Rabies☆

WH Wunner, The Wistar Institute, Philadelphia, PA, USA

ã 2014 Elsevier Inc. All rights reserved.

Defining Statement 2
Rabies and Rabies-Related Viruses 3
Virus Structure 3
Viral Protein Function 4
Viral Genome Structure 7
Gene Transcription and Genome Replication 7
Virus Life Cycle 7
Epidemiology of Animal and Human Rabies 8
Control and Prevention of Rabies in Domestic and Wildlife Animals 11
The Blueprint for Rabies Prevention and Control 12
Human Rabies 12
Clinical Signs and Symptoms 12
Rabies Diagnosis 13
Rabies Pathogenesis and Histopathology 14
Rabies Vaccine Prophylaxis 16
First Line of Protection Against Rabies – Preexposure Prophylaxis 16
First Line of Defense After Exposure to Rabies – Postexposure Prophylaxis 17
Second Line of Defense: Establishing Active Adaptive Immunity 17

Glossary Negri body Cytoplasmic matrix composed of rabies viral

Acinus A minute terminal lobule in the salivary glands.
Apoptosis A process by which cells undergo a natural
programmed cell death or physiological cell death in
response to diverse stimuli.
Encephalomyelitis Acute demyelinating disease caused by
virus without invoking an immunopathologic mechanism.
Epitopes Structural sites on proteins that bind antibodies
and T-cell receptors.
Fixed rabies virus Laboratory strains that grow in tissue
culture at a predictable rate, with predictable biological
properties, and regularly kill inoculated animals within a
predictable period.
Guillain–Barré syndrome An acute inflammatory
demyelinating polyradiculoneuropathy that in the majority
of cases follows a ‘virus-like’ illness.
Hydrophobia The unique characteristic of rabies involving
both severe thirst, accompanied by a desire to drink and at
the same time intense fear of water, which is regarded as the
most specific manifestation of clinical rabies in humans.
Leptomeninges The two innermost layers of the meninges;
cerebrospinal fluid circulates between these innermost
layers.
Lyssavirus Rabies virus and variant viruses known as
‘rabies-related’ viruses, which share with rabies virus the
unique capability to produce a rabies-like
encephalomyelitis.
nucleocapsid proteins.
Nodes of Ranvier Tiny gaps formed between myelin
sheaths that wrap and insulate axons, which themselves (the
gaps) are not insulated allowing them to generate electrical
activity and pass nutrients and waste into and out of
neurons.
Rhabdoviridae Taxonomic family of rhabdoviruses in the
order Mononegavirales that distinguishes itself as
identifying with bacillus or ‘rod- or bullet-shaped’ virus
particles, including Vesicular Stomatitis virus as the
prototypical member of the Vesiculovirus genus and Rabies
virus as the prototypical member of the Lyssavirus genus.
Street rabies virus Virus isolated from natural infection that
has not been serially passaged in animals or tissue culture.
Superantigen A class of proteins that bind class II major
histocompatibility proteins and stimulate powerful
non-specific proliferation of T-cells bearing particular Vb
sequences as part of their ab antigen receptor.
Viropexis Entry of virus particles into cells by engulfment,
followed by fusion of membrane-bound vesicles with
lysosomes.
Xoplasmic transport Bi-directional (anterograde or
retrograde) flow of proteins, particles, and organelles along
the length of the neuronal cell’s axon or nerve track to reach
intraneuronal destinations.

☆
Change History: September 2014. WH Wunner, updated some of the text, added new Figure 6 and updated Figure 4 and added new references.

Reference Module in Biomedical Research http://dx.doi.org/10.1016/B978-0-12-801238-3.02517-4 1

2 Rabies

Abbreviations LC8 Light chain 8

ABLV Australian bat lyssavirus Le Leader
ACIP Immunization Practices Advisory Committee MAb Monoclonal antibody
AGP Antigenome promoter MIT Mouse inoculation test
ARAV Aravan MOKV Mokola virus
BBLV Bokeloh bat lyssavirus Mr Relative molecular mass
BSR A clone of BHK (baby hamster kidney) cells mRNA Messenger ribonucleic acid
CCV Cell culture-based vaccine nAChR Nicotinic acetylcholine receptor
CD Cytoplasmic domain NC Nucleocapsid
CD44 Cell determinant 44 (a cell marker protein) NCAM Neural cell adhesion molecule
CNS Central nervous system NTR Neurotrophin receptor
cRNA Complementary (antigenome) RNA nts Nucleotides
CRU Cellular receptor unit NTV Nerve tissue vaccine
CSF cerebrospinal fluid ORF Open reading frame
CTL Cytotoxic lymphocyte PCECV Purified chick embryo cell vaccine
CVS Challenge virus standard PEP Postexposure prophylaxis
DFA Direct fluorescent antibody PET Postexposure treatment
DNA Deoxyribonucleic acid PNS Peripheral nervous system
DUVV Duvenhage virus PrEP Preexposure prophylaxis
EBLV-1 European bat lyssavirus type 1 PRP Partners for rabies prevention
ED Ectodomain RABV Rabies virus
EM Electron microscope RIFFIT Rapid fluorescent focus inhibition test
ER Endoplasmic reticulum RIG Rabies immunoglobulin
ERA Evelyn–Robitnicki–Abelseth RNA Ribonucleic acid
ERIG Equine rabies immunoglobulin RNP Ribonucleoprotein
GP Genome promoter rNTPs Ribonucleoside triphosphates
HDCV Human diploid cell vaccine RT-PCR Reverse-transcription polymerase chain
HEP High egg passage reaction
HRIG Human rabies immunoglobulin SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel
hsp70 Heat-shock protein 70 SHIBV Shimoni bat virus
IB Inclusion body TM Transmembrane
ID Intradermal(ly) Tr Trailer
IFA Indirect fluorescent antibody TUNEL Terminal deoxynucleotide transferase dUTP
IFN Interferon nick end labeling
IKOV Ikoma lyssavirus VAP Viral attachment protein
IM Intramuscular(ly) VNA Virus-neutralizing antibody
IRKV Irkut vRNA Viral ribonucleic acid or genome RNA
IU International unit VSV Vesicular stomatitis virus
KHUV Khujand WCBV West Caucasian bat virus
LBV Lagos bat virus

Defining Statement

Rabies is an acute neurologic disease caused by a lyssavirus (of the Rhabdoviridae family) that attacks the central nervous system of
all warm-blooded animals and humans and is normally a fatal viral infection. The first written record of the disease dates to twenty-
third century BC, and in fourth century BC Plato used the word ‘Lyssa’ to describe the ‘erotic passion’ or ‘madness’ (viciousness)
associated with the disease at the time. Outbreaks of rabies in Europe were rare until the Middle Ages, when rabies epizootics
occurring in dogs, rabid wolves, and foxes were reported in many countries. By the 1700s, rabies was common throughout Europe,
particularly in central Europe, and was beginning to be reported in the New World.
Rabies virus (RABV) is a highly neurotropic virus in infected animals and humans. Virus infection in humans causes alternating
symptoms of depression and agitation and almost invariably leads to extreme types of violent behavior and death. Hydrophobia is
a unique characteristic of rabies in humans. RABV is transmitted to humans from wild and domestic animals, through bites and
deposition of virus-laden saliva. The predominant animal reservoir for RABV continues to be in dogs, which causes a sizable clinical
problem for humans, especially in those countries, mainly of Asia and Africa and to a lesser extent in Central and South America,
where canine rabies is largely uncontrolled.
 Rabies 3

Rabies and Rabies-Related Viruses

Rabies virus (RABV) is the prototype virus of the genus Lyssavirus in the family Rhabdoviridae. RABV, which is distributed worldwide,
is a highly neurotropic virus in infected animals and humans, invariably causing a fatal encephalomyelitis, commonly referred to as
rabies. Until 1956, RABV was considered the only virus associated with the oldest known disease, recognized in ancient times (as early
as the twenty-third century BC) as ‘mad dog’ disease. Variants of RABV, known as ‘rabies-related’ or non-RABV lyssaviruses, have since
been identified after the initial discovery of two rabies-related viruses originating from Africa: Lagos bat virus (LBV) and Mokola virus
(MOKV) in 1956 and 1968, respectively. Rabies-related viruses share with RABV the unique ability to elicit the clinical disease of
rabies, producing a rabies-like encephalomyelitis, and also share other RABV structural characteristics. There are now fourteen
recognized lyssaviruses, 12 of these have been differentiated into three phylogroups based on phylogenetic analyses. RABV (formerly
identified as genotype 1) belongs to phylogroup I along with Duvenhage virus (DUVV) (genotype 4), European bat lyssavirus, type
1 (EBLV-1) (genotype 5), and type 2 (EBLV-2) (genotype 6), and Australian bat lyssavirus (ABLV) (genotype 7). Three putative species,
Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), and Bokeloh bat lyssavirus (BBLV) the newest of species to be
characterized are considered independent species of phylogroup I. Phylogroup II includes LBV (genotype 2), MOKV (genotype 3), and
Shimoni bat virus (SHIBV), the newest of species to be characterized and considered an independent species within phylogroup
II. West Caucasian bat virus (WCBV) and the Ikoma lyssavirus (IKOV), isolated from an African civet, which do not cross-react
serologically with any members of the two phylogroups, could tentatively belong to a third phylogroup (phylogroup III). Phyloge-
netic analyses suggest that all lyssaviruses originate from a precursor bat virus.

Virus Structure
RABV and the non-RABV lyssaviruses belong to the class of RNA viruses that contain a linear, single-strand, negative-sense
(noninfectious) RNA genome that is approximately 12 kb or 12 000 nucleotides (nts) in length (Mr 4.2
106). The viral genome
(2–3% of the virion weight) forms the backbone of the tightly coiled helical RNP (ribonucleoprotein/RNA plus protein) core,
which extends along the longitudinal axis of the bullet-shaped virus particle (Figure 1). The RNA genome contains five genes that
code for the five structural proteins of the virion, which are designated N (nucleoprotein), P (phosphoprotein), M (matrix protein),
G (glycoprotein), and L (RNA transcriptase/polymerase or large protein). The five genes are organized in the genome in order,
starting at the 3’ end, N, P, M, G, and L. The first four genes are separated by short (di- or penta-) intergenic nt stretches (N–P, P–M,
and M–G) and a longer 19–28-nt sequence between the last L gene and the G gene (Figure 2). The full nucleotide sequence of the
RNA genome includes a noncoding leader (Le) sequence at the 3’ terminus and a noncoding trailer (Tr) sequence at the 5’ end.
These short (58–70 nt) sequences provide specific cis-acting signals (a specific nucleotide sequence ‘acting within’ the genome
RNA) to initiate specific functions in genome RNA transcription and replication.
The five structural proteins of RABV (as well as non-RABV lyssaviruses) comprise 67–74% of the virion by weight. The three viral
proteins associated with the viral RNP core include the N (the most abundant protein in the RNP core) and the noncatalytic
polymerase-associated P and the catalytic L components of the RNA polymerase complex. All three proteins are involved in the
RNA polymerase activity of the virion, which generates monocistronic mRNA transcripts of each of the viral protein genes and a
full-length complementary (plus-sense) transcript of the viral genome that serves as a template to produce a progeny (negative-
sense) RNA genome. The remaining two structural proteins of RABV, G and M, are associated with the lipid bilayer envelope that
surrounds the RNP core. M is a small-size nonglycosylated protein that lines the viral envelope forming an inner leaflet between the
envelope and the RNP core. G is the only glycoprotein. It forms the trimeric spikes on the outer surface of the viral envelope. The
mature viral G is glycosylated with branched-chain oligosaccharides, which account for 10–12% of the total mass of the protein.
There are approximately 450 trimeric spikes distributed on the outer surface of each virion. Other host-derived minor protein
components such as actin and heat shock proteins of the hsp70 type, CD44 and CD99-related glycoprotein (VAP21), are also
found in rabies virions. The role of these proteins is unclear; it is possible that some of them act to chaperone the viral proteins
during synthesis and are incorporated into virions after binding to and assisting in G folding. Cytoskeletal proteins normally
expressed on the host cell surface may also be incorporated into virions as a consequence of their proximal location and function in
virus budding. Some of these proteins ensure intracellular transport of viral RNP along the pathogenetic pathway of the virus.
A lipoprotein bilayer that forms the viral envelope around the helical RNP core is therefore a mixture of viral and host proteins and
lipids, which constitute 20–26% of the viral envelope.
RABV particles or virions are best described as bacilliform or bullet-shaped with one end rounded (hemispherical) and the other
end flattened (planar) (see Figure 1). The physical appearance of this rigid rod-like, bullet-shaped virion was first described from
images seen using the electron microscope (EM). The average length of standard-size, infectious virions is 180 nm (130–250 nm)
and the average diameter is 75 nm (60–110 nm). Besides standard-size virions, other shorter, often cone-shaped virions are
sometimes observed. These are defective virions that have genomes approximately one-third to one-half the size of the full-
length genome of standard-size virions. These shorter genomes are the result of internal nucleotide sequence deletions that occur
during virus replication, particularly in virus-infected cell cultures.
4 Rabies

