Rabies Virus – Structure, Genome Organization, Pathogenesis, Replication

Rabies is a viral disease that causes inflammation of the brain in humans and other mammals. It is transmitted through the saliva of infected animals, usually through a bite. The virus attacks the central nervous system, leading to symptoms such as fever, muscle spasms, and hallucinations. If left untreated, rabies is almost always fatal. It is important to seek medical attention as soon as possible after being bitten by an animal, as treatment is most effective when given before symptoms appear.

Epidemiology of Rabies Virus

• Rabies is found in many parts of the world, but it is most common in countries with a high number of stray dogs. It is also found in wild animals such as bats, foxes, and raccoons.
• In developed countries, rabies is relatively rare due to effective vaccination programs for domestic animals and public education about the importance of seeking medical attention after an animal bite.
• The World Health Organization (WHO) estimates that tens of thousands of people die from rabies each year, with the majority of cases occurring in Africa and Asia. However, it is difficult to accurately estimate the number of rabies cases, as many cases go unreported, especially in areas with limited access to healthcare.
• Rabies is a preventable disease, and efforts to control the disease include vaccination programs for animals, pre-exposure prophylaxis for high-risk individuals, and post-exposure treatment for people who have been bitten by an infected animal.

Rabies Virus Structure

• Rhabdoviruses are roughly 180 nm in length and 75 nm in width.
• There are five proteins encoded by the rabies genome: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (P) (L).
• All rhabdoviruses contain two basic structural components: a ribonucleoprotein (RNP) core and an envelope. The nucleoprotein securely encases genomic RNA within the RNP.
• The RNP is coupled with two additional viral proteins, the phospoprotein and the large protein (L-protein or polymerase).
• On the surface of the virus, the glycoprotein forms around 400 trimeric spikes that are densely packed. The M protein is connected with both the envelope and the RNP and may be the key protein in the assembly of rhabdoviruses. The structure and composition of the rabies virus are represented in the diagram below.
• Rabies is an RNA virus. There are five proteins encoded by the genome, named N, P, M, G, and L. The order and relative size of the genome’s genes are depicted in the following diagram.
• Rabies virus structure is determined by the configuration of these proteins and the RNA genome.

Rabies Virus Shape

The rabies virus is a bullet-shaped virus with a diameter of about 75 nanometers and a length of about 180 nanometers. It is a member of the Rhabdovirus family and is classified as a negative-sense single-stranded RNA virus. The virus is enveloped, meaning that it has a protective outer layer made up of lipids that surrounds the viral genome. The virus is covered in spikes made up of a protein called glycoprotein, which helps it to bind to and infect host cells. The rabies virus is highly infectious and is transmitted through the saliva of infected animals, most commonly through bites or scratches. It can infect all warm-blooded animals, including humans, and is usually fatal once symptoms appear.

Genome organization of Rabies Virus

• The rabies virus genome is roughly 12 kb of single-stranded, antisense, nonsegmented RNA. There is an approximately 50 nucleotide leader sequence (LDR) followed by N, P, M, G, and L genes.
• The virion of a rhabdovirus is an enveloped, rod- or bullet-shaped structure that contains five protein species.
• The nucleoprotein (N) coats the RNA at a rate of one protein monomer every nine nucleotides, forming a helical nucleocapsid.
• P (phosphoprotein) and L (large protein) duplicates are associated with the nucleocapsid. The L protein is well called, as its gene occupies over half of the genome. Its vastness is justified by the fact that it is a protein with multiple functions.
• M (matrix) protein forms a layer between the nucleocapsid and envelope, while G (glycoprotein) trimers create protruding spikes from the envelope.
• All rhabdovirus genomes encode these five proteins, and in the case of Rabies Lyssavirus, all five are encoded.

Transmission of Rabies Virus

• People are typically infected via a deep bite or scratch from a rabid animal, and rabid dogs responsible for up to 99 percent of human infections.
• Bats are currently the leading cause of human rabies mortality in the Americas, as dog-mediated transmission has been mostly eliminated in this region.
• In addition to Australia and Western Europe, bat rabies is a rising public health problem in Australia. Human mortality caused by exposure to foxes, raccoons, skunks, jackals, mongooses, and other wild carnivore host species are extremely uncommon, and it is unknown if rodent bites transmit rabies.
• Transmission is also possible if infected animal saliva comes into close contact with human mucosa or fresh skin wounds. Rabies transmission through inhalation of virus-containing aerosols or organ transplantation is described, but highly uncommon.
• Theoretically, human-to-human transmission by bites or saliva is possible, but has never been confirmed. The same holds true for transmission to humans through eating of raw animal meat or milk.

