Rhabdovirus: Definition, Classification, and Transmission

1. CHAPTER 18 Rhabdovirus

2. Definitions of the virus: • The family Rhabdoviridae encompasses six genera of viruses that infect a broad range of hosts. • The family contains animal and human pathogens, including rabies, vesicular stomatitis, and bovine ephemeral fever viruses. • The family Rhabdoviridae includes four genera that contain animal

4. Definitions of the virus: viruses: the genera Lyssavirus, Vesiculovirus, Ephemerovirus, and Novirhabdovirus. The family contains animal and human pathogens, including rabies, vesicular stomatitis, and bovine ephemeral fever viruses. The genus Lyssavirus (‘lyssa’ meaning rage) includes rabies virus and closely related viruses.

6. Each of these viruses is capable of causing rabies-like disease in animals and humans. Certain terrestrial mammals are reservoir hosts of rabies virus, and bats are reservoirs of both rabies and the rabies-like viruses. The genus Vesiculovirus includes vesicular stomatitis Indiana and vesicular stomatitis New Jersey viruses. The genus Ephemerovirus contains bovine ephemeral fever virus.

7. Rhabdovirus virions are approximately 45–100 nm in diameter and 100–430 nm long, and consist of a helically coiled cylindrical nucleocapsid surrounded by an envelope with large (5–10 nm in length) glycoprotein spikes. The precise cylindrical form of the nucleocapsid is bullet or conical shape. The genome is a single molecule of linear, negative sense, single-stranded RNA, 11–15 kb in size. The rabies virus encodes five genes in the order 3’-N-P-MG-L-5’.

8. N is the nucleoprotein gene that encodes the major component of the viral nucleocapsid; P is a cofactor of the viral polymerase; M is an inner virion protein that facilitates virion budding by binding to the nucleocapsid and to the cytoplasmic domain of the glycoprotein; G is the glycoprotein that forms trimers that make up the virion surface spikes; L is the RNA-dependent RNA polymerase that functions in transcription and RNA replication.

9. The glycoprotein (G) contains neutralizing epitopes, targets of vaccine-induced immunity; it and the nucleoprotein include epitopes involved in cell-mediated immunity. Virions contain lipids and carbohydrates as side chains on the glycoprotein. Rhabdoviruses are stable in the environment, when the pH is alkaline—vesicular stomatitis viruses can contaminate water for many days but the viruses are thermolabile and sensitive to the sunlight.

11. Virus entry into host cells occurs by receptor-mediated endocytosis via coated pits, and subsequent pH-dependent fusion of the viral envelope with the endosomal membrane releases the viral nucleocapsid into the cytoplasm, where replication occurs. The viral glycoprotein G is responsible for receptor recognition and cell entry. Specific cell receptors have not clearly been identified for rhabdoviruses; several non-essential receptors have been identified for rabies virus, including:

12. neurotrophin receptor p75NTR, the muscular form of the nicotinic acetylcholine receptor, neuronal cell adhesion molecule, and gangliosides. Phosphatidyl choline is a receptor for vesicular stomatitis virus, and fibronectin for viral hemorrhagic septicemia virus. Replication first involves messenger RNA transcription from the genomic RNA via the virion polymerase. When sufficient quantities of the nucleocapsid (N) and phosphoprotein (P) have been expressed, there is a switch from transcription of

13. of mRNA to positive-sense antigenomes, which serve as the template for synthesis of negative-stranded, genomic RNA. Using virion RNA as a template, the viral transcriptase transcribes five subgenomic mRNA species. There is only a single promoter site, located at the 3’ end of the viral genome; the polymerase attaches to the genomic RNA template at this site and, as it moves along the viral RNA, it encounters stop–start signals at the boundaries of each of the viral genes.

14. Only a fraction of the polymerase molecules move past each junction and continue the transcription process. This mechanism, called attenuated transcription, results in more mRNA being made from genes that are located at the 3’ end of the genome and a gradient of progressively less mRNA from downstream genes N>P>M>G>L. This allows large amounts of the structural proteins such as the nucleocapsid protein to be produced relative to the amount of the L (RNA polymerase) protein.

16. Attachment of nucleocapsid protein to genomic RNA molecules leads to the self-assembly of helically wound nucleocapsids. Through the action of the matrix protein (M) protein, nucleocapsids are in turn bound to cell membranes at sites where the envelope spike glycoprotein is inserted. Virions are formed by the budding of nucleocapsids through cell membranes. Budding of rabies virus occurs from intracytoplasmic membranes of infected neurons, whereas the same process occurs

17. almost exclusively on plasma membranes of salivary gland epithelial cells. Vesicular stomatitis viruses cause remarkably rapid cytopathology in cell culture, whereas the replication of rabies and bovine ephemeral fever viruses is slower and non-cytopathic, because these viruses do not shut down host-cell protein and nucleic acid synthesis. Rabies virus produces prominent cytoplasmic inclusion bodies (Negri bodies) in infected cells.

