases of animal rabies in the United States, 1955-1999
(All images from CDC)
Reported cases of rabies in foxes in the United States, 1999
Reported cases of rabies in dogs in the United States, 1999
Rabies in domestic animals by month in the United States, 1999
Figure 6 PCR test results for the presence of rabies virus. The arrows indicate positions of positive
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 is determined by multiple
(neutralizing serum or cerebrospinal fluid antibodies in an unvaccinated
person are diagnostic but usually are only detectable late in disease).
- Immunofluorescence antigen determination using biopsy skin, brain or corneal
specimens (figure 8). A full thickness nuchal skin biopsy (skin biopsy from the
nape of the neck in which the observer looks at the nerves at the base of
the hair follicles) or brain biopsy can be examined for rabies antigen using
a direct fluorescent antibody test.
- Saliva may be tested for rabies virus RNA by RT-PCR (reverse
transcription-polymerase chain reaction) or by isolation of the virus.
- Histologically very characteristic is the presence of Negri bodies.
These are eosinophilic intracytoplasmic inclusions formed by aggregates of nucleocapsids in
neurons of about 50 to 80% of infected humans (table 3 and figure 7). They are
typical of rabies, but the results need to be read by someone experienced
with rabies and there can be false positives - so all such results need to
be confirmed by another method.
- 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).
Histopathologic evidence of rabies encephalomyelitis (inflammation) in brain tissue and meninges
| Mononuclear infiltration
Perivascular cuffing of lymphocytes or polymorphonuclear cells or inflammation around a blood vessel CDC
| Lymphocytic foci
Babes nodules consisting of glial cells Image: CDC
| Negri bodies
Figure 7 Neuron without Negri bodies CDC
Negri body in infected neuron CDC
Negri body in brain cell © Bristol Biomedical Image
Archive. Used with permission
Histopathology of rabies, brain. Characteristic Negri bodies are present within a Purkinje cell of the cerebellum in this patient who died of rabies.
CDC/Dr. Makonnen Fekadu firstname.lastname@example.org
Rabies virus budding from an inclusion (Negri body) into the endoplasmic reticulum in a nerve cell.
A. Negri body.
Ribonucleoprotein. Notice the abundant strands of coiled RNP (almost
everything in the image is
B. Notice the abundant RNP in the inclusion.
C. Budding rabies virus. CDC
Rabies virus-infected neuronal cell with intracytoplasmic
inclusions (Negri bodies). The red stain indicates areas of rabies viral antigen by using IHC or
avidin-biotin complex CDC
Figure 8 Direct fluorescent antibody test
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
PREVENTION AND TREATMENT OF A PERSON WHO MAY HAVE BEEN EXPOSED
The wound should be immediately
and thoroughly washed with soap and water, then treated with 40-70% ethyl
alcohol or an antiseptic such as benzyl ammonium chloride. The State Health
authorities should be promptly informed. The risk of exposure to rabies and
whether prophylactic treatment should be given are determined in consultation
with the State Health Department. If the animal is available, the brain should
be examined for rabies virus antigen by fluorescent antibody. (In some cases, if
the bite was from a domesticated cat or dog, the animal may be kept under close
This is an inactivated vaccine and is strongly immunogenic. It is grown in human
diploid cells or rhesus monkey lung cells and is more potent and has fewer side
effects than the vaccine used in the early 1980’s. A purified chick embryo cell
grown vaccine is also available. The vaccine is administered as a series of
injections over a 4-week period. HRIG (human rabies immunoglobulin) is also
rabies immunoglobulin (HRIG)
HRIG is prepared from the plasma of
hyperimmune donors. Up to half of the recommended dose is infiltrated into the
wound area if possible. The remainder is given as an intramuscular injection. A
separate syringe and a separate site are used for the HRIG and the vaccine so
that the HRIG does not neutralize the vaccine.
So far there has never been a
case of someone who received appropriate post-exposure prophylaxis in the US
developing rabies. (About 40,000 people per year are treated in the US).
People at risk for rabies
infection may be vaccinated as a preventive measure. Such individuals include
certain people in areas with
enzootic rabies who are at risk for exposure to rabid animals: veterinarians
and their staff, wildlife control workers, spelunkers (mainly those cave
explorers who go into undeveloped caves with bat colonies); travelers who
will be spending more than a month in areas with enzootic rabies.
People at high risk for exposure
to rabid animals should have regular serologic testing and booster vaccinations
If a vaccinated person is
exposed to rabies, they still need to get post-exposure prophylaxis, but
the number of post-exposure vaccination shots is reduced and HRIG is not used.
If symptoms are localized to the
site of the bite, aggressive antiviral therapy (vaccine, HRIG, ribavirin,
interferon, monoclonal antibodies, etc) may be tried. There is no specific
anti-viral treatment once CNS symptoms develop. Intensive supportive care is
given. Five of the six known survivors of rabies infection received prophylaxis
prior to developing clinical symptoms. There has been one documented case of a
non-vaccinated survivor of rabies. (Willoughby RE Jr, et al. Survival after
treatment of rabies with induction of coma. N Engl J Med. 2005 352:2508-14.
Jackson AC. Recovery from rabies. N Engl J Med. 2005 16;352:2549-50).
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