Figure 1 Schematic representation of the rabies virion. The drawing shows the internal ribonucleoprotein (RNP) core consisting of the single-strand,
negative-sense genome RNA encapsidated with nucleocapsid protein (N), the virion-associated RNA polymerase (L), and polymerase cofactor
phosphoprotein (P). The RNP core in association with the matrix protein (M) is condensed into the typical bullet-shaped particle that is characteristic of
rhabdoviruses. A lipid bilayer envelope (or membrane) in which the surface trimeric glycoprotein (G) spikes are anchored surrounds the RNP-M
structure. The membrane ‘tail’ depicted in the drawing represents the trailing piece of envelope that is frequently observed attached to the virus as it buds
from the plasma membrane of the infected cell when viewed under the electron microscope (EM). Reproduced from Wunner, W. H., Larson, J. K.,
Dietzschold, B., Smith, C. L. (1988). The molecular biology of rabies viruses. Review of Infectious Diseases 10(Suppl. 4): S771–S784, with permission.

Figure 2 Organization of the rabies virus (RABV) RNA genome. The nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and
large RNA-polymerase protein (L) genes (orange) are separated by four intergenic nucleotide sequences and flanked by the leader (Le) RNA and trailer
(Tr) RNA sequences (green) at the 30 and 50 termini, respectively. Numbers shown above the five genes and below represent nucleotides per gene and in
the Le, Tr, and intergenic nucleotide sequences, of the RABV Evelyn–Robitnicki–Abelseth (ERA) strain. Reproduced with modification from Wunner,
W. H. (2007). Rabies virus. In: Jackson, A. C., Wunner, W. H. (eds.) Rabies, 2nd edn., pp. 23–68. San Diego, CA: Elsevier, Academic Press; From Wunner
and Conzelmann (2013) Rabies virus. In: Jackson, A.C. (ed.) Rabies, 3rd edn., pp. 17–60. San Diego, CA: Elsevier, Academic Press, with permission.

Viral Protein Function

The nucleoprotein (N; 450 amino acids) is the most abundant of the viral proteins produced in RABV-infected cells. The model for
rhabdovirus genome transcription proposes that the viral RNA polymerase will produce the greatest number of transcripts (mRNA
copies of a gene) from the gene closest to the 30 end of the RNA genome. A gene that is closer to the 30 end of the genome is
transcribed more frequently than the next gene and so on along the genome. One of the primary functions of N is to ‘encapsidate’
the nascent (newly synthesized) viral RNA that is made during virus replication, which protects it from ribonucleases that degrade
naked single-stranded RNA in the cell. The most conserved amino acids of N stabilize certain key protein-RNA interactions. N is
also the major protein of cytoplasmic inclusion bodies (Negri bodies) that are prominent as morphologically distinct lesions found
in infected neurons of the diseased brain (‘rabies pathogenesis and histopathology’). Although the amino acid sequence of N is the
most conserved of the viral proteins among the lyssaviruses, the noted amino acid differences provide unique, genotype-specific
epitopes on the N making it a useful protein for comparative antigenic analysis and genetic typing based on its nucleotide sequence
in diagnostic and phylogenetic analyses. Determination of a stretch of nucleotide sequence is a commonly applied method for
comparing a collection of lyssaviruses and allowing by sequence alignment a direct comparison of virus isolates to reference
sequences leading to the phylogenetic classification of a particular virus.
 Rabies 5

N is the second most extensively analyzed of the RABV proteins (after G) with respect to its antigenic and immunogenic
structure and function. The RNP of RABV induces protective immunity against peripheral challenge, with lethal RABV (infection by
a virulent form of RABV) in animals due to dominant B- and T-cell-specific epitopes (amino acid regions) on N. Three antigenic
epitopes (antibody binding sites) on N have been mapped to amino acids 358–367 (antigenic site I) and three linear epitopes
(antigenic site IV) have been mapped to two independent regions, amino acids 359–366 and 375–385. The apparent paradox that
N appears to have two independent antigenic sites (I and IV) recognized by antibodies that do not compete with each other for
binding to the N antigen suggests that the two overlapping epitopes are recognized independently on different forms of N. The
paradox was resolved when it was discovered that N, which is diffusely distributed in the cytoplasm and available to encapsidate
RNA, represents one form and the N that is associated with cytoplasmic inclusion bodies represents the other form. N is also the
major target for T-helper (Th) cells that cross-react among rabies and rabies-related lyssaviruses. N has other properties that enable
it to function as an exogenous viral ‘superantigen’, and is perhaps the only viral superantigen that has been identified in humans.
Some of the properties include its potent activation of peripheral blood lymphocytes in vaccinated humans and its ability to
produce a more rapid and heightened virus-neutralizing antibody (VNA) response upon injection of inactivated rabies vaccines.
N is also capable of inducing early T-cell activation steps and expansion and mobilization of CD4+ Vb8T-cells to trigger and support
production of VNA and bind to HLA class II antigens expressed on the surface of cells.
Phosphoprotein (P; 297 amino acids), which is derived from the second gene (next after the N gene) in the RABV and
non-RABV lyssavirus genome, is a multifunctional protein and a key component and regulatory protein of the virion-associated
RNA polymerase complex in viral genome replication. In the cell, one of the functions of P is to act as a chaperone specifically
directing N to encapsidate viral RNA, by interacting with newly synthesized (nascent) soluble N (N ) to maintain N in a
competent form for RNA encapsidation, preventing N from polymerizing (self-aggregating) and nonspecifically binding to
cellular RNA. P also plays a pivotal role as a noncatalytic cofactor in the transcription and replication of the viral genome.
It serves to stabilize L and place the polymerase complex on the RNA template, which L alone is unable to do. These two functions
of P are accomplished via domains of P that are specific for each of these two proteins. P is present in the infected cell in a form that
is distinguishable from its presence in the virion and these forms can be differentiated by their degree of hypo- to hyperpho-
sphorylation when resolved by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Phosphate groups are
added to RABV P by two distinct protein kinases, one of which phosphorylates the protein at serine (S)63 and S64 in the N-terminal
portion of the molecule altering the migration of P in SDS-PAGE. The other kinase, which appears to be selectively packaged in
mature virions, does not alter the migration of P in SDS-PAGE. Other protein–protein interactions that involve RABV P are equally
important, such as its interaction with cellular proteins that influence or help regulate virus tropism and cell-to-cell spread. Dynein
light chain 8 (LC8) is a cellular protein, part of cytoplasmic dynein and the myosin V microtubule-associated motor protein
complex that facilitates the transport of RABV through neurons. Other interactions include the inhibition of innate immune
responses, such as the interferon (IFN) response, that typically prohibit productive virus replication.
The rabies virion-associated RNA polymerase or large protein (L; 2142 amino acids) is the catalytic component of the
polymerase complex of RABV. It is encoded in the fifth or most distal gene from the 30 end of the RNA genome, which accounts
for more than half, 54%, of the viral genome-coding potential. Along with noncatalytic P, L is responsible for the majority of
enzymatic activities involved in viral RNA transcription and replication. The virion-associated RNA polymerase has the unique
function at the start of infection to initiate the primary transcription of the genome RNA after the RNP core is released into the
cytoplasm of the infected cell. The transcripts, which include the 30 Le RNA and monocistronic mRNAs of the N, P, M, G, and
L genes, are initiated, elongated, and in the case of mRNAs cotranscriptionally modified to include 50 capping, methylation, and 30
polyadenylation. These enzymatic activities that are assigned to L are identified with but not precisely mapped within one (block
III) of four blocks of highly conserved amino acid sequences distributed noncontiguously in L. Block III, the catalytic domain,
between amino acid residues 530 and 1177 in the central part of the primary sequence of L, consists of four motifs (A–D) that
represent regions of highest similarity and are thought to constitute the polymerase module of L. These structurally conserved
motifs are engaged in the transcriptional activity of L that requires binding of substrate ribonucleoside triphosphates (rNTPs),
polyadenylation, and protein kinase activity for specific phosphorylation of the P in transcriptional activation. At least two other
consensus sequences bind ATP that is utilized in the essential activities of L.
The matrix protein (M; 202 amino acids) of RABV and the non-RABV lyssaviruses is derived from the third gene in the RNA
genome. It is the smallest of the virion proteins. There are two isoforms of M: a major form, Ma, which accounts for 70–75% of
total M in lyssa virions, and a minor form, Mb, which accounts for the rest of M in the virion, but only 10–15% of total M in the cell.
M plays a critical role in the morphogenesis of lyssa virions as it binds to and condenses the nascent RNP complex (nascent virus
core) into a tightly coiled, helical RNP-M structure, forming a sheath around the RNP core. This structure, which assumes a ‘bullet’
shape, is referred to as the virus ‘skeleton.’ At the time that M binds to the RNP core, it also interacts with the host plasma membrane
at the marginal region of the cytoplasm where it initiates the virus budding process from the plasma membrane. The virus budding
process appears to be triggered by a proline-rich motif (PPEY), referred to as a ‘late-budding domain’ (L-domain), located within
the highly conserved 14-amino acid sequence near the N-terminus of the RABV M. Similar proline-rich motifs identified in other
types of budding viruses are also associated with virus budding from the plasma membrane. While the release of virus particles
from the plasma membrane of infected cells requires the M of the RNP-M skeleton structure, the efficiency of virus budding is
greatly enhanced by the interaction of the skeleton structure with the viral envelope glycoprotein (G).
The glycoprotein (G; 505 amino acids) of RABV, like non-RABV lyssaviruses, is translated from mRNA that is transcribed from
the fourth gene in the RNA genome. The G gene encodes a 524-amino acid protein, which includes a signal peptide of 24 amino
6 Rabies

