Virion morphology

The lyssavirus genus includes rabies virus and ten other defined species differentiated according to their genomic sequence. Two further isolates, Shimoni bat virus (SHIBV) and Bokeloh bat virus (BBLV), are awaiting official classification (Table 58.1). First established by Louis Pasteur as a transmissible agent, the rabies virion is ‘bullet-shaped’ in appearance, with an average diameter of approximately 75 nm (60–110 nm), an average length of 180 nm (130–200 nm), and has helical symmetry (Fig. 58.1).

Table 58.1 Lyssaviruses

Fig 58.1 A pictorial representation of RABV.

(Adapted from an image designed by Dr AR Fooks, AHVLA-UK and produced by A Featherstone, Research Graphix, UK.)

Virus genome structure

The rabies virus genome is composed of approximately 12 000 nucleotides, although variations in length exist between rabies virus strains (Fig. 58.2). The genomic RNA contains all the information necessary to produce the five different viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the large polymerase protein (L). The viral ribonucleoprotein (RNP) core consists of the viral RNA encapsidated by N proteins and associated with the P and L proteins. This RNP is the minimal replicative unit for these viruses. The other viral proteins, M and G are involved in virion structure and attachment to cellular receptors, respectively. The intergenic region (G-L) is approximately 450 nucleotides in length and does not appear to encode any polypeptides. Studies on the G-L region of a wide range of RABV isolates have shown that these viruses do not have the first termination signal identified in the Pasteur virus (PV) strain but instead have a second termination signal, conserved in all RABV genomes, and closer to the beginning of the L-gene.

Fig 58.2 Schematic representation of rabies virus genome, with approximate genome sizes (in nucleotides) for representatives of 7 lyssavirus species. ∇ indicates the intergenic region (G-L).

Virus adsorption and cell entry

Infection is initiated as soon as the virus envelope glycoprotein (G) attaches to the host cell. This process of adsorption involves the interaction of the glycoprotein spikes, the major determinants for virus neuropathogenicity, with cell surface receptors. The mechanisms by which rabies virus targets cells for infection, the nature of the cell receptor(s) and the determinants of cell fusion remain unclear. Neuron-specific receptors for the virus including p75 neurotrophin receptor (p75NTR), the nicotinic acetylcholine receptor (nAChr) and the neuron adhesion molecule (NCAM), may promote efficient entry into neurons. However, it is unlikely that these are the only receptors utilised by the virus, as efficient viral replication may occur in a number of different cell lines including chick or duck embryo cells, baby hamster kidney cells, mouse neuroblastoma cells, human diploid lung fibroblasts, chick embryo fibroblasts, and Vero cells, although with minimal cytopathic effects. Therefore, cell receptors must exist on a number of different cell types that lyssaviruses can exploit and use for cell entry and replication.

Virus penetration and uncoating

After the virion has bound to its cellular receptor, viral entry (internalization) usually proceeds by fusion of the viral envelope with the cellular membrane. The virus may also enter the cell through coated pits and uncoated vesicles (viropexis or pinocytosis), which often incorporate several virions at once. Following internalization, the viral glycoprotein mediates fusion of the viral envelope with the endosomal membrane and releases the viral RNP in an endosomal vesicle. The endosome then fuses with a lysosome where the enzymes allow the ribonucleocapsid to discharge into the cytoplasm.

Transcription

The RNP complex contains all of the components required to activate viral transcription. Complete uncoating of the nucleocapsid is not necessary as the viral RNA polymerase is capable of copying the virus RNA while it is still in the RNP form. This provides the genomic RNA with protection from cellular ribonucleases. Each of the five viral proteins is encoded on individual monocistronic messenger RNAs (mRNA). These mRNAs are capped, methylated and polyadenylated by the enzymes packaged in the virion. Transcription proceeds following attachment of the polymerase complex to the genome promoter at the terminal 3′ end of the genome. The transcriptase complex then produces transcripts of each virus gene with a transcriptional gradient being produced through polymerase ‘fall-off’ at each gene boundary where intergenic gene start and stop signals exist. This results in a much greater expression of 3′ proximal genes. This transcriptional gradient is seen in many non-segmented negative strand viruses and is thought to be an evolutionary mechanism to ensure appropriate expression of each gene.