19. Rabies virus can infect all mammals and infection results in death. The disease occurs throughout extensive portions of the world, although certain regions have never reported domestic rabies. • In parts of the world, bat rabies represents an unique problem, as it occurs in areas where there is no other transmission of the virus. Bats also transmit rabies-like viruses that cause sporadic cases of fatal human encephalitis.

20. Different rabies viruses can be grouped into distinct genotypes by genome sequencing or reactivity against a panel of monoclonal antibodies. These genotypes reflect the evolutionary consequence of host preference: “raccoons-bite-raccoons-bite-raccoons” and after an unknown number of passages the virus becomes a distinct genotype, still able to infect and kill other species, but transmitted most efficiently within its own reservoir host population. Thus, when spill-over does occur from a reservoir species.

21. The features of rabies are similar in most species, but there is great variation between individuals. After the bite of a rabid animal, the incubation period is usually between 14 and 90 days, but it may be longer. Human cases have 2–7 years before the onset of clinical disease. Two clinical forms of the disease are furious rabies, and dumb (or paralytic) rabies. In the furious form, the animal becomes restless, nervous, aggressive, and dangerous as it loses fear of humans and bites at anything that gains its attention.

22. The animal cannot swallow water because of pharyngeal paralysis, giving rise to “hydrophobia.” Other signs include excessive salivation, exaggerated responses to light and sound, and hyperesthesia. As the encephalitis progresses, fury gives way to paralysis, and the animal presents the same clinical picture as seen in the dumb form. Terminally, there are convulsive seizures, coma, and respiratory arrest, with death occurring 2–14 days after the onset of clinical signs.

23. The animals develop rabies depends on the dose and genotype of virus, the location and severity of the bite, and the species of animal involved. The bite of a rabid animal usually delivers virus deep into the musculature and connective tissue, but infection can occur after superficial abrasion of the skin. From its entry site, virus access peripheral nerves directly or is amplified by first replicating in muscle cells (myocytes). The virus invades the peripheral nervous system through sensory or motor nerve endings and virus binds to the receptor for the neurotransmitter acetylcholine at neuromuscular junctions.

24. Neuronal infection and centripetal passive movement of the virus within axons result in infection of the central nervous system. An ascending wave of neuronal infection and neuronal dysfunction then occurs. Virus reaches the limbic system of the brain, where it replicates extensively, leading to the fury seen clinically. Progressive spread within the central nervous system changes to the dumb or paralytic form of the disease. Depression, coma, and death from respiratory arrest follow.

26. Late in the infection, rabies virus spreads centrifugally from the central nervous system through peripheral nerves to a variety of organs including the salivary glands. In the nervous system most virus is formed by budding on intracytoplasmic membranes in the salivary glands, virions bud on plasma membranes at the apical (luminal) surface of mucous cells and are released in high concentrations into the saliva. Thus the saliva is highly infectious at the time when virus replication within the central nervous system causes the infected animal to become furious and bite indiscriminately.

27. The brains of animals with rabies exhibit variable inflammation and only modest histological evidence of neuronal injury; the presence of eosinophilic intracytoplasmic inclusions (Negri bodies) in neurons is characteristic and diagnostic, these being especially common in neurons in the hippocampus and Purkinje cells in the cerebellum. This remarkable of lethal neurological dysfunction despite minimal target destruction in many rabid animals suggests that the primary neuronal lesion is functional rather than structural.

28. Negri bodies in the neurons are characteristic of rabies, but not infrequently are difficult to identify. It is important to determine whether an animal have bitten a human is rabid. If rabies is suspected, the animal must be killed and brain tissue collected for testing. Post-mortem diagnosis most commonly involves direct immunofluorescence or immunohistochemical staining to demonstrate rabies virus antigen in frozen sections of brain tissue (medulla, cerebellum, and hippocampus).

29. Antemortem diagnosis is attempted in suspected human rabies cases using RT-PCR of saliva or immunofluorescence staining of skin biopsy or corneal impression. Only positive results are of diagnostic value. Rabies virus proteins are highly immunogenic, and different types of vaccines have been developed. Virus-specific responses are not detected in infected animals during the stage of movement of the central nervous system, because little antigen is delivered to the immune system as most is sequestered in muscle cells or within axons.

30. Infectious rabies virus is susceptible to antibody-mediated neutralization and clearance during this early stage, hence the efficacy in exposed humans of the classical Pasteurian post-exposure vaccination, when combined with the administration of hyperimmune globulin. Immunologic intervention is effective for some time during the long incubation period because of the delay between the initial virus replication in muscle cells and the entry of virus into the nervous system.

31. Oral vaccination against rabies has been used either live-attenuated or recombinant vaccines that are delivered in baits to target wildlife species. Recombinant vaccinia viruses that express the rabies virus glycoprotein have proven effective in immunizing foxes and raccoons.