acids at the N-terminus that is posttranslationally cleaved before the mature 505-amino acid G is integrated into the envelope of the
virion. G is a type I glycoprotein, which indicates that the protein is anchored in the membrane envelope by a transmembrane
(TM) domain located near the C-terminus of the protein. The short C-terminal portion (22 amino acids) of G that remains beneath
the membrane (toward the cytoplasm) is the cytoplasmic domain (CD) and the large N-terminal portion (439 amino acids) of the
mature G, the ectodomain (ED), extends outward from the membrane surface of the infected cell and released virion. The CD of
G interacts with M of the skeleton particle to complete virion assembly and enables efficient release of virions. During posttrans-
lational modification (glycosylation) of G in the endoplasmic reticulum (ER) and Golgi apparatus, three molecules of G form
trimers (aggregates of nascent G molecules presumably held together at the TM domain), which later become the trimeric spikes on
the surface of the virus. The ED of each molecule in the trimeric spike is responsible for virus binding to cell surface receptors and
the entry of virus into cells in the early phase of the RABV life cycle and for induction of anti-rabies VNA, when engaging the host’s
defense against the virus.
Glycosylation of RABV G is important for its expression on the membrane surface and proper function. Complex carbohydrates
(oligosaccharides) are linked to asparagine residues in the tripeptide sequence (sequon) asparagine-X-serine or asparagine-X-
threonine, where X is any amino acid except proline. While RABV G can have one, two, or three (or sometimes four) sequons
(N-glycan sites) per molecule, not all sites are occupied on all molecules, which depends on various factors that influence efficiency
of glycosylation. At least one site (N319 in RABV G) is required, however, and it is a sequon that is highly conserved in all the
lyssavirus genotypes and other rhabdoviruses such as vesicular stomatitis virus (VSV). The sugar residues of N-glycan are
presynthesized in the cell and transferred from a lipid precursor by the enzyme oligosaccharyltransferase as a precursor core
oligosaccharide unit (Glc3Man9GlcNAc2) to the specific sequons of the nascent G molecule, as the protein is processed in the
lumen of the ER and Golgi apparatus. There the high-mannose core oligosaccharide unit is trimmed with the help of the chaperone
calnexin, which assists in the correct folding of G to achieve its biological activity, stability, and antigenicity.
A key biological role of G is its ability to facilitate virus entry into host cells after targeting the binding of the virus to its
receptor(s) on host cells. Acting as a fusion protein, the lyssavirus G enables the virus to become internalized as it triggers a low
pH-dependent fusion with the plasma membrane and endosomal membranes to enter cellular endosomes. During this process,
G assumes at least three structurally distinct ‘conformational’ states. The G on the virion surface assumes the ‘native’ state prior to
the virus binding to the cellular receptor. After the virus attaches to the receptor and is internalized, the G on the virion surface is
‘activated’ to a hydrophobic state, enabling it to interact with the hydrophobic endosomal membrane. After entering the
endosomal compartment and its low-pH environment, the fusion capacity of G is further activated via a major conformational
change in the G that exposes the fusion domain, which interacts with and destabilizes one or both of the participating membranes.
Once activated to fuse with endosomal vesicles, the viral uncoating process begins; at pH 6.2–6.3, G assumes a reversible ‘fusion-
inactive’ conformation, which makes the G monomer appear longer than the ‘native’ form and assume selective antigenic
distinctions and be highly sensitive to cellular proteases. RABV spreads from cell to cell in two ways. It either buds from one
infected cell into the culture medium (in vitro) or extracellular space (in vivo) to infect another cell by attaching to the next cell’s
surface receptor or spreads directly from cell to cell without budding into the culture medium or extracellular space. The precise
method of direct cell-to-cell spread of virus in vitro and in vivo has not been determined. But the fact that the virus can enter a cell
without first attaching to the cell surface receptor in cell culture (direct cell-to-cell spread) points to the importance of the
pH-dependent and pH-independent fusion functions of G in virus spread.
Another important biological role of G is its ability to define the pathogenicity of the virus and determine the neuroinvasive
pathway the virus takes to reach the central nervous system (CNS) and the brain, and cause a lethal infection. The RABV and
non-RABV lyssavirus strains are defined by their ‘neurotropism’ as having a pathogenic (virulent) or nonpathogenic (avirulent)
phenotype. The virulent virus causes a lethal infection, while the avirulent virus does not cause a lethal disease. The virulent and
avirulent phenotypes are often defined by a single amino acid substitution in G. The position most often associated with a single
amino acid substitution causing the change from virulent to avirulent phenotype is the position 333 of the 505-amino acid
sequence. Normally, arginine occupies position 333 in the G of the virulent phenotype, while glutamine or lysine occupies the
same position in the G of the avirulent phenotype. Although substitution of other amino acids at position 333 can make this
transition in phenotype happen, there are still other identifiable factors that can play a role in the pathogenicity of RABV and
non-RABV lyssaviruses. These may include the transcription activity of L, the length of the long intergenic nucleotide sequence
between the protein-coding or open reading frame (ORF) regions of the G and L genes, and optimal protein–protein (e.g., G–M)
interactions. Nevertheless, at least with ‘fixed’ strains of pathogenic and avirulent RABVs that distinguish themselves by having an
arginine and glutamine at position 333 of their G, respectively, all else being the same, it has been shown that the two phenotypes
invade the CNS from a peripheral site at different rates. It is clear that not only does the G allow entry into the nervous system by
selecting the appropriate receptor it facilitates and enhances retrograde axonal transport of the virus to the CNS. By selecting
different neuronal pathways to the brain from a peripheral site of inoculation, it is also clear that the pathogenic virus reaches the
brain faster than the avirulent phenotype. Still, other studies have found that the two virus phenotypes showed no difference in
the rate of speed and pathway of spread to the CNS. So, it remains controversial and requires further investigation into whether the
viruses, depending on their amino acid constituents in the G and phenotype, use different receptor sites on the cell surface or use
the same receptor sites with different efficiencies in determining the neuronal pathways for the viruses.
Finally, RABV G is highly immunogenic and of major importance for the induction of VNA and T-cell-mediated immunity, two
of the host’s adaptive immune responses against virus infection. G induces VNAs that recognize both conformational and linear
antigenic epitopes and stimulates helper as well as cytotoxic T-cell activity. The various epitopes that bind VNAs delineate a total of
 Rabies 7

eight antigenic sites that have been physically mapped (located) on the ED of G. Epitopes that bind T-cells have also been mapped
on G. To visualize and understand the precise topography of the antibody and T-cell binding sites on the G, it will be necessary to
study the three-dimensional structure of the antigen (derived from G crystals). So far, this has denied investigators, who have made
numerous attempts to crystallize the RABV G for X-ray diffraction analysis, any success.

Viral Genome Structure

The linear single-strand negative-sense RNA (vRNA) of lyssavrus genomes of about 12 kb in length includes the 5 genes that appear
in a strictly conserved order 30 -N-P-M-G-L-50 flanked by the short 30 - and 50 - terminal regulatory extragenic regions known as leader
(Le) and trailer (Tr) regions. These two extragenic regions are terminated by unmodified 30 -hydroxyl (30 -OH) and 50 -triphosphate
(50 -PPP) ends, respectively (Figure 2). The Le region, acting as a genetic promoter (GP), initiates transcription of five monocistronic
mRNAs from the genes of the vRNA genome as well as the synthesis of full-length antigenome RNA (cRNA). The 30 -end of the cRNA
functions as the antigenome promoter (AGP) that directs synthesis of full-length N-encapsidated vRNAs representing synthesis of
progeny vRNAs in viral genome replication. Much as recently been learned from X-ray diffraction studies of crystal ring structures of
helical N-viral RNA (N-vRNA) or RNP complexes containing as few as 10 N molecules how the genome vRNA is encapsidated by
N. One should not lose site of the role that P plays in directing N encapsidation of vRNA (see description of P above) and that an
N-P complex is involved in vRNA encapsidation process. The vRNA in these structures is sequestered in a cavity at the interface
between the N- and C-terminal lobes of N that seem to ‘clamp down’ onto the bound RNA. The complete occlusion of the vRNA by
N (N-vRNA) explains the excellent protection of the vRNA against high salt, attack by RNases, and silencing by siRNAs. During
transcription and replication of the viral genome RNA, the tight clamping of the N-vRNA interaction opens exclusively and
transiently to allow the polymerase complex access to the vRNA. Easiest access to the N-vRNA genome would be at the 30 -end of the
genome as well as the 30 -end of the antigenome (N-cRNA) to allow transcription and replication of the downstream copying of
successive genes to occur.

Gene Transcription and Genome Replication

The Le RNA and mRNAs of the five genes are sequentially transcribed off the N-vRNA genome according to the widely accepted
stop/start model of transcription for all single-strand (non-segmented) negative-sense RNA viruses. The first transcript initiated by
the 30 -terminal GP is the Le RNA, however recent studies suggest that transcription of the downstream protein-encoding genes
starting with the N gene can proceed independent of Le RNA synthesis. Directing the activity of the viral RNA polymerase, acting as
a transcriptase, are conserved stop/start signals separated by non-transcribed intergenic sequences at the gene borders that control
the observed transcription. A short run of mostly 7 U residues signal the polymerase to stop transcription while the polymerase
continues to move back and forth on the N-vRNA template to synthesize a 50–150 nt-long poly(A) tail that is added onto
the mRNA. Without fully separating but purposely dissociating from the N-vRNA template at the gene borders, it is thought that
the polymerase releases the newly synthesized polyadenylated mRNA and re-associates with the template to initiate transcription of
the next downstream gene at the restart signal without transcribing the border signals between sequential genes. Since the
polymerase enters exclusively at the 30 -end of the genome and dissociates from the genome at every gene junction, upstream
mRNAs are produced more abundantly than downstream mRNAs. This stop/restart process of the polymerase as it travels along the
genome represents an efficient method of attenuation of transcription creating a differential accumulation of transcription
products for translation. The mRNAs typically have a 50 -terminal 7-methylguanylate cap structure (m7G) for translation initiation
and a short 50 -noncoding region of 20–30 nts, as well as a longer 30 -noncoding region of 100–500 nts prior to the poly(A) tail.
A particularly long 30 -noncoding region exists in the RABV G mRNA corresponding to the long intergenic region in the RABV
genome between the G and L genes (initially described as a pseudogene). Differences in the 30 -noncoding region of genes mostly
account for the differences in the overall length of lyssavirus genomes. The mRNAs produced are efficiently translated by host cell
protein synthesis machinery into the viral proteins. Replication of the negative-sense (-strand) genome requires synthesis of the
full-length positive-sense (-strand) antigenome (cRNA) or replicative intermediate. The cRNA is produced by the viral polymerase,
acting as a replicase ignoring the stop/start signals that delineate the mRNAs. Synthesis of the cRNA starts at the 30 -end of the
N-vRNA by primer-independent synthesis to produce the 50 -end of the cRNA antigenome (complementary to the vRNA) including
a sequence corresponding to that of the short leader RNA. Like the N-vRNA, the newly formed positive-strand cRNA is encapsidated
by N (N-cRNA) as the cRNA sequence is elongated in the 5–30 direction. The resulting antigenome cRNA by definition has a 30 -end
that is identical in the first 11 nts to that of the genome vRNA and can efficiently direct the synthesis of abundant amounts of
progeny genome N-vRNAs from the N-cRNA template. Since progeny genome vRNAs makes up 98% of full-length RNAs in virus-
infected cells, it is obvious that the AGP is much more active in directing genome replication than GP is in directing full-length
genome transcription.