Replication of Rabies Virus

• The infection process is initiated by the fusion of the rabies virus envelope with the host cell membrane (adsorption). Possibly implicated is the interaction between the G protein and certain cell surface receptors.
• After adsorption, the virus enters the cytoplasm and penetrates the host cell. The virus particles congregate in the endosomes (cytoplasmic vesicles). The fusion of viral membranes with endosomal membranes releases viral RNP into the cytoplasm (uncoating). Because lyssaviruses have a linear, single-negative-stranded ribonucleic acid (RNA) genome, viral replication requires the transcription of messenger RNAs (mRNAs).
• The genomic strand of rabies RNA is transcribed by a viral polymerase (L gene) into leader RNA and five capped and polyadenylated mRNAs, which are then translated into proteins. Translation, which involves the production of the N, P, M, G, and L proteins, happens on cytoplasmic ribosomes.
• Although G protein synthesis is initiated on free ribosomes, the endoplasmic reticulum (ER) and Golgi apparatus are responsible for its completion and glycosylation (processing of the glycoprotein). The ratio of intracellular leader RNA to N protein controls the transition from transcription to replication. When this switch is triggered, viral genome replication commences.
• The initial step in viral replication is the production of complete copies of the viral genome (positive strands). During the transition to replication, RNA transcription becomes “non-stop” and stop codons are disregarded.
• The viral polymerase enters a single spot on the 3′ end of the genome and then synthesises complete copies of the genome. These positive rabies RNA strands act as templates for the creation of complete negative strands of the viral genome.
• During the assembly process, the N-P-L complex envelopes negative-stranded genomic RNA to create the RNP core, while the M protein forms a matrix or capsule surrounding the RNP.
• The RNP-M complex migrates to a region of the plasma membrane with glycoprotein inserts, where the M-protein commences coiling. The M-RNP complex binds to the glycoprotein, causing the virus to emerge from the plasma membrane.
• Within the central nervous system (CNS), plasma membranes are favoured for viral budding. Viruses in the salivary glands, on the other hand, bud largely from the cell membrane into the acinar lumen.
• Viral budding in the salivary gland and virus-induced aggressive biting in the host animal increase the likelihood of viral transmission to a new host.

Pathogenesis of Rabies Virus

• The saliva of a rabid animal inoculates muscle and subcutaneous tissues with the rabies virus. During the incubation period, which lasts weeks to months, there is a delay in the movement of the virus at the inoculation site.
• Rabies virus attaches to nicotinic acetylcholine receptors at the neuromuscular junction and travels at a rate of 50–100 mm per day within the axons of peripheral nerves toward the spinal cord via retrograde rapid axonal transport.
• The virus disseminates along neuroanatomical channels within CNS axons. Rabies virus replicates in neurons and produces neuronal dysfunction via unidentified mechanisms, which presumably accounts for the disease’s clinical manifestations and deadly result.
• Changes in behaviour are characteristic of rabies, which is typically transmitted by biting infected animals. The virus then spreads centrifugally (away from the CNS) along nerves to many organs, including salivary glands, in animals that transmit the virus.
• The rabies virus is secreted in vectors’ saliva and transmitted to new hosts via biting. In North America, bats, raccoons, skunks, and foxes are significant rabies vectors, while canines are the most significant vector worldwide. There may be no recognition of bat bites, and there may be no known encounter with bats.

