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NEW THIS SUNDAY: TWO ON RABIES

Saturday, 6th of July 2013

TWO ON RABIES

 

• RABIES VACCINATION IN DEVELOPING COUNTRIES

http://www.childsurvival.net/?content=com_articles&artid=486

 

·       HUMAN RABIES: NEUROPATHOGENESIS, DIAGNOSIS, AND MANAGEMENT

Original Text

Prof Thiravat Hemachudha MD a    , Gabriella Ugolini PhD b, Supaporn Wacharapluesadee PhD a, Witaya Sungkarat PhD c, Prof Shanop Shuangshoti MD a, Jiraporn Laothamatas MD c 

The Lancet Neurology, Volume 12, Issue 5, Pages 498 - 513, May 2013

From the conclusions: “Physicians and neurologists need to promote awareness of the absolute requirement of prompt administration of evidence-based prophylactic treatment to all individuals exposed to RABV. This approach is still deficient or even non-existent in many parts of the world. Equally, if not more importantly, is the need to control this disease in its main vector, the dog. The knowledge and means to do so are available, but the will and appropriate support from societies and governments are often not.”

 

Best viewed at http://www.thelancet.com/journals/laneur/article/PIIS1474-4422(13)70038-3/fulltext#bib14

Copyright © 2013 Elsevier Ltd All rights reserved.

Rabies is an almost invariably fatal disease that can present as classic furious rabies or paralytic rabies. Recovery has been reported in only a few patients, most of whom were infected with bat rabies virus variants, and has been associated with promptness of host immune response and spontaneous (immune) virus clearance. Viral mechanisms that have evolved to minimise damage to the CNS but enable the virus to spread might explain why survivors have overall good functional recovery. The shorter survival of patients with furious rabies compared with those with paralytic rabies closely corresponds to the greater amount of virus and lower immune response in the CNS of patients with the furious form. Rabies virus is present in the CNS long before symptom onset: subclinical anterior horn cell dysfunction and abnormal brain MRI in patients with furious rabies are evident days before brain symptoms develop. How the virus produces its devastating effects and how it selectively impairs behaviour in patients with furious rabies and the peripheral nerves of patients with paralytic rabies is beginning to be understood. However, to develop a pragmatic treatment strategy, a thorough understanding of the neuropathogenetic mechanisms is needed.

Rabies caused by rabies virus (RABV) genotype 1 is one of the most common fatal infections worldwide. It is mainly associated with dog bites in Europe, Asia, and Africa and with bats in the Americas.1 Two-thirds of patients infected with dog RABV variants present with classic furious rabies (characterised by fluctuating consciousness and changed mental status, phobic or inspiratory spasms, and autonomic stimulation signs). The remaining third develop paralytic rabies, which resembles Guillain-Barré syndrome, although progression to coma, myoedema, and bladder incontinence clearly differentiate these two disorders (table).2, 3 Rabies associated with bat RABV variants has atypical features, such as focal brainstem signs, myoclonus, hemichorea, and signs and symptoms of Horners syndrome.3, 4

  

TableTable image 

Features of furious and paralytic rabies in patients infected with dog RABV variants and in naturally infected dogs

In other infectious encephalitides, damage can be shown by gross pathology, whereas in rabies the CNS seems healthy post mortem, with only minimal neuronal loss and varying degrees of evidence of inflammatory reactions. This finding might be explained by the unique multilevel strategy that RABV has evolved to prevent viral clearance; RABV uses replication mechanisms that delay severe compromise of host cell metabolism and has the capacity to prevent apoptosis and evade innate and adaptive immune responses, which otherwise would promote changes in blood—brain barrier permeability and combat viral progression.10 MRI of the brain of patients with rabies reveals only subtle changes and the absence of gadolinium-enhanced lesions.6, 7 With contrast imaging, mild to intense inflammation is noted mainly in patients with rabies contracted after organ transplantation and in comatose patients infected with dog RABV variants.6, 7, 11, 12

Attempts to treat symptomatic patients infected with dog RABV variants with therapeutics and intensive care support are usually unsuccessful. A few patients who survived infection with bat RABV variants with good functional recovery had evidence of an early immune response, measured by neutralising or non-neutralising antibody to RABV in blood and CSF, with no RNA or virus detected in samples of biological fluids or hair follicles.13—18 Therefore, a good outcome might depend on the promptness of the host response in the eradication of virus in both the CNS and periphery.14