Virus Life Cycle

Following the bite of a rabid animal, infectious virus in the saliva is introduced into the bite wound and enters susceptible cells in
the muscle or skin at the wound site and the virus’s life cycle begins. In the virus-infected cell the virus begins to multiply (replicate)
8 Rabies

to produce more (progeny) virus. RABV and non-RABV lyssaviruses maintain their life cycles in selected (targeted) cells of the
infected host. After the virus enters the first cell and replicates, the progeny virions bud from the cell and spread to other susceptible
cells. As more progeny virions are produced, more and more (the growth can be exponential) infectious particles populate the
extracellular space (in vivo) or enter the medium of cell culture (in vitro). Typically, RABV does not enter or circulate in blood. The
sequence of events in the RABV life cycle can be divided into three phases. In the first, or early phase, the virus particle either
attaches to the surface of a targeted or susceptible host cell via a cell surface receptor or enters the cell via direct virus fusion
(externally) with the plasma membrane through coated pits (viropexis) and subsequently (internally) with endosomal membranes
of the cell. During the endosomal fusion process (called endocytosis), virus particles are stripped (uncoated) of their viral envelope
components and the helical RNP is released into the cytoplasm. When the virus attaches to the surface of a target cell via a ‘specific
receptor,’ it most likely binds to a receptor molecule or cellular receptor unit (CRU) that leads to or permits direct virus entry into
the cell. Various ‘receptor’ molecules have been implicated in binding RABV to cells in culture including lipid, gangliosides,
carbohydrates, and proteins of the plasma membrane, but none have been proven to be ‘specific’ receptors. However, three specific
in vivo CRUs appear to correlate with the defined neurotropism of the virus. The nicotinic acetylcholine receptor (nAChR), the first
identified receptor for RABV found at neuromuscular junctions, where RABV can also be found colocalized in situ, is regarded as a
likely candidate for a receptor for the virus. It was thought that RABV binding to AChRs would localize and concentrate the virus on
post-synaptic cells at neuromuscular junctions facilitating uptake and transfer of virus to peripheral neurons and ultimately the
CNS. Another possibility is the neural cell adhesion molecule (NCAM) CD56, a cell adhesion glycoprotein, which is found on the
cell surface of RABV-susceptible cell lines (in vitro) as well as in vivo. Blocking the NCAM receptor with the physiological inhibitor
heparin sulfate or specific anti-NCAM receptor polyclonal or monoclonal antibodies, which inhibited RABV infection of suscep-
tible cells expressing NCAM receptor helped identify this receptor molecule as a cell surface receptor for RABV. A third possibility is
the low-affinity p75 neurotrophin receptor (p75NTR), a nerve growth factor receptor expressed on the surface of cultured BSR cells.
Whether p75NTR is an obligatory receptor for RABV has been questioned since the challenge virus standard (CVS), a fixed
laboratory strain of RABV infects mice that lack p75NTR. Whatever receptor is present on the cell surface in vivo, it seems the
virus is not limited to choosing only one type of receptor in order to initiate the virus life cycle in the infected animal or human.
In the second or middle phase of the RABV life cycle, after the virus is internalized by endocytosis in the cell, the tightly coiled
transcriptionally ‘frozen’ viral RNP core, which is released into the cytoplasm, relaxes to form a loosely coiled RNP helix to facilitate
N-vRNA genome transcription and replication in the cytoplasm. The viral proteins are synthesized from the viral mRNA transcripts.
Four of the mRNAs, N-, P-, M-, and L-mRNA, are translated on free ribosomes in the cytoplasm and the G-mRNA is translated on
ribosomes bound to the ER (referred to as the rough ER). Before the nascent G is completely synthesized, it enters cotranslationally
into the lumen of the ER where the protein is folded with the help of molecular chaperones and undergoes modification at the
asparagine-X-serine or -threonine sequons by core glycosylation and N-glycan processing, that is, trimming of high-mannose
triglucosylated oligosaccharides in the rough ER and Golgi stacks to form the ‘complex type’ N-linked monoglucosylated
oligosaccharide of the mature G molecule. After final processing of the N-linked carbohydrate side chains in the Golgi apparatus,
viral G trimers are formed and appear on the plasma membrane surface of the infected cell. While protein synthesis is happening,
the incoming genome vRNA is transcribed into full-length positive-strand cRNA (antigenome RNA or replicative intermediate),
which in turn is copied into full-length negative-strand progeny genome RNA or vRNA (Figure 3).
In the late phase of the RABV life cycle, once sufficient pools of vRNA and viral N, P, and L proteins have accumulated, vRNA is
encapsidated by N (directed by the N–P complex) and L is added and nascent rabies viral RNP complexes are formed. ‘Skeletons’ of
virus particles and finally mature virus particles are assembled with the addition of M and G, respectively, to the RNP complexes.
During RABV morphogenesis, an amorphous, intracytoplasmic ground substance or ‘matrix’, known as inclusion bodies (IBs), long
known as Negri bodies commonly found in brain tissue as well as tissue culture, is formed. The formation of IBs comprising
filamentous matrix substance (generally N–P complexes), which precedes virus particle formation in neurons of the infected brain,
is often a hallmark for the diagnosis of rabies. The association of M with the newly formed transcriptionally active RNP selectively
inhibits further transcription and replication of the viral genome and localizes the RNP at the cellular membrane where the
glycosylated trimeric G is concentrated and M interacts with G. Finally, M begins to alter the structure of the RNP by condensing it
into a tightly coiled ‘skeleton-like’ (RNP + M) form in which the polymerase activity is ‘frozen’ in place. Thereby, M plays an
important role by covering the RNP and giving the virus particle its ‘bullet’ shape. In the final stages of virus assembly the mature
virion acquires its lipid envelope derived from the host cell’s plasma membrane, with the trimeric G spikes embedded in it as the
virion buds through the membrane.

Epidemiology of Animal and Human Rabies

The natural course of RABV transmission is from animal to animal and hence it is principally a zoonotic disease. On the occasions
that RABV spreads to humans it is primarily the consequence of an attack by an aggressive infected animal biting the human and
transmitting the virus present in saliva, which comes from the animal’s salivary glands. The reservoir for all RABV and non-RABV
lyssaviruses is the animal species pool that circulates the various lyssavirus species. But not all populations of terrestrial carnivores
serving as reservoir hosts for rabies are affected proportionately. Transmission of RABV and the non-RABV lyssaviruses is usually
among animals of the same species with cross-species transmission to other mammals usually depending upon direct interactions
with infected individuals of the reservoir host species. Such virus spillover from one host species to another can and has led to viral
 Rabies 9

Figure 3 Rabies virus (RABV) life cycle in the cell. Virus enters the cell following attachment through coated pits (viropexis) or via cell surface
receptors, mediated by the viral glycoprotein (G) fusing with the cellular membrane (endocytosis). After internalization, the viral G mediates low
pH-dependent fusion with the endosomal membrane and the virus is uncoated, releasing the helical nucleocapsid (NC or N-vRNA) of the
ribonucleoprotein (RNP) core. The five structural genes (N, P, M, G, and L) of the genome RNA (vRNA) in the NC are transcribed into five positive (+)
strand monocistronic messenger RNAs and a full-length (+) strand (antigenome) cRNA (replicative intermediate). The antigenome cRNA serves as the
template for replication of progeny genome () strand vRNA. The proteins N, P, M, and L are synthesized from their respective mRNAs on the free
ribosomes in the cytoplasm and G is synthesized from the G-mRNA on ribosomes bound to endoplasmic reticulum (rough ER). Some of the N–P
molecular complexes produce cytoplasmic inclusion bodies (Negri bodies) in vivo and some N–P complexes encapsidate the (+) strand and () strand
viral RNAs. After progeny genome RNA is encapsidated by the N–P protein complex, and L protein is incorporated to form progeny RNP (both full-length
standard and shorter defective) structures, the M protein binds to the RNP and condenses the RNP into the ‘skeleton’ structures. The skeleton structures
interact with the trimeric G protein structures anchored in the plasma membrane and assemble into virus particles that bud from the plasma membrane
of the infected cell into adjacent extracellular or interstitial space. Reproduced from Wunner, W. H. (2007) Rabies virus. In: Jackson, A. C., Wunner,
W. H. (eds.) Rabies, 2nd edn., pp. 23–68. San Diego, CA: Elsevier, Academic Press, with permission.

evolution and adaptation in a new host species over broad timescales. Bat lyssavirus variants, for example, sporadically spill over to
infect wild and domestic animals. New host species, such as red foxes on Prince Edward Island, Canada, and Nova Scotia, Canada,
and striped skunks in Arizona, have sustained transmission of bat variants of RABV within terrestrial carnivore populations until
possible natural extinction. Extinction can occur because of disappearance of the virus or the virus reaching undetectable levels
within the local species population, or by vaccination control programs as has occurred with skunk rabies in Arizona. Initial
epidemics of rabies in animals are typically the largest in a series of epidemics that emerge over time. As ‘wildlife’ rabies enters into a
new region to infect previously naive populations, a series of successively smaller epidemics may occur at increasing frequency. This
has occurred with red fox rabies in regions of Europe and with a raccoon-adapted variant of RABV in regions of the USA. In the
mid-1970s, the raccoon-adapted variant was introduced into the mid-Atlantic region of the United States as a consequence of
transporting raccoons unsuspected of incubating rabies from a couple of southeastern states for the purpose of restocking
dwindling local populations. These animals were moved, apparently while incubating the raccoon-adapted RABV variant, and
when placed among naı̈ve animals in state parks and game reserves soon spread the virus to other raccoons that over time and
distances transported the virus northward to Maine and Ontario, Canada and westward to Ohio, creating the most expansive
outbreak of animal rabies so far recorded. Typically, regions of higher population densities of wildlife species suffer greatest
population depression due to rabies transmission and the highest incidence of disease during the initial epidemic. In the United
10 Rabies

States, raccoons have been the wild animal species most frequently diagnosed as rabid since the early 1990s, whereas skunks and
foxes were the most frequently reported rabid cases during previous decades, 1960–90s and 1950–60s, respectively (Figure 4).
Rabies among skunks in North America has a long history, since the 1800s, as a source of human rabies. Currently, two RABV
variants that circulate within specific wildlife species circulate among skunks in the central United States; one of these, the North
Central Skunk variant, also occurs in central and western Canada, and the other RABV variant circulates among skunks in
California. Periodic epizootics (periods of increased disease activity) or epidemics of rabies in skunk populations that occur in
other states tend to drive the cyclic variations in skunk population numbers (Figure 5).
Bats are perhaps the most unique group of wildlife mammals to transmit RABV and non-RABV lyssaviruses to carnivores and
humans. Currently, there are 12 uniquely identifiable lyssavurus species and a further two recently described viruses that appear to
cause a rabies-like pathogenesis in humans and terrestrial carnivores. Only two of these lyssaviruses, MOKV and IKOV, have not yet
been detected in bat species. Among the many families, subfamilies and genera of bats that comprise the order Chiroptera, it is the
vampire bat that preferentially attacks livestock and transmits rabies sporadically to cattle. Observations linking vampire bats to

Figure 4 Rabies among wildlife animal species in the United States, 1951–2010. The number of reported cases of rabies among selected wildlife
animals in the United States, 1951–2010, as reported through national surveillance (data obtained from annual CDC surveillance reports).
Reproduced from Hanlon, C. A., Childs, J. E. (2013). Epidemiology. In: Jackson, A. C. (ed.) Rabies, 3rd edn., pp. 61–121. San Diego, CA: Elsevier,
Academic Press, with permission.