Translation

Following transcription, the individual viral mRNAs are processed using the host cell translation system. Free polyribosomes, present in the cytoplasm, translate the N, P, M and L mRNAs, and the G protein is constructed as a transmembrane molecule in the rough endoplasmic reticulum (RER), followed by transportation via the Golgi apparatus to the plasma membrane of the cell. In neuronal cells, visible changes are sometimes apparent and large accumulations of viral protein occur with the continuation of the transcription translation process. These dense areas of protein are referred to as ‘Negri bodies’.

Replication, assembly and budding

Once the mRNAs have been translated into proteins a change in composition of the viral polymerase complex occurs that leads to a switch from transcription to replication. This switch is thought to be associated with the accumulation of N protein in the cell but the exact mechanism remains unclear. Once the polymerase is in a replicative mode, the complex initially drives the production of a full-length anti-genome (positive-) sense strand of RNA, with the viral polymerase ignoring transcriptional signals present at each of the gene boundaries. The anti-genome strand serves as an intermediate for the formation of nascent genome (negative-) sense RNA.

The negative sense genomic RNA is then encapsulated by the M protein which forms a matrix surrounding the RNP core. Interactions between M and the RNP are thought to enable migration of the RNP to areas of the plasma membrane in which the glycoprotein is embedded. Condensation and coiling of the RNPs is then initiated by the matrix protein. Once associated with the glycoprotein, the condensed M-RNP forms a complete virion which buds from the plasma membrane and is released from the cell.

Rabies virus

Rabies infection in man is generally acquired from the bite of an infected animal. The domestic dog (Canis familiaris) is the most important vector, although bat rabies continues to cause human infection across much of Central and South America. Extensive vaccination campaigns in dog and terrestrial wildlife populations have reduced the incidence of rabies across the globe. However the virus still remains endemic across much of the developing world, where the majority (99%) of human deaths due to rabies occur, mainly in Africa and Asia. The World Health Organisation (WHO) estimates an annual toll of 55 000 deaths following human infection with rabies virus, although this is likely to be a gross underestimate. Despite having a devastating impact on human life, rabies infection is preventable either through pre-immunisation, or in the event of exposure, post exposure prophylaxis (PEP) involving vaccination and the administration of rabies immunoglobulin. Importantly, a high proportion of infections occur in children under the age of 16, often in poor rural areas where vaccine and PEP are not readily available.

The UK is currently classified as ‘rabies free’, with 25 human cases of rabies reported between 1946 and 2010. The majority of these cases were acquired in countries other than the UK. However, the death of a bat worker in Scotland in 2002 due to infection with an indigenous bat rabies virus, European bat lyssavirus type-2 (EBLV-2), was a notable exception. Of the human cases reported in the UK, 19 had not received any PEP, two had started PEP, and three had no known history of pre- or post-exposure vaccination.

In endemic areas, urban rabies occurs among scavenger dogs and cats living in close proximity to human populations, and poses a more immediate threat. Sylvatic rabies, which refers to the spread of disease amongst wildlife, poses less of an immediate threat to the human population. The main focus of sylvatic spread is confined to different species depending on the region in which an outbreak occurs. Examples include: fox rabies in continental Europe, Canada and northern parts of the USA; rabid racoons along the US eastern seaboard; rabies in skunk populations in the US mid-western states; rabies in coyotes in the southern states of the USA; and rabies in the mongoose populations of the West Indies and in Africa. As there is limited contact between these wild species and humans, spread is mostly to other susceptible wildlife.

Control of the spread of rabies in dogs has been achieved using surveillance projects and the initiation of vaccination programmes. These approaches have been successful in the USA and many European countries, resulting in a concomitant reduction in the number of rabies cases in humans in these countries. The red fox (Vulpes vulpes) was the principal reservoir of sylvatic rabies virus in many of these countries but oral vaccination campaigns have eliminated the virus across Western Europe and the USA. However, reintroduction from areas of endemicity in Eastern Europe and the Americas has occurred; in France in 2008 after importation of a rabid dog, and in Italy in 2008 when rabies was isolated from red foxes. Both Italy and France had been classified as rabies free since 1997 and 1998, respectively, and remained so up until these recent re-introductions.