In this Review, we summarise relevant data acquired from our studies of patients infected with dog RABV variants and from animal models associated with RABV genotype 1 infection. We extrapolate these data to explain the various clinical phases in furious and paralytic rabies caused by infection with dog RABV variants. We use a dog model with naturally acquired rabies to explain clinical diversities; this model exhibits clinical patterns similar to those seen in man, with an incubation period of weeks to months, and it allows in-vivo studies of CNS abnormalities and blood—brain barrier status by MRI. Results suggest that the immune system is unlikely to have a role in the acceleration of death, especially in furious rabies.3, 19

The clinical stages of rabies are: incubation, prodrome, acute neurological signs, coma, and death.3 Average survival (from clinical onset to death, with partial or no intensive care support) of 80 patients with furious rabies and 35 with paralytic rabies after infection with a dog RABV variant in Thailand between 1988 and 2004 was 5·7 and 11 days, respectively (data compiled from previously published reports; table).2 These survival times did not substantially differ from those reported in India between 1980 and 2007.20, 21 Survival can be extended to 1 month or longer in patients receiving intensive care support.12

Weakness at the bitten extremities might be evident at initial presentation, although subsequent progression can be in the form of either furious or paralytic rabies.5, 22 Although all cardinal features of furious rabies (eg, fluctuating consciousness, hydrophobia or aerophobia, inspiratory spasms, signs of autonomic dysfunction) are seen in most patients with this form of the disease, they might not be evident at the same time, and disappear during coma. Comatose patients with furious rabies (or those near to coma) develop flaccid limb weakness, which has been frequently misinterpreted as paralytic rabies. Conversely, lower motor neuron ascending weakness with only motor disturbance is the initial manifestation of paralytic rabies,5 in which consciousness is preserved until the preterminal phase (table).2

Atypical signs and symptoms of rabies associated with infection with either bat or dog RABV variants have been increasingly recognised.3, 4, 13, 23 Presentation of transverse myelitis as neuromyelitis optica or tetanus-like symptoms with locked jaw have been reported.24, 25

The transfer of RABV-containing saliva from a bite from an infected animal is the most efficient route of transmission. Other routes of transmission include: inhalation of aerosolised RABV; tissue and organ transplants; handling and skinning of infected carcasses; and contamination of an open wound, scratch, abrasion, or mucous membrane by infected saliva or neural tissue.3 The efficiency of bite transmission depends on virus inocula and viral tissue tropism. The likelihood of infection with dog RABV variants is highest after deep bites that reach the muscle, because the virus can only infect motor endplates in the muscle, and uptake by sensory and sympathetic nerve endings in the muscle does not occur.26—34 Entry via the motor route is determined by the presence at the neuromuscular junction (but not at sensory or autonomic endings) of the nicotinic acetylcholine receptor (on the postsynaptic site), which binds RABV, promoting neural cell adhesion molecule-mediated uptake by motor endplates.28, 35 Viraemia does not occur.36 Whether a sensory pathway occurs after infection with dog RABV variants through a cutaneous lesion (ie, no access to motor endplates) is still unclear, because confirmatory experiments have not yet been done. Postexposure prophylaxis (PEP) with vaccine alone is needed in patients with minor skin lesions inflicted by a dog infected with RABV.

By contrast, patients with a negligible scratch to the skin inflicted by a bat infected with RABV are at very high risk of infection3 because bat RABV variants, unlike dog RABV variants, are able to multiply in epidermal cells in vitro.37 The higher incidence of local neuropathic pain in patients with bat RABV variant rabies (70%) than in dog-related cases (30%),3, 4 and signs and symptoms of Horners syndrome and other atypical rabies features, might suggest additional (or alternative) transmission of bat RABV variants via sensory or sympathetic skin innervation, which should be investigated.28