Figure 5 Distribution of rabies virus (RABV) variants in the United States. Common species of terrestrial carnivores among wildlife serving as reservoir
hosts. The geographic occurrence of two skunk rabies variants is represented as Northcentral Skunk and Southcentral Skunk. In addition to this
distribution of RABV variants is the reservoir present in species of bats, which covers the entire continental United States. Reproduced from Childs, J. E.,
Real, L. A. (2007). Epidemiology. In: Jackson, A. C., Wunner, W. H. (eds.) Rabies, 2nd edn., pp. 123–199. San Diego, CA: Elsevier, Academic Press, with
permission.
 Rabies 11

rabies epidemics among terrestrial animals, particularly in South and Central America, causing paralytic rabies in cattle, the
preferential target species of vampire bats, go back more than 90 years. Occasionally, vampire bats bite and infect humans,
particularly in many South and Central American countries, but other chiropteran hosts historically associate more with trans-
mission of rabies to animal and human populations. Clinically, RABV transmitted by vampire bats to cattle, humans, and other
bats cause ‘paralytic’ disease and virtually never ‘furious’ rabies (‘Human rabies’). Paralytic rabies in cattle starts with signs of
posterior unequal coordination followed in later stages with anorexia, bellowing, weakness in the hind legs, and other signs of
brain damage. Finally, ascending paralysis, temporary cessation of breathing, and death occur. Humans can experience similar
clinical signs and fatal outcome because of contact with infectious saliva from the bite or by inhalation of aerosolized saliva of a
rabid vampire bat. It is not known whether some specific property or properties of the RABV variant circulating among vampire bats
might be responsible for this rather oblique presentation of the disease. Bats are the second most common transmitters of rabies to
humans. Human rabies resulting from insectivorous bat species, particularly in the New World, did not appear until the 1950s. The
first human case to be particularly identified with an insectivorous bat occurred in 1953, in Florida, and since then the number of
rabid bats reported in the United States has increased dramatically. The association of classical RABV with insectivorous bat species
across the New World over the past 60 years as been as diverse as there are different insectivorous bat species that appear to serve as
virus reservoirs for lyssaviruses in the New World compared to that of the Old World. This diversity of RABV variants that has
emerged through natural infections of insectivorous bats has manifested itself in different clinical signs of the disease, i.e.,
aggression, weakness, and anorexia with relatively short morbidity periods (4–20 days). With highly sophisticated genetic analyses
of the different lyssaviruses isolated from insectivorous bats, it has been possible to differentiate the virus variants that associate
with bats, as well as terrestrial hosts such as red fox, skunks and raccoons, so that the virus variant can now be correlated with a
particular bat species and probably the country where the transmission occurred. Human infection caused by RABV variants from
insectivorous or suspected insectivorous bats has been increasing in North America (at least 69 cases identified between 1950 and
2010) now that rabies in the domestic dog population has been eliminated.
The dog is the most common animal to transmit rabies to humans as well as to other carnivores in all regions of the world in
which public health concern is highest. When dog rabies is reduced by effective disease control measures, such as mass vaccination
of both domestic and stray dogs, human rabies cases also decline. Implementation of control programs and policies for bat rabies,
on the contrary, has not been possible, largely because regimens for administration of vaccines in bats have not been adequately
developed, or have been much less widely implemented. In contrast to RABV variants found predominantly in wildlife in North
America (the United States and Canada) and Western Europe, the greatest dissemination of RABV in many locations in the world
has been by dogs. The domestic dog continues to be the major reservoir species for RABV throughout Africa and Asia, and to a lesser
extent in South America and Latin America. The similarity of canine RABV isolates from South America, Africa, Eastern Europe, and
parts of Asia suggests that rabies in dogs arose from a common source, perhaps from a single origin in the Palearctic region that
includes Europe, the Middle East, and northern Africa. Canine rabies was imported from Europe to the New World in the
eighteenth century during the time of the Spanish dominion in southern parts of North America and in Mexico as early as 1709.
Outbreaks of dog rabies in South America followed early in the nineteenth century in Peru (1803) and Argentina (1806). Later,
epidemics of canine rabies came in 5- to 6-year cycles in South America. Feral, stray, and neighborhood dogs contribute to free-
ranging populations in both developed and developing countries. Interestingly, young dogs, frequently less than 1 year of age,
which have not received a complete course of rabies vaccination, contribute in a major way to rabies outbreaks, particularly during
major mass vaccination campaigns in canine rabies endemic regions.
The greatest number (>99%) of human rabies deaths result from infection by RABV maintained by domestic dogs and
transmitted by dogs that bite humans and these occur mostly in tropical areas in developing countries of the world, where accurate
estimates of human rabies deaths are impossible to obtain by surveillance methods. Regional laboratories are also inadequate or
nonexistent for systematic detection and laboratory confirmation of human or animal rabies cases. Recent estimates by the World
Health Organization (WHO), nevertheless, suggest that the highest incidence of human rabies deaths is 55 000 annually in Africa
and Asia from being bitten by RABV-infected dogs. The global public health burden and cost of canine rabies in Africa and Asia
alone, based on direct medical expenses and indirect costs borne by patients seeking postexposure vaccine treatment (PET),
amounts to roughly US$196 million/year and US$289 million/year, respectively. Worldwide each year an estimated 7 million
people bitten by suspect rabid dogs increases the high demand for expensive postexposure prophylaxis (PEP) and PET.

Control and Prevention of Rabies in Domestic and Wildlife Animals

Control and prevention of rabies in domestic animals has been most successful in countries that advocate strong veterinary and
public health programs and have the cooperation of people who take their responsibility for animal pets seriously. Nationwide
vaccination programs together with rules or laws to enforce the elimination of stray dogs and cats and use of quarantines in rabies-
free countries have done the most to keep rabies from spreading. To be effective, these measures as well as the oral immunization
approach for wild animals must reduce the number of unvaccinated animals to a level at which rabies transmission is interrupted
and this level must then be maintained.
Preventing the spread of rabies to susceptible animals and humans continues to be a major battle for biologists and public
health officials throughout the world. Persistence of multiple variants of RABV and non-RABV lyssaviruses in wildlife animal
reservoirs presents a continuing challenge to those dealing with medical, veterinary, and wildlife management. To control rabies at
12 Rabies

its source is to control the disease in wildlife vector species, and this may be the most challenging and difficult task of all. Many
techniques have been tried over the past three decades, beginning with the control of rabies in foxes in Western Europe using the
concept of oral vaccination and baits carrying vaccine to immunize free-ranging animals. Almost every species of mammal that acts
as a vector for the disease requires a unique approach depending on many factors, including density of wildlife populations,
restriction of movement, food sources, and functions of the species to prevent a particular species from becoming infected. On the
contrary, the impact that vaccines have had on protecting domestic animals, causing a dramatic reduction in the incidence of
canine and human rabies, clearly holds great promise for the prevention and ultimate control of rabies in wild animals.
The main transmitters of wildlife rabies throughout the world are the wild carnivores. These animals are highly susceptible to
rabies: they excrete virus in the saliva and live in high-density populations, and the long incubation period of the virus ensures
maintenance of the infection. The most significant terrestrial wild carnivores transmitting rabies in the world are the red fox (Vulpes
vulpes) in Central and Western Europe and the red fox and raccoon dog (Nyctereutes procyonoides) in Eastern Europe. Foxes (V. vulpes
and Urocyon cinereoargenteus), skunks (predominantly Mephitis mephitis), and raccoons (Procyon lotor) are the major animal vectors
in various parts of North America, and wolves, stray dogs, mongooses (Herpestes auropunctatus), meerkats, and polar foxes in other
areas and countries of Africa, Asia, and Central and South America. Establishing well-organized and well-managed programs with
extensive national and international cooperation among veterinary and public health personnel who share as a common goal the
control of rabies in wild animals, from local eradication to inhibition of spread into uninfected areas, will undoubtedly be more
difficult than controlling rabies in domestic animals. Several European and North American team efforts, nevertheless, have made
significant progress in evaluating the efficacy and safety of vaccines and vaccine delivery systems for oral immunization of wild
carnivores. The progress is such that it can be said that the oral immunization method can be an effective and practical approach for
the elimination of enzootic rabies in terrestrial animals, one species at a time.

The Blueprint for Rabies Prevention and Control

Experts in rabies management worldwide formed the ‘Partners for Rabies Prevention’ (PRP) initiative and developed a ‘blueprint’ to
provide government officials and their public health professionals, veterinarians, scientists, medical doctors, and other experts in
diagnostics, virology, epidemiology, ecology, immunology, animal welfare, outreach health educators and communicators with
advice in the form of a comprehensive set of guidelines and the necessary encouragement and support to develop their own
national canine rabies elimination programs. This online document of operating procedures is designed to help governments plan,
implement, evaluate, and sustain canine rabies elimination programs as described in ‘The Blueprint for Rabies Prevention and
Control’ (available at http://www.rabiesblueprint.com). Information to help governments focus their planning, implementation,
evaluation, and ability to sustain canine rabies elimination programs, in particular, is contained in one section of the blueprint
(www.caninerabiesblueprint.org). The Canine Rabies Blueprint, which was established in 2010, is a powerful toolkit in simple
language that is freely accessible to a wide range of users. This type of ‘one health’ approach to canine rabies control and
elimination is expected to grow in interest and have considerable impact on local endemic canine rabies in high-risk countries.

Human Rabies
Clinical Signs and Symptoms
Humans infected with RABV develop classical encephalomyelitis, often referred to as ‘furious’ or ‘encephalitic’ rabies, in approx-
imately 80% of the cases and the rest (20%) exhibit a paralytic form of the disease. Rabies in humans usually develops 20–90 days
after exposure, although occasionally the disease develops after only a few days and rare cases have occurred after a year or more
following exposure. The clinical illness in humans, which follows such a variable and sometimes prolonged nonsymptomatic
incubation period, usually begins with a prodromal phase, which may last 2–10 days. At this point the symptoms are almost
entirely nonspecific, comprising fever, malaise, anorexia, nausea, vomiting, diarrhea, sore throat, cough, myalgia, and headache.
After the prodromal period, signs of nervous system involvement develop, leading to an acute neurological phase, including
intermittent hyperactivity marked by agitation lasting 1–5 min, occurring spontaneously or triggered by a variety of tactile,
auditory, and visual stimuli, disorientation, hallucinations, seizures, bizarre behavior, nuchal stiffness, and paralysis. Other
abnormalities include muscle fasciculation (particularly near the site of the exposure), hyperventilation, and hypersalivation.
Hydrophobia, a term often used to describe rabies involving both severe thirst and fear of water, is regarded as a unique
characteristic and most specific manifestation of rabies in humans, and appears to develop in 50–80% of patients with encephalitic
or furious human rabies. The mental status of the patient gradually deteriorates during a period of 2–12 days until the onset of
coma, which may last for hours or months, or an abrupt death due to cardiac or respiratory arrest. With intensive medical support,
cardiac and respiratory arrest may not occur, and the patient may experience rare recovery. The incubation period (from exposure to
onset of clinical signs of disease) in rabies has a greater length and variability than in most other infectious diseases, which can
cause considerable stress to the patient. Severe facial bites and multiple bites to other areas of the body tend to have shorter
incubation periods. In cases of long incubation periods, it is often difficult for the patient to recall a bite exposure, particularly if the
bite was from an insectivorous bat, or the patient becomes unintelligible from the disease. From cases of untreated humans who
developed rabies following known animal bites, the highest number (50–80%) occurred from bites to the head, the next highest
 Rabies 13