Clinical manifestations of Rabies Virus

• In humans, there are five distinct stages of rabies: incubation, prodrome, acute neurologic period, coma, and death (or, very rarely, recovery).
• Once clinical symptoms of rabies occur, no specific antirabies medicines are effective. The incubation period for rabies is more variable than that of any other acute illness, ranging from as few as 5 days to more than 2 years following initial exposure.
• Children and those bitten close to the central nervous system may experience somewhat shorter incubation times (e.g., the head). The prodromal stage, which typically lasts between 2 and 10 days, is when clinical symptoms initially manifest.
• These symptoms are frequently nonspecific (malaise, fever, and fatigue) or suggest involvement of the respiratory system (sore throat, cough, and dyspnea), the gastrointestinal system (anorexia, dysphagia, nausea, vomiting, abdominal pain, and diarrhoea), or the central nervous system (anorexia, dysphagia, nausea, vomiting, abdominal pain, and diarrhoea) (headache, vertigo, anxiety, apprehension, irritability, and nervousness).
• More notable abnormalities (agitation, photophobia, priapism, increased libido, insomnia, nightmares, and depression) may also occur, indicating the presence of encephalitis, psychiatric disorders, or brain diseases.
• The presence of pain or paresthesia at the site of viral injection, along with a recent history of animal bite, may prompt evaluation of rabies.
• The acute neurologic phase begins with objective symptoms of malfunction of the central nervous system. The disease may be categorised as furious rabies if hyperactivity (i.e., hydrophobia) predominates, or as dumb rabies if paralysis predominates.
• In both variants of the condition, fever, paresthesia, nuchal stiffness, muscle fasciculations, focal and widespread convulsions, hyperventilation, and hypersalivation are possible.
• At the conclusion of the acute neurologic phase, episodes of fast, erratic breathing may ensue, followed by paralysis and coma.
• Unless the patient is getting ventilatory assistance, which may prolong survival for days, weeks, or longer, death may occur as a result of additional complications.
• Although life support techniques can prolong the course of rabies, they rarely alter the disease’s outcome. However, the prospect of recovery must be acknowledged, and when resources allow, every effort must be made to assist the patient. At least seven known incidents of human “recovery” exist.

Diagnosis of Rabies Virus

Steps Summery

1. absorbance (receptors and virion interation).
2. Penetration (virus entry).
3. De-coating (envelope removal).
4. Transcription (synthesis of mRNAs).
5. Interpretation (Synthesis of structural proteins).
6. Procedure (G-protein gycosylation). Replication (synthesis of genomic RNA from the intermediate strand).
7. construction
8. Budding (complete virions).

A. Laboratory Diagnosis in Humans

Specimens

For the diagnosis of rabies, saliva, serum, cerebrospinal fluid (CSF), blood, urine, and skin and brain biopsies are often utilised specimens.

Microscopy

Demonstration of Negri bodies by microscopy is the typical histological feature of rabies. Impression smears of the human brain tissue taken at postmortem are dyed by Seller’s procedure for demonstration of Negri bodies. The stain contains methylene blue alcohol as fixative and basic fuchsin as staining reagent.

B. Direct Antigen Detection

For the diagnosis of rabies, viral antigens can be seen in the brain tissue after death or in antemortem saliva, skin biopsy taken from the face or neck, and corneal smear. A specialised technique to show rabies antigen in clinical specimens is the direct fluorescent antibody (DFA) test, which utilises certain monoclonal antibodies.

Isolation of the virus

By cultivating viruses in cell lines or on animals, viruses can be extracted from brain tissue, CSF, saliva, and urine. 

• Culture: A delicate technique for isolating viruses is to do it in cell lines (such as WI-38, BHK-21, and CER). Since the virus has a negligible cytopathic effect, viral antigen is used to demonstrate virus presence in implanted cell cultures within 2-4 days. The DFA test is a regularly employed specific technique to demonstrate the antigen and uses monoclonal rabies antibodies labelled with fluorescein isothiocyanate. 
• Animal inoculation: Mouse is the preferred animal for the animal vaccine. Injections of CSF, saliva, and urine are made intravenously. The mice who received the vaccine are watched for symptoms of clinical disease. The brain tissues are tested for the existence of Negri bodies by microscopy or for viral antigen by the DFA test after the mice have died or have been inoculated for 28 days.

Serodiagnosis

• Antibodies to rabies are present in the serum and CSF of human patients. The concentration of antibodies in the cerebrospinal fluid (CSF) is high.
• In fifty percent of instances, a quick fluorescence focus inhibition test titer demonstrates the presence of antibodies in the serum.
• After the first week of sickness, positive antibody titers (2–25% of serum titer) are detected in the cerebrospinal fluid (CSF).

Other tests

• Normal to increased white blood cell count with 6–8% atypical monocytes. It is possible to see albuminuria and sterile pyuria. In the prodromal and early acute neurologic episodes, hyperventilation causes respiratory alkalosis, which is followed by respiratory acidosis when respiratory depression advances.
• After one week of illness, 80% monocytosis is found in the cerebrospinal fluid (CSF). The findings of protein and glucose tests are normal.