Despite elimination of terrestrial rabies, lyssaviruses present in bat populations continue to pose a further, albeit low risk, threat to human life. In 1916, in Brazil, a number of cattle and also humans were diagnosed with rabies following bites from infected haematophagus vampire bats. Further infection was then reported in many Central and South American countries, and also in the West Indies, with considerable mortality in cattle. Numerous reports have shown that many species of insectivorous and fruit-eating bats may harbour rabies virus, with excretion and transmission occurring at different rates. Exposure appears to occur readily in some bat colonies from the close contact among large numbers congregating together, evidenced by the presence of rabies virus neutralising antibodies in bat populations.

While rabies virus has a worldwide distribution, other species appear to be restricted to particular geographic regions and hosts. Lagos bat virus (LBV), first isolated from a fruit bat colony on the island of Lagos in Nigeria, is distributed throughout Africa and has been isolated from domestic cats and dogs, but not from humans. Mokola virus, distributed across Africa, has been isolated from shrews and humans, but not from bats. Duvenhage virus (DUVV), first isolated in South Africa, has been shown to cause disease in both bats and humans.

In Europe, four related lyssaviruses exist within bat populations. European bat lyssavirus type-1 (EBLV-1) is present in Europe as two distinct genetic lineages, EBLV-1a and EBLV-1b. EBLV-1a is thought to be the most recently introduced strain from North Africa via southern Spain, and it exhibits an east–west European division. The distribution of EBLV-1b follows a north-south division. EBLV-1a and EBLV-1b have only been reported together in France and the Netherlands. Most of the EBLV-1 cases in European bats have been identified in one bat species, Eptesicus serotinus, which habituates both the UK and mainland Europe, and should be regarded as the host species for EBLV-1. European bat lyssavirus type-2 (EBLV-2) was first isolated in 1985 from a Swiss bat biologist who had been working with bats in Finland, Switzerland and Malaysia. A year later this virus was isolated from bats in Denmark (Myotis daubentonii and M. dasycneme) and from M. daubentonii in Germany, the only known natural wild hosts of this virus (apart from a single case in N. noctula). In total, there are only 19 records of this virus (17 in bats, two in humans) from Denmark, Finland, Germany, the Netherlands, Ukraine, Switzerland and the UK. Both EBLV-1 and EBLV-2 have been shown to cause disease in mammals including companion animals, wildlife and domestic livestock. EBLV-1 has been shown to cause disease in sheep, foxes, a stone marten, ferrets, cats and dogs. In contrast, EBLV-2 has not been detected naturally in mammals other than bats and humans.

West caucasian bat virus (WCBV) was isolated in 2002 from Miniopterus schreibersii bats in southeastern Europe. This is the only isolation of this virus. Antigenically this isolate represents the most divergent member of the lyssaviruses, and shows a lack of serological cross reactivity to the other lyssavirus species. Bokeloh bat virus (BBLV) a new ‘putative’ bat lyssavirus isolated in Bokeloh, Germany, in 2010 from a Myotis nattereri bat, has yet to be classified.

Virus transmission

The inability of rabies virus to penetrate intact skin means that it has to gain entry to the body through broken skin (often as a result of a biting incident) or through the mucous membranes (eyes, nose and mouth). In many developing countries, where canine immunisation programs are minimal or non-existent, the predominant source of human infection is from dogs (99% of cases worldwide). In regions of the world where canine rabies has been controlled by vaccination, wild animal bites represent the main source of human infection. Bat bites are also a source of concern throughout the world, as these bites are often small and may go unnoticed.

There have also been reports of rabies infection due to aerosolised virus, either following potential exposure to virus in caves containing large numbers of potentially infected bats, or following accidental laboratory exposure. Experimental aerosol transmission has been reported using a murine model. Transfer of infection via infected corneal transplants has been reported on at least eight occasions in five different countries. A number of organ recipients have acquired rabies having received organs from infected donors. In 2004 in the USA, transplantation of kidneys, liver and an arterial segment from a donor who died from unexplained encephalitis, resulted in the deaths of the transplant recipients from rabies virus. A history of bat bite was subsequently identified in the donor. A similar situation occurred in Germany in 2005. Even though virus has been shown to be excreted in both saliva and conjunctival exudates, direct transmission of virus from person-to-person spread is extremely rare, although possible transmission from mother-to-child has been reported.