number (15–40%) from bites to the hand or arm, and the fewest (3–10%) from bites to the leg. The risk from scratches or
contamination of minor wounds with saliva is about 0.1%.
In paralytic (or ‘dumb’) rabies, muscle weakness develops early in the course of the disease. The paralysis may start in the bitten
extremity and spread to other extremities, but is not necessarily related to the anatomical site of the animal bite (exposure).
Consequently, rabies is often misdiagnosed in this clinical form, especially if there is no history of animal exposure. At the onset of
this clinical form of rabies, patients are alert with a normal mental status. They may have local pain, paresthesias, or pruritus at the
bite site even though sensory perception upon examination may be normal. Muscle fasciculations may be present and facial
muscles become weak bilaterally. The clinical picture may be confused with the Guillain–Barré syndrome. Unlike Guillain–Barré
syndrome, urinary incontinence is common and pain and sensory disturbances may occur in paralytic rabies.
Other lyssaviruses, in addition to RABV, have also caused rabies. Only LBV, which was first isolated from fructivorous bats in
Nigeria, has not been associated with human rabies. Four other lyssaviruses recently isolated from bats, ARAV and KHUV, which
were isolated from bats in Central Asia, and IRKV and WCBV, which were isolated from bats in Russia, have also not been
associated with human disease. MOKV, which was isolated from shrews in Nigeria, was associated with meningoencephalitis,
without the typical features of brainstem involvement seen in classical rabies, in a 6-year-old girl who died in Nigeria in 1971 after a
6-day illness. Another patient, a 3 1/2-year-old girl from Nigeria, who presented with a sudden onset of fever and convulsions,
made a complete recovery. In this case, MOKV was isolated from her cerebrospinal fluid (CSF), although it is questionable whether
cross-contamination of her CSF might have occurred since the shrew isolate of MOKV was handled in the same laboratory. In 1970,
a 31-year-old man from rural South Africa developed an illness that was indistinguishable from rabies. The patient’s illness was
characterized with fever, excessive sweating, hydrophobia, and spasms of his face, arms, and torso. He was confused, irritable, and
periodically aggressive. He died 5 days after the onset of illness. The man had been bitten on the lip by a bat 4 weeks earlier. The
virus that was isolated from the patient’s brain after death was DUVV. In 1985, two cases of rabies-like illness occurred in Europe;
one was an 11-year-old girl from Belgorod, Russia, who was bitten on the lip by an unidentified bat, and the other was a 30-year-old
man, who was a zoologist from Finland. Both died with signs indistinguishable from that of rabies. The virus isolated from the girl
was called Yuli virus and classified as EBLV-1, and the virus isolated from the man who died 23 days after the onset of illness was like
other enzootic European bat RABV isolates and was classified as EBLV-2. In 1996, a 39-year-old woman from Australia died after a
20-day illness. She sustained numerous scratches to her left arm over 4 weeks while she cared for fruit bats and apparently had been
bitten by an insectivorous bat prior to the onset of her illness. Her illness was marked by progressive left arm weakness that later
developed into progressive limb and facial weakness with reduced tendon reflexes and fluctuations in her level of consciousness
prior to her death. The virus she was infected with was identified by the reverse-transcription polymerase chain reaction (RT-PCR)
assay, perhaps the most frequently applied test presently available, and found to be identical to ABLV, which had been identified in
flying foxes (fruit-eating bats). This patient had rabies that typically involved the brainstem and quickly progressed to diffuse brain
involvement. Two years later, in 1998, a 37-year-old woman from Queensland, Australia, experienced fever and paresthesia in her
left hand and pain in her left shoulder, along with a sore throat and difficulty in swallowing for 5 days before she was admitted to
hospital. She had pharyngeal spasms, evidence of autonomic instability, and progressive neurologic deterioration before she died
19 days after onset of her illness.

Rabies Diagnosis
An estimated 7–10 billion diagnostic tests of all types for rabies are performed in the laboratory each year in the United States
alone, most frequently for the postmortem examination of animals that have either bitten a person or in some other way
potentially caused human exposure to the disease. Evidence of RABV infection in a contact animal (and sometimes in an exposed
person subjected to an antemortem diagnosis of rabies), based on a positive diagnostic test, prompts administration of rabies PET
and PEP to the exposed person and any other persons who had direct contact with the exposed person, respectively. A skin biopsy
from the nape of the neck that includes several nerve-innervated hair follicles is often the best sample for a human antemortem
diagnosis of rabies using a direct fluorescent antibody (DFA) test or RT-PCR assay. Fresh saliva is another sample source for RT-PCR
assay, while serum and CSF are suitable for the detection of anti-rabies antibodies that can be assayed in a rapid fluorescent focus
inhibition test (RFFIT). Often, a positive diagnosis of RABV in animals, as in the observation of rabies viral antigen in the brainstem
at the level of the medulla, pons, or midbrain, also initiates the appropriate management of exposed domestic animals, including
booster immunization of previously immunized animals and euthanasia or quarantine of unvaccinated animals.
While various types of diagnostic tests may be performed, diagnosis of rabies based upon clinical presentation alone may be
difficult to discern. Clinical symptoms or signs of human rabies often occur long after exposure to the virus and can be difficult to
distinguish from encephalitis caused by other viral infections, such as poliovirus or canine distemper virus in humans, or may be
confused with Guillain–Barré syndrome. RABV infection can be inferred in some cases from indirect evidence, such as the
demonstration of RABV-neutralizing antibody in the serum of an unvaccinated individual or antibody in CSF. Specific histopath-
ologic changes in postmortem examination, namely the presence of Negri body inclusions in neurons of the CNS, can provide
microscopic evidence of RABV infection, but only with a low degree of sensitivity. A more specific indication of RABV infection
from a postmortem examination of humans suspected of having rabies is the direct visualization of RABV particles in brain tissue
and CSF or saliva using EM. Similarly, specific indications of rabies can be used to determine the treatment of humans based on
examination of brain specimens taken from contact animals suspected of having rabies after they have been euthanized by any
humane method that does not damage the head. Other indications can be made in postmortem tissue (from animal or human)
14 Rabies

prepared as a smear, impression, or section or from CSF or cultured cell monolayer infected with the suspected virus. These are
detection of the viral proteins (antigens) by DFA and indirect fluorescent antibody (IFA) tests and viral genome RNA using genetic
hybridization probes and RT-PCR techniques. Immunoglobulin (or viral antigen-specific monoclonal antibody (MAb)) molecules
labeled by covalent chemical attachment of a fluorescent tag to produce a fluorescent-MAb conjugate permit visualization and
localization of the target protein in tissue preparations or saliva. These viral antigen-specific antibody diagnostic reagents, which
interact specifically with the desired viral antigens (targeting specific epitopes on the proteins) in the DFA and IFA tests, provide a
more definitive diagnosis of rabies infection in the contact animal and exposed human. The most common confirmatory test is
cultivation of virus by inoculation of laboratory (test) animals or cell culture. The mouse inoculation test (MIT) is a sensitive and
reliable procedure to confirm the presence of virus in pieces of the brain that test positive by the DFA or IFA methods. Direct
detection of the viral genome or virus-specific mRNAs by hybridization with nucleic acid probes applied to tissue sections (in situ
hybridization) requires close attention to the specificity of the probe sequences that are used to anneal with its viral positive or
negative RNA strand target sequence. Since no commercial sources of RABV-specific probes are available, all probes must be
developed for the laboratory, and special attention must therefore be paid to the diversity of lyssavirus strains that might be present
in the specimen.

Rabies Pathogenesis and Histopathology

The pathogenetic process of rabies usually begins with the introduction of virus-laden saliva into a bite or scratch wound inflicted
by a rabid animal. The incubation period after the bite can be extremely variable depending on the relative susceptibility of the
species, the location of the wound (to the head vs. extremities), and the depth and location of virus deposit in the wound.
In humans, the incubation period is usually 20–90 days, more generally 15 days to 1 year, but cases have been described recently in
which the extremely sophisticated, highly sensitive, and precise antigenic (using MAbs) and genetic (employing the RT-PCR
technique) diagnostic methods established that the incubation period could last several years. Other routes of infection have
included intranasal (in animals that sniff ) and oral routes (among bats that spray in high-density cave dwellings) as well as corneal
transplants (in human cases). Because the neural routes from mucosal membranes and the eye to the CNS are relatively short, the
virus has almost direct access to the brain from these tissues. RABV in natural infections does not establish a viremia.
Perceptions of what happens to the virus at the site of entry in vivo and the events that precede the transit of virus toward the CNS
(the ‘neural phase’) are still vague. One puzzling aspect of preneural RABV infection is the sequestering of virus at the site of
inoculation. Macrophages in vitro can be persistently infected with RABV, but it has not been demonstrated in animal models that
macrophages in vivo sequester RABV. Evidence of viral antigen accumulating in striated muscle in experimental animals after
intramuscular inoculation of RABV has suggested that muscle becomes a potential initial site of virus replication although this has
not been observed in humans. The ‘preneural phase’ of virus infection in muscle is usually confined to a small number of myocytes
close to the inoculation site and produces few infectious particles. Any shedding of virus into extracellular spaces around myocytes,
of course, makes this virus vulnerable to the PET with anti-rabies antibody and vaccine.
Exposed sensory nerve endings of neuromuscular or neurotendinal spindles deep in muscle are the most likely sites of virus
entry into the peripheral nervous system after virus replication in muscle. The primary site of virus attachment might actually be
nerve endings leading directly to neuronal infection in badly torn muscles of a bite wound. Similarly, the many sensory nerve
endings of epithelial and subepithelial tissues exposed by superficial abrasions of skin and mucous membranes may also serve as
primary sites of virus entry. The entry of RABV into peripheral nerves begins the ‘neural’ or ‘neuronal’ phase of infection, which is
characterized by the centripetal movement of virus exclusively within axons to the CNS. Schwann cells and perineural elements do
not appear to be infected.
Virus migration from peripheral sites on the body to the brain is suspected to occur passively in the axoplasm of peripheral
motor and perhaps sensory nerves (centripetal passage to the brain) at the estimated rate of retrograde axoplasmic transport
(3 mm h1 or between 50 and 100 mm day1) in peripheral nerves. Evidence that the RABV P (phosphoprotein) of the RNP
complex interacts with LC8, a component of both cytoplasmic myosin V and dynein, which are involved in actin-based transport
(important in early steps of viral entry) and microtubule-based transport in neurons, respectively, has led to speculation that the
RABV P–dynein interaction may be fundamentally important in axonal transport of RABV. Other mechanisms of virus transport
may facilitate its spread (e.g., movement of virus across cell-to-cell junctions, including synaptic junctions). Observations of virus
budding on axonal membranes and virus particles accumulating intra-axonally at nodes of Ranvier as the virus progresses to the
brain support the possibility of other mechanisms of virus transport. The ascending paralysis seen in many infected animals and
humans reflects the progressive infection of dorsal root ganglia of the CNS. The virus ascent toward the brain has been confirmed
by sequential immunofluorescence studies. The ability of virus to spread to the CNS from a peripheral site (its neuroinvasiveness) is
an important component of the ‘neurovirulence’ characteristic of the virus. Studies have shown that while the spread of pathogenic
(virulent) and nonpathogenic (avirulent) viruses in vitro (spreading from cell to cell in cell culture) reflects their ability to spread
in vivo, the pathogenic virus spreads more rapidly in the CNS and infects more neurons than the avirulent virus in vivo. Once RABV
infection enters the brain, the virus spreads rapidly along neuroanatomical pathways, spreading within the CNS as in the peripheral
nerves.
Inflammatory lesions caused by perivascular infiltration of lymphocytes, macrophage, and occasionally polymorphonuclear
cells are the most common and frequently noted histological signs of change in the spinal cord and brainstem of animals and
humans infected with RABV. The ganglionic changes observed in rabies, although not exclusively confined to rabies, could serve as
 Rabies 15