Antemortem diagnosis of human rabies

• Until recently, the majority of rabies cases were lethal, and patients typically succumbed to the disease.
• With extensive patient support and improved patient management, however, few cases have survived.
• Consequently, laboratory procedures for antemortem diagnosis of rabies in people are proving to be more useful in such instances.

C. Laboratory Diagnosis in Animals

The laboratory diagnosis of rabies in dogs and other animals is essential for assessing infection risk and monitoring postexposure treatment in humans bitten by the animals.

Specimens

The brain of the deceased animal is the preferred specimen. The brain is carefully extracted from a deceased animal. For virus isolation, a portion of the specimen is collected in 50% glycerol saline (preservative). The hippocampus and cerebellum, which are plentiful in Negri bodies, are collected in Zenkers’ fixative for the demonstration of Negri bodies.

Microscopy

Seller’s approach stains brain tissue impression smears to demonstrate the presence of Negri bodies. In laboratories lacking equipment for cell culture and antigen detection, this procedure is nonetheless useful.

Direct antigen detection

The demonstration of viral antigens in infected brain tissue and saliva by DFA is an effective way for diagnosing rabies in animals. Detection of antigen in the saliva indicates whether or not the animal excreted the virus in its saliva.

Isolation of the virus

As explained previously for the diagnosis of human rabies, viruses can be isolated from animal brain matter and saliva by cell line culture or in animals.

Treatment of Rabies Virus

There is no antirabies agent available. With extensive supportive care and management of sequelae, it has been proved that complete recovery from established rabies is possible. Previously, it was believed that rabies was always deadly.

Management, Prevention and Control of Rabies Virus

Pre-exposure prophylaxis and post-exposure prophylaxis can be used to describe rabies prophylaxis, depending on whether it was administered before or after exposure.

Pre-exposure prophylaxis

Pre-exposure prophylaxis is administered to high-risk individuals, including dog handlers, other animal handlers, and veterinarians. This is accomplished by employing safer cell culture vaccinations. Preexposure prophylaxis necessitates the administration of three doses of cell culture vaccinations on days 0, 7, 21, or 0, 28, and 56. A booster dose is indicated after one year, followed by an annual dose. In reducing the spread of rabies, animal control and immunisation techniques now play larger roles than postexposure prophylaxis.

Postexposure prophylaxis

Postexposure prophylaxis is initiated immediately after infection exposure. After exposure to a potentially infected dog or other rabid animals, urgent preventive measures consist of (a) local treatment, (b) confirmation that the animal is rabid, (c) delivery of hyperimmune serum, and (d) administration of an antirabies vaccination.

• Local treatment: Local treatment consists of a timely wound cleansing. The virus is transmitted through animal bites to the wounds. The wound should be cleaned quickly with soap and water, and then treated with quaternary ammonium compounds, such as Cetavlon, tincture, or aqueous solution of iodine or alcohol.
• Confirmation of the animal’s rabies status: This is possible through clinical observation of the questionable dog. If the dog is healthy 10 days after biting a human, rabies is extremely unlikely to have spread. In addition to the evidence of Negri bodies in brain tissue at autopsy or viral antigens in brain tissue or saliva, the diagnosis can also be made by seeing Negri bodies in brain tissue at autopsy. Treatment with hyperimmune serum: By injecting purified equine rabies immune globulin (ERIG) and human rabies immune globulin, passive immunisation is performed (HRIG). HRIG is rapidly administered to achieve passive immunisation against rabies. The suggested dose of HRIG is 20 IU per kilogramme of body weight. Fifty percent of the medication is administered through the incision and the remaining fifty percent is administered intramuscularly. It is often administered before to or simultaneously with the initial dose of rabies vaccine. It is not administered after rabies vaccination.
• Antirabies vaccine: Rabies is the only illness for which postexposure vaccination is widely and successfully employed. This is due to the disease’s lengthy incubation period. The likelihood of preventing rabies increases when persons are vaccinated as soon as possible following exposure. Since Pasteur’s successful use of a rabies vaccine derived from a dehydrated preparation of rabbit spinal cord in 1885, a wide variety of vaccines are used to protect humans against rabies, even after infection with the virus. Recent years have witnessed the gradual replacement of neural-tissue vaccinations with cell culture vaccines that are produced in human diploid cells and subsequently inactivated with propiolactone (refer the box Vaccines).

Rabies virus under microscope – Rabies virus image

FAQ

References

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