Incubation period

The incubation period or eclipse phase can vary from weeks to years, but lasts 1—2 months on average. The incubation period is an intriguing feature in rabies and does not depend on bite location or clinical form.3 During most of the incubation, RABV lies in the muscle as a so-called smouldering, or low-replication-rate, infection38that remains confined to the inoculated portion of the muscle.28, 31 Immune recognition at this focal site might not be adequate. Dendritic cells—ie, antigen-presenting cells that have a key role in triggering both innate and adaptive immune responses39—are insufficiently activated by naturally acquired (street) RABV infection.40—42The long incubation period is probably caused by low titre inocula and by the existence of endogenous RNA-silencing mechanisms or microRNAs (miRNAs) that slow down viral replication in the muscle.43 In fact, the muscle-specific miRNA mir-133, which is predicted to bind to both the nucleoprotein and glycoprotein transcripts of RABV,44 has been shown to substantially reduce expression of rabies viral protein in transfected Neuro-2a cells.45 With high titre inocula, however, RABV is able to infect motor endplates without previous replication in the muscle, as shown in rodent and primate models;28—31 moreover, with high titre inocula, uptake without previous replication can also occur by motor axons after inoculation directly into nerves.46, 47 This finding might explain the exceptionally short incubation times in human rabies that usually occur in association with penetrating nerve injury.3

Mechanisms for clinical diversity in furious and paralytic rabies in man

By the time a patient develops the first prodromal symptoms, such as fever, flu-like symptoms, and gastrointestinal disturbances, the virus is already widely disseminated throughout the CNS.5, 6 A more localised prodrome or neuropathic pain (eg, paraesthesia, allodynia, burning sensation) is a sign of dorsal root ganglia dysfunction as a result of immune attack (table).5 A case study of the clinical features of a patient with furious rabies is described in panel 1 and figure 1, and of a patient with the paralytic form in panel 2.

Panel 1

Clinical features of a patient with furious rabies

A man aged 50 years who had been bitten by a dog on his left wrist 7 weeks earlier5 had prodromal symptoms of severe aching pain and paraesthesias on his left hand and arm (figure 1). MRI of the brain, 3 days after onset, showed an ill-defined mild hyperintensity change involving the deep and subcortical white matter, hippocampal gyri, brainstem, and cervical cord.6 Slight progression of MRI disturbances was noted on day 7. CSF examination showed two lymphocytes and 40 mg/dL protein. Rabies viral RNA was detected in his saliva 2 days after admission, but not in the CSF specimen.48 He survived for 8 days.

   

  

Figure 1 Full-size image (44K) Download to PowerPoint

Clinical diversity in a patient with furious rabies

See panel 1 for background information. On day 3 after onset (A), the patients mental state was clear but he had pain in his left arm (arrows). The only abnormality on electrodiagnostic studies was the presence of abundant fibrillations and positive sharp waves in left C5—C8 limb and cervical paraspinal muscles (+). His motor and sensory functions were intact. Diminished-to-absent deep tendon reflexes were noted in the left arm. On day 5 (not shown), sensory nerve action potential (SNAP) amplitudes had reduced by about 50% in the left upper limb nerves compared with those on the right side. Diminished pinprick sensation up to his elbow was noted (shaded area in B) and the pain became intense. By day 6 (B), SNAP amplitudes had reduced further on the left upper extremity, and fibrillations and positive sharp waves involving bilateral C5—C7 limb and paraspinal muscles had progressed (+). Results of motor conduction studies, including F-waves, remained normal. Mild weakness of left hand and wrist muscles was detected. Pain was less severe than in previous days and tolerable. The diminished sensation in the area up to the left elbow (shaded area in B) had progressed slightly, along with absence of deep tendon reflexes and joint position sense of the left arm. He was confused and disoriented (circle in B), and he died on day 8. Reproduced from Hemachudha and colleagues,23 by permission of Springer.

Panel 2

Clinical features of a patient with paralytic rabies

A man aged 34 years who had been bitten by a dog on his right ankle 2 months earlier had severe itching and piloerection on his right leg that progressed to his left leg within 2 days after presentation.5 On admission (day 3 after onset), he showed no detectable weakness. Reflexes were 1+ (diminished) in the right lower limb and 2+ (normal) in the other limbs. Hypoaesthesia to pinprick sensation was present on his right leg up to the groin. By day 4, paraparesis was noted, and reflexes were absent in both lower limbs and highly diminished in the upper limbs. On day 6, facial diaparesis and bulbar dysfunction were noted, and he was later intubated. He became agitated on day 7 and died on day 9. His CSF was acellular with 70 mg/dL protein. RNA was detected in the CSF on day 3 but not on day 7; saliva was negative for RNA on days 3 and 7.48