diagnostic lesions for rabies, because they are not encountered in diseases that must be considered in the differential diagnosis of
rabies. The morphologically distinct lesions that are significant in rabies diagnosis are the specific eosinophilic inclusion bodies
(Negri bodies) found within neurons of peripheral and basal ganglia of the spinal cord, in the pons and medulla of the brainstem,
in the cerebral cortex and thalamus and Purkinje cells of cerebellum, and in ganglion cells of Ammon’s horn of the hippocampus.
Signs of active transport of virus in the brain include replication in the neurons, budding from the plasma membrane, and release
into tissue interstitial spaces. The virus appears to infect susceptible cells by viropexis or direct cell-to-cell transmission. Once
delivered in the extracellular spaces of the brain, the virus can presumably spread by passive transport over relatively long distances
through intercellular spaces large enough to pass virus by interstitial fluid movement. Virus that is transmitted directly from one
nerve cell to a contiguous nerve cell could also spread and result in the same long-distance movement because of the many long,
intertwined neuronal processes that connect in the brain.
Surprisingly, despite the severe neurological signs of clinical rabies (Human rabies) the neuropathological findings are generally
mild (see below). This suggests that RABV-infected neurons become dysfunctional without developing major morphological
changes or positive terminal deoxynucleotide transferase dUTP nick-end-labeling (TUNEL) staining, indicating fragmented cellular
DNA that is normally associated with apoptosis. If true, what is the fundamental underlying basis for the proposed neuronal
dysfunction, if not apoptosis, that would account for the severe clinical neurological signs observed in rabies? One hypothesis has
been that apoptotic cell death in the CNS of humans and in experimental animals in which apoptotic changes have been examined
is caused by RABV infection. After finding evidence of oligonucleosomal DNA fragmentation typical of apoptosis and that cell
death was concomitant with expression of the rabies viral G, an (unexpected) inverse relationship was found between the
pathogenicity of the virus (comparing the virulent phenotype with the avirulent phenotype) and apoptosis. In other words,
apoptosis may be a protective rather than a pathogenic mechanism in RABV infections both in vitro (studies with cell culture) and
in vivo. The less pathogenic (avirulent) viruses induce more apoptosis than the more pathogenic (virulent) viruses in vitro as well as
in vivo, using peripheral routes of inoculation. In RABV infection the mechanisms involved in the ensuing cell death or survival of
neurons, both in vitro and in animal models using different viral strains and routes of inoculation, are clearly complex.
Once the virus begins to spread from the brain in the final phase of rabies infection, the tropism of infectious virus changes and
the virus is delivered to a variety of extraneural and nonneural tissues. The centrifugal (outward) spread of virus generally occurs via
the same axoplasmic transport routes that were used for the centripetal (inward) passage of virus inward to the CNS. The spread of
RABV in the absence of CNS infection is virtually nonexistent. Among the most heavily infected tissues following propagation of
virus in the CNS are the end organs in oral and nasal cavities, and the head and neck. Infection may be found in free sensory nerve
endings of tactile hair follicles or in epithelial cells of hair follicles in the skin. To take a skin biopsy from the nape of the neck is one
of the best diagnostic methods of confirming an antemortem diagnosis of rabies in humans (‘Human rabies’) (see Rabies
Diagnosis). The fact that rabies antigen is occasionally detectable in skin biopsy and in corneal impressions is evidence of its
nerve-mediated dissemination.
Infection of the salivary glands by dissemination of virus from the CNS is extremely important in the life cycle of the virus
because it is required for effective transmission of disease. Virus titers in the salivary glands often exceed those in the brain. In some
animal species, the salivary glands become infected before the signs of CNS infection become apparent, and at the height of salivary
gland infection, large amounts of fluorescent viral antigen may be detected in individual acinar cells, in cell clusters, and
occasionally in the entire acinus.
Presently, the molecular basis of RABV pathogenesis is linked to surface G, which also plays an important role in the initiation of
RABV infections as the viral attachment protein that interacts with the cell surface receptor. Through selection of neutralization-
resistant variants of RABV (using virus-neutralizing MAbs against G) that lost their virulence phenotype and by identification of
single amino acid changes in G, the pathogenicity of the virus appears to correlate with the presence of an epitope on G within
antigenic site III. The amino acid substitution in the mutant that renders the virus nonpathogenic for adult mice was located at
position 333 of RABV G where arginine is normally found. Isoleucine at position 333 in the fixed RABV Evelyn–Robitnicki–
Abelseth (ERA) strain variant and glutamine or glycine in the fixed rabies CVS strain variants replaced arginine at position 333 of
RABV G in the respective pathogenic parent viruses. Several other fixed RABV strains including attenuated viruses that were not
selected by MAbs but resisted neutralization by specific MAbs (194-2 and 248-8), such as the Flury high egg passage (HEP) and
Kelev strains, also substituted isoleucine, glutamine, or glycine for arginine 333. Other discrete amino acid substitutions for
arginine 333 include methionine and serine. The biological relevance of arginine at position 333 of RABV G or its influence on the
pathogenic mechanism responsible for the restricted neurotropism of RABV infection is not completely understood. Experiments
have suggested that the neural routes of entry to the CNS and the rate of virus spread may differ depending on the amino acid at
position 333, but as yet the complete story has not been told.
Many of the dominant pathologic features of RABV infection were first described in the early 1870s to early 1990s. With the
introduction of EM and immunohistochemistry, pathology studies have continued to provide key insights into our understanding
of rabies. Although the clinical outcome of RABV encephalomyelitis might suggest severe degrees of destruction, the histopatho-
logical changes observed in the CNS are typically relatively mild. Some inflammatory cell infiltration of the leptomeninges usually
occurs in human rabies cases. Infiltrates are composed of lymphocytes and monocytes, small numbers of plasma cells, and a
predominance of neutrophils when inflammation is intense. Perivascular ‘cuffing’ is seen predominantly in gray matter, especially
in the brainstem and spinal cord, marked with infiltrates of lymphocytes and monocytes. The development of neuronophagia
(accumulations of activated microglia/macrophage in the process of phagocytosing degenerating and/or dying neurons) is typical
in many rabies cases. Microscopic accumulations of proliferating microglia/monocytes known as Babes nodules (first described by
16 Rabies

Figure 6 Negri bodies stained with hematoxylin and eosin (HE) in perikarya of (A-C) cerebellar Purkinje cells and (D, E) pyramidal neurons in the
cerebral cortex of human rabies cases. The arrow in (D) indicates a Negri body in an apical dendrite. (magnifications, A
315, B
460, C
550, D
730,
E
865). Reproduced from Rossiter, J. P., Jackson, A. C. (2013). Pathology. In: Jackson, A. C. (ed.) Rabies, 3rd edn., pp. 351–408. San Diego, CA:
Elsevier, Academic Press, with permission.

Babes in 1892) can be observed in gray matter of the medulla oblongata in human rabies cases. However, because Babes nodules
can be present in other viral encephalitides and other infectious disorders they are less reliable for rabies diagnosis. On the other
hand, Negri bodies, described in the 1903 by Adelchi Negri are more of a classical hallmark of cytological features in rabies. Stained
with hematoxylin- and eosin, they appear as dense, well-defined, oval or round (typically 2–10 mm in diameter, but can be
0.5–27 mm) eosinophilic intracytoplasmic inclusions (Figure 6). Although found in 50–90% of human rabies infections, especially
in hippocampal neurons and cerebella Purkinje cells, their significance in the pathogenesis of rabies is not as obvious. The
ultrastructure of Negri bodies includes randomly orientated viral NC (source of RABV N for antigen diagnosis) resembling large
aggregates of granulo-filamentous matrix material occasionally interspersed with bullet-shaped virus particles giving the bodies an
overall granular, electron dense manifestation. They are usually found in areas of the CNS where there is little inflammation, and
less frequently seen in degenerating neurons and/or in association with inflammatory foci.
Histopathology in peripheral nerves as virus spreads through the peripheral nervous system (PNS) is associated with inflam-
matory, reactive, and degenerative changes in structural components of the PNS. Changes in sensory and autonomic ganglia as well
as a neuronopathy (degeneration and loss of sensory and autonomic neurons) leading to degeneration of spinal and peripheral
nerve fibers and routes have particular diagnostic utility.

Rabies Vaccine Prophylaxis

First Line of Protection Against Rabies – Preexposure Prophylaxis
Rabies is a preventable disease in humans if the right precautions are taken to ensure pre-exposure prophylaxis (PrEP). Those who
consider themselves to be at increased risk of possible exposure to rabid animals, especially unvaccinated free-roaming dogs should
be administered PrEP vaccination. Veterinarians and animal handlers because of their frequent and often unsuspecting contacts
 Rabies 17

with domestic as well as wildlife animals, and those persons who by the nature of their work, hobbies (e.g., caving) or travels to
rabies endemic areas put themselves at high risk of exposure and infection, particularly where access to timely PEP is not an option
(e.g., no medical clinics) should receive the full regimen of inoculations. PrEP consists of a series of three doses of vaccine, given
either intramuscularly (IM) or intradermally (ID) on each of 3 days; 0, 3, and either day 21 or 28. Antibody titers of 0.5
international units (IU)/ml or higher are considered to be protective against rabies. A single booster dose of vaccine would be
necessary should the titer fall below this minimum titer.