Diagnostic methods

Vaccination

It was the French scientist Louis Pasteur who first introduced vaccination following rabies virus exposure. The original vaccine was a crude extract of infected rabbit spinal cord, which had been desiccated and passaged several times to produce a ‘fixed’ strain of the virus. The success of this vaccination regime in cats and dogs was announced at the Academies des Sciences in 1885. The successful treatment of a young boy who experienced multiple bites from a rabid animal subsequently established this approach. The Semple vaccine, a modification of Pasteur’s original vaccine, was a suspension of 4% inoculated rabbit brain attenuated with 5% phenol. This was used in the UK from 1919–1966, but due to the high rate of allergic encephalitis following its administration, was replaced by a duck embryo-derived vaccine in 1966. This was not produced in neuronal cell-lines and there was a decrease in the incidence of adverse reactions following its introduction. This type of vaccine has since been replaced by cell culture-derived inactivated vaccines, several types of which are currently available, including: human diploid cell vaccine (HDCV), purified chick embryo cell vaccine (PCEV) and Vero cell vaccine. While each of these current vaccines has proven to be both safe and immunogenic, they are not necessarily available in developing countries, and they do not protect against all lyssavirus species.

Pre-exposure prophylaxis

In the UK, the HDCV and PCEV vaccines are licensed and available. These are normally administered to those who require pre-exposure vaccination, e.g. laboratory staff working directly with the virus. For those people travelling to rabies-endemic countries, pre-exposure vaccination may be recommended depending on the local incidence of rabies in the country to be visited, the availability of appropriate anti-rabies biologicals, and the intended activity and duration of stay of the traveller. The protocol requires administration of three intramuscular doses of vaccine at days 0, 7 and 28, following which the levels of neutralising antibody are assessed using a certified test. Response to these vaccines can persist for at least two years post-vaccination. These vaccines are safe and well tolerated, with adverse reactions being extremely rare.

There are a number of different animal vaccines available, mostly live-attenuated preparations that would not be acceptable for use in humans. The animal is generally inoculated intramuscularly, with boosters required every 1–3 years depending on the vaccine type.

Post-exposure prophylaxis

Wound cleansing and immunizations, undertaken as soon as possible after suspected contact with an animal and following WHO recommendations, can prevent the onset of rabies in virtually 100% of exposures, although some exceptions to this have been recorded. The wound should be washed immediately with copious amounts of water and preferably cleansed with soap. Alcohol, quaternary ammonium compounds, iodine or povidone should be used, if available. The level of treatment or care required depends on the category of the contact as outlined by the WHO (category 1, 2 or 3, see Table 58.2). Post-exposure anti-rabies vaccination should always include administration of both passive antibody (human rabies immunoglobulin, HRIG) and vaccine in patients without any history of rabies pre-vaccination, for both bite and non-bite exposures, regardless of the interval between exposure and initiation of treatment. HRIG provides immediate availability of neutralising antibodies at the site of exposure before the patient elicits an endogenous antibody response following vaccination.

Table 58.2 WHO categories of rabies exposure

Depending on vaccine type, the post-exposure schedule requires four to five intramuscular doses over four weeks. According to the WHO, two intramuscular doses of a cell-derived vaccine separated by three days are sufficient for rabies-exposed patients who have previously undergone complete pre-vaccination or PEP with cell-derived rabies vaccines. Rabies immune globulin treatment is not necessary in such cases.

There have been seven reports of recovery from rabies once clinical signs have developed, five of whom had received pre-exposure vaccination, and two of whom had no pre-exposure vaccination, but were treated according to variations of the Milwaukee protocol. The first was a 15-year-old girl who developed clinical rabies one month after she had been bitten by a bat. A ‘therapeutic’ coma was induced allowing an immune response to be elicited. A cocktail of drugs including ketamine, midazolam, ribavirin and amantadine was then used as part of the treatment regimen. Lumbar puncture after 8 days showed a raised level of anti-rabies antibodies and the girl was brought slowly out of the coma. Signs of paralysis and sensory denervation were observed. After nearly 5 months following initial hospitalisation she was alert and communicative but with involuntary movements, difficulty speaking and an unsteady gait. The second was reportedly in Brazil in 2008; a young boy was treated following a similar protocol and survived. Similar regimens have been attempted for at least 12 other rabies patients without success.