Pathways of propagation of RABV and relation with clinical prodrome

The spinal propagation sequence of RABV in man after a deep bite to the left wrist (as in the patient with furious rabies in panel 1 and figure 1) has been extrapolated from a combined analysis of studies of RABV propagation in animal models28,29,31,32,38,49—55 (ie, intramuscular inoculation of naturally acquired RABV in skunks38,53 and hamsters,50—52 and intramuscular inoculations of fixed RABV challenge virus standard [CVS] strain or derived recombinants in rodents29, 54, 55 and primates28, 31, 32, 49) and knowledge of spinal connectivity (figure 2).56—58 The relation between the spinal propagation sequence and prodromal symptoms and signs in the patient with furious rabies described in panel 1 and figure 1 is summarised (figure 3). Notably, when classic experimental studies of naturally acquired RABV propagation50—53 are revisited in the context of recent neuroanatomical investigations, taking into account current knowledge on CNS connectivity, the propagation properties of naturally acquired RABV of canine origin seem to be indistinguishable from those of fixed RABV (CVS strain), which is consistent with retrograde transneuronal transfer via the motor route.

   

  

Figure 2 Full-size image (211K) Download to PowerPoint

Pathways of propagation of RABV to and from the spinal cord

The diagram depicts propagation after a deep bite by a rabid animal on the left wrist (as in the patient described in panel 1 and figure 1). Spinal cord line drawing (top left) shows location of spinal segments. Fine dashed black lines in main image show borders of spinal laminae (I—X). Rabies virus (RABV) uptake from the muscle occurs exclusively via the motor route (first-order neurons [1°]), with no infection of sensory (proprioceptive dorsal root ganglia) and autonomic neurons innervating the same muscle.28, 29, 49 Centripetal propagation to the spinal cord is represented by solid blue lines (solid green for antagonists at C8) and coloured circular markers (colour-coded according to synaptic order, see key on left side of figure); black arrows show transport direction. RABV propagates exclusively by retrograde transneuronal transfer, from infected motor neurons (1°, grey-coloured circular marker, C8 left) to monosynaptically connected spinal interneurons and dorsal root ganglia populations (second-order neurons [2°], black-coloured circular markers).29, 54, 55 Dorsal root ganglia populations include: large proprioceptive dorsal root ganglia neurons (group Ia and II afferents) of low cervical or first thoracic segments ipsilaterally;54, 56, 57 short propriospinal neurons ipsilaterally in laminae V—VII (lateral part) of cervical or upper thoracic segments, which receive both dorsal root ganglia afferents and corticospinal, rubrospinal, and reticulospinal inputs and innervate arm muscles ipsilaterally;56, 57 and long propriospinal neurons bilaterally in laminae VII, VIII, and X (including cholinergic populations in lamina X29 and partition cells54), which connect nearly the entire length of the spinal cord and innervate motor neurons of axial, proximal, and distal muscles bilaterally. The long propriospinal neurons receive both dorsal root ganglia afferents and reticulospinal, vestibulospinal, and corticospinal pathways, are involved in a variety of reflex pathways, and are part of spinal locomotor networks.56—58 Among 2° in lamina VII are Renshaw cells, Ia inhibitory interneurons, and Ib and II interneurons (in VII—VI). Further steps of retrograde transneuronal transfer from 2° result in infection of higher-order interneurons and dorsal root ganglia populations. Proprioceptive dorsal root ganglia innervating motor neurons of antagonist muscles (3°, solid green pathway at C8) are also infected (via Ia interneurons, 2°, which are pathways for reciprocal inhibition). Centrifugal (anterograde) propagation of RABV (open coloured circular markers and dashed red lines, or dashed green lines for antagonists at C8) is a slow (distance-dependent) process (see key on right side of figure), causing infection of other interneurons and motor neurons. Centrifugal transport of RABV from the spinal cord occurs in motor and sensory axons50—52(large red arrows), leading to infection of extraneural organs via their sensory innervation.38,50—52,59DRG=dorsal root ganglia. Ia IN=Ia inhibitory interneurons. IML=intermediolateral cell group. LPN=long propriospinal neurons. MN=motor neuron. pc=partition cell. rc=Renshaw cells. SPN=short propriospinal neurons.