First Line of Defense After Exposure to Rabies – Postexposure Prophylaxis

Since the incubation period may be variable and often sufficiently long following the peripheral inoculation of RABV from an
animal bite, depending on the location and severity of the bite exposure, it is possible to neutralize the virus at the wound site with
anti-rabies antibody before the virus enters and spreads along neuronal pathways to the CNS. According to WHO recommenda-
tions, PEP must be administered to every patient that has been exposed to rabies. Immediately or as soon as possible after an
exposure to a potentially rabid animal, it is essential to start PET and PEP by first cleaning the wound using proper wound cleansing
procedures as a first line of defense against RABV infection. It is important in this procedure to inactivate as much as possible of the
RABV that may have entered the wound. Proper wound cleansing involves thorough washing of the wound or mucous membrane
for at least 15 min using soap, water, detergent, povidone iodine, or another antiviral substance. If soap and detergent are not
available the wound should be flushed with copious amounts of water. After the wound has been thoroughly cleansed, rabies
immunoglobulin (RIG) (virus neutralizing antibody) must be injected into and around the bite (wound) or mucous membrane
sites to neutralize virus deposited with saliva from the animal’s bite. Two types of RIG are administered to patients globally; human
rabies immunoglobulin (HRIG) is used more frequently in industrialized countries whereas equine rabies immunoglobulin
(ERIG) is more frequently administered to patients in developing countries. To administer the RIG properly, the full dose of
20 IU kg1 of body weight of HRIG and 40 IU kg1 of body weight of ERIG must first be injected directly into as well as around the
wound site in as many places as anatomically feasible. Any HRIG or ERIG that is left over after infiltrating the wound sites should be
administered deep intramuscularly at a site that is distant from the vaccine inoculation site so as not to interfere with (neutralize)
the vaccine dose that is also given at this time for prophylaxis (infection prevention).
Rabies vaccines available in developing countries for PEP are mostly nerve tissue vaccines (NTVs) produced from the brain tissue
of infected sheep or mice although these are gradually being replaced by more immunogenic and less reactogenic cell culture-based
rabies vaccines (CCVs), particularly in large cities. The problem with the modern cell culture vaccines is they cost more to produce,
limiting their use to those at the highest risk of RABV infection, who ironically are often among the poorest of society. The
postexposure vaccination regimens recommended for CCVs use either IM or ID routes of inoculation of which the latter is an
option for reducing the dose administered and the cost of treatment. The first dose of vaccine is given with RIG administered in and
around the wound site. It is important to note that there is sufficient data to insure that when used in conjunction with prompt
appropriate wound care along with RIG, the postexposure vaccination regimens recommended by the global WHO Expert
Committee on rabies can prevent death from rabies. There are two IM and two ID postexposure vaccination regimens recom-
mended for use with cell culture rabies vaccines. In the United States, the Advisory Committee on Immunization Practices (ACIP)
recommends a four-dose rabies vaccination regimen with human diploid cell vaccine (HDCV) or purified chick embryo cell
vaccine (PCECV) administered IM on days 0 (as soon as possible after exposure), 3, 7, and 14 instead of the Essen regimen, which
stipulates five IM doses, previously recommended for use in North America. The Essen regimen is also used in Europe and in those
parts of Asia and Africa where ID administration of rabies vaccines is not yet approved. The Essen regimen specifies one full dose IM
on days 0, 3, 7, 14, and 28. The Zagreb regimen, which specifies two doses IM on day 0 (one in each deltoid) and one additional
dose on days 7 and 21, reduces the number of doses required from five to four, and, importantly, it reduces the number of visits to
get the rabies vaccine from five to three. The Zegreb regimen is used in some European countries.
The WHO-recommended ID vaccination regimens, which use reduced doses of vaccine, were developed to reduce the cost of
expensive CCVs that were introduced to replace NTVs in Asia. Thailand was the first country to begin using ID regimens to
administer CCVs. The Thai Red Cross or ‘2-2-2-0-2’ regimen and the Eight-site or ‘8-0-4-0-1-1’ regimen are two ID regimens
currently recommended for use by the WHO, the Thai Red Cross regimen being the most widely used ID vaccination regimen. The
updated ‘2-2-2-0-2’ regimen is administered with 0.1 ml doses of vaccine ID over the deltoid region on days 0, 3, 7, and 28. In the
‘8-0-4-0-1-1’ regimen, vaccine is administered ID in a 0.1 ml dose at eight different sites (upper arms, lateral thighs, suprascapular
region, and lower quadrant of abdomen) on day 0, another 0.1 ml dose of vaccine at four different sites (each upper arm and each
lateral thigh) on day 7, and another 0.1 ml dose of vaccine in the upper arm on days 28 and 90. It is important to note that all of the
PEP regimens have been developed with the administration of RIG to provide initial passive immunity.

Second Line of Defense: Establishing Active Adaptive Immunity

Because RABV is strictly neuroinvasive and little viral antigen is present in tissue in the early stages of the disease, the humoral
response to viral antigens in the infected host is negligible until later in the course of infection, usually after clinical signs appear,
and remains low until the terminal phase of the disease. High levels of antibody appear in serum and in CSF only at death and in
cases where illness is naturally or artificially prolonged. In the rare cases of chronic and abortive RABV infections, virus-specific
antibodies have been detected in the serum and CSF, and in the brain of animals. VNA is produced in response to the massive
18 Rabies

amount of viral antigen (G in particular) that is generated through widespread infection of the CNS and made accessible to the
host’s reticuloendothelial system.
The critically important cell-mediated immune response to RABV infection is perhaps the most puzzling of the host’s total
immune response to RABV infection. T-helper lymphocytes, which play an essential role in supporting B-cell production of
antibodies, and cytotoxic lymphocytes (CTLs), which function independently in cell-mediated viral clearance, are both key
responses in the cell-mediated immunity derived from viral antigens. However, in spite of their importance in viral clearance,
T-lymphocytes appear to be suppressed in animals infected by pathogenic ‘street’ viruses. As a result of virus-induced immuno-
suppression, the disease increases in severity and mortality rises. The strongest indications that T-lymphocytes play a role in
immune protection against RABV infection comes from studies in which athymic (nude) mice died when infected with the
attenuated Flury HEP strain that normally causes nonlethal infection of the CNS. Normal mice, similarly infected, show no signs of
the disease. Likewise, when normal mice infected with Flury HEP virus are treated with the immunosuppressive agent, cyclophos-
phamide or antithymocyte serum, the infection becomes as lethal as street virus (mortality in groups of mice increases to 50% and
occasionally to as high as 100%). Mice that survive avirulent virus infection develop CTLs, whereas the mice treated with
immunosuppressants and mice infected with virulent virus fail to develop CTLs. Further evidence for the protective role of CTLs
has come from adoptive transfer experiments in which G-specific CTL clones were observed to protect mice against RABV infection.
Since the function of CTLs is to destroy target cells that display virus-induced changes on their surfaces, it is clear that a strong CTL
response is essential in mice that survive RABV infection. It is clear from these studies and others that both B- and T-cells play an
important role in virus clearance.
Unlike the response to virulent RABV infection, immunization with attenuated live and inactivated RABV vaccines induces
humoral and cell-mediated immune responses that develop to functional levels in 7–10 days and persist for a year or more.
Vaccine-induced antibody and T-cell-mediated responses in animals and humans, known as adaptive immune responses, are
regarded as key and are of paramount importance in the prophylatic protection of animals and humans. Also, acute infections
caused by live virulent RABV strains, as well as the less virulent strains, injected into a peripheral site (e.g., the hind leg of an
animal), that progress to and within the spinal cord and the brain are accompanied by the production of the inflammatory
cytokines IL-1, TNF-a, INF-b, and IL-6, as well as chemokines such as CCL-5 and CXCL-10. This indicates that the nervous system
can sense RABV entry and mount a reactive innate, as well as an adaptive immune response. An adaptive innate immune response is
helpful in PEP and PET if, as proposed, chemokines and inflammatory cytokines attract lymphocytes that are activated in the
periphery. It is to be emphasized that even with a relatively rapid adaptive immune response of 7–10 days, the requirement in PET
for the administration of RIG is critical. RIG is the fraction of anti-rabies serum produced in immunized humans (HRIG) or equines
(ERIG), which provides immediate passive immunity. It is administered with proven effectiveness over the initial few days and even
weeks after RABV exposure in humans. RIG must be administered to humans exposed to RABV as a passive immune treatment in
order to provide immediate access to VNAs until the patient’s immune system can begin to produce its own antibody. The
importance of antibodies in controlling the spread of RABV infections is clear when one considers that antibodies are capable of
effectively neutralizing virus that is present in intercellular spaces or in body fluids. Antibodies may bind to virus expressed on the
cell surface, allowing complement- or antibody-dependent cellular cytotoxicity to mediate killing of infected cells.
Antibodies, however, are also implicated in an immunopathologic role in regard to the ‘early death’ phenomenon, which is
associated with rabies in animals and possibly humans. If animals have low levels of virus- or vaccine-induced rabies-specific
antibody at the time they are challenged with RABV, they frequently die earlier than those without antibodies. In an analogous
situation, where humans appear to develop clinical rabies with shorter incubation periods as a result of postexposure immuniza-
tion, compared with individuals who were exposed to rabies but not immunized, the early death phenomenon may also apply.
Inasmuch as the mechanism of early death may resemble the immune cytolysis observed in RABV-infected cells treated with
antiserum and complement in vitro, the immunopathologic effect of antibody would be to accelerate the pathogenesis of the virus.
The efficacy of postexposure immunization and long-term effects of vaccine-induced prophylaxis against rabies seems also to be
linked to the stimulation of a strong CTL response. Induction of CTLs and other effector T-cells during infection with live
attenuated RABV vaccine strains and in response to immunization with inactivated virus is consistent with the observation that
T-lymphocytes, and CTLs in particular, are essential for protection against a lethal dose of RABV. There is no evidence that early
death is associated with rabies-specific T-cells or does any other form of immunopathology appear to be mediated through rabies
immune T-cells.
For further information regarding practical approaches toward rabies prevention, including the rationale for PrEP and PEP,
treatment of wounds, vaccines and vaccine dosage, booster vaccination, and serologic testing, the reader is referred to the
recommendations of the ACIP.

Further Reading
Banyard AC, Hayman DTS, Freuling CM, Műller T, Fooks AR, and Johnson N (2013) Bat rabies. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 215–267. San Diego, CA: Elsevier.
Briggs DJ, Nagarjan T, and Rupprecht CE (2013) Rabies vaccines. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 497–526. San Diego, CA: Elsevier.
Centers for Disease Control and Prevention and Human Rabies Prevention – United States, 2010. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent
human rabies, Recommendations of the Advisory Committee on Immunization Practices (ACIP), Morbidity and Mortality Weekly Report, 59 (RR-2), pp.1–8.
Centers for Disease Control and Prevention, Compendium of Animal Rabies Prevention and Control, 2011, Morbidity and Mortality Weekly Report, 60 (RR-6), pp. 1–17.
Hanlon CA (2013) Rabies in terrestrial animals. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 179–213. San Diego, CA: Elsevier.
 Rabies 19

Hanlon CA and Childs JE (2013) Epidemiology. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 61–121. San Diego, CA: Elsevier.
Hanlon CA and Nadin-Davis SA (2013) Laboratory diagnosis of rabies. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 409–459. San Diego, CA: Elsevier.
Jackson AC (2013) Human disease. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 269–349. San Diego, CA: Elsevier.
Jackson AC and Fu ZF (2013) Pathogenesis. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 299–349. San Diego, CA: Elsevier.
Knobel DL, Lembo T, Morters M, Townsend SE, Cleavland S, and Hampson K (2013) Dog rabies and its control. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 591–615. San Diego, CA:
Elsevier.
Lafon M (2013) Immunology. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 387–408. San Diego, CA: Elsevier.
Lembo T (2013) Blueprint for rabies prevention and control. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 671–681. San Diego, CA: Elsevier.
Moore SM, Gordon CR, and Hanlon CA (2013) Measures of rabies immunity. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 461–495. San Diego, CA: Elsevier.
Nadan-Davis SA (2013) Molecular epidemiology. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 123–177. San Diego, CA: Elsevier.
Rosatte RC (2013) Rabies control in wild carnivores. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 617–670. San Diego, CA: Elsevier.
Rossiter JP and Jackson AC (2013) Pathology. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 351–386. San Diego, CA: Elsevier.
Taylor LH, Costa P, and Briggs DJ (2013) Public health management of humans at risk. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 543–573. San Diego, CA: Elsevier.
World Health Organization (2005) WHO Expert Consultation on Rabies: First Report (2004: Geneva, Switzerland). Geneva: WHO.
World Health Organization (2010) Rabies vaccines: WHO position paper-recommendations. Vaccine 28(44): 7140–7142. http://dx.doi.org/10.1016/j.vaccine.2010.08.082.
World Health Organization (2011) The immunological basis for immunization series: Rabies. Geneva: WHO pp. 1–23.
Rabies in the Americas. Wunner WH (ed.) (2005) Virus Research 111: 1–4.
Wunner WH and Conzelmann K-K (2013) Rabies virus. In: Jackson AC (ed.) Rabies, 3rd edn., pp. 17–60. San Diego, CA: Elsevier.