   

  

Figure 3 Full-size image (99K) Download to PowerPoint

Association between prodromal symptoms and signs and spinal propagation of rabies virus in a patient with furious rabies

1°=first-order neuron. 2°=second-order neuron. 3°=third-order neuron. 4°=fourth-order neuron. 5°=fifth-order neuron. DRG=dorsal root ganglia. SNAP=sensory nerve action potential.

Centripetal propagation

From the infected muscles, centripetal propagation of RABV occurs only via the motor route and is mediated exclusively by retrograde transneuronal transfer from motor neurons, which begins 2 days after uptake from motor endplates and proceeds at high speed (12-h intervals for each synaptic step, irrespective of distance).28,49 By day 4 after onset of infection in the motor neurons, connected spinal interneurons and ipsilateral (low cervical and upper thoracic) dorsal root ganglia innervating the bitten arm are heavily infected.53, 54 Dorsal root ganglia infection first involves large neurons in dorsal root ganglia (proprioceptive Ia and II afferents and other large myelinated afferents targeting infected motor neurons and interneurons; figure 2); proprioceptive dorsal root ganglia innervating motor neurons of antagonist muscles are also infected (via Ia inhibitory interneurons). Small dorsal root ganglia neurons (unmyelinated and small myelinated afferents), which target higher-order interneurons in the dorsal horn, are infected later in the course of infection (figure 2). Via spinal interneuronal pathways, the infection rapidly involves bilateral cervical and thoracic dorsal root ganglia supplying the contralateral arm, the neck and back, and, later, lumbosacral dorsal root ganglia (figure 2).51, 53 In parallel, retrograde transneuronal transfer leads to infection of brainstem and corticospinal pathways targeting the infected spinal motor neurons and interneurons, and higher-order CNS neurons.28, 29, 32, 49, 50, 53, 55

Centrifugal propagation

A slow phase of centrifugal (anterograde) propagation can only begin 2 days after replication in each infected neuronal population, and leads to viral transport to the ventral and dorsal roots and centrifugal spread to extraneural organs via their sensory innervations38,50—52,59—ie, to muscle spindles (via large dorsal root ganglia neurons), skin (via cutaneous afferents from large and small dorsal root ganglia neurons), and to immune and visceral organs, including the salivary glands, heart, and blood vessels (via small dorsal root ganglia neurons). Centrifugal viral propagation to visceral organs59 is probably mediated by dorsal root ganglia neurons that supply, via dichotomising axons, both somatic and visceral nerves and organs (eg, left ulnar nerve and the heart) and several visceral organs.60, 61 These pathways form an anatomical substrate for referred pain and cross-organ sensitisation. Centrifugal propagation to extraneural organs is related to the topography of viscerotopic sensory innervation60 and is distance-dependent; because anterograde axonal transport of RABV is inefficient,28, 49 it might take weeks to reach remote organs. Progressive functional changes in infected sensory innervation of extraneural organs (including the heart and autonomic plexuses) might explain organ dysfunction and possibly even dysautonomia in patients with rabies. Although extraneural organs from which RABV can be detected also receive autonomic innervation, the autonomic nerve supply is unlikely to contribute to centrifugal spread. Experimental evidence after RABV inoculation into skeletal muscles shows that autonomic involvement (of spinal preganglionic neurons in the central autonomic area) is a rare and indirect event that is not indicative of peripheral uptake and might only begin quite late, via intraspinal pathways.28, 29 Similarly, post-mortem ultrastructural findings in a case report of a patient with furious rabies showed that cytoplasmic virus inclusions were abundant in sensory ganglia but rarely detected in sympathetic ganglia.62

During centrifugal spread, RABV antigen can also be carried to lymph nodes,63 both directly via their sensory innervation64 and indirectly via virus budding from axons51 and draining of antigen to the lymph nodes. We postulate that high virus load in extraneural organs due to centrifugal spread38,50—52,59 helps dendritic cell activation and migration, triggering T-cell stimulation in lymph nodes and the adaptive immune response. In both cases (ie, direct or indirect transport of virus to lymph nodes), virus and activated dendritic cells will first reach regional lymph nodes, which explains why T-lymphocyte activation occurs earlier in regional lymph nodes than in lymph nodes at other locations.65 Although the clinical stages of rabies are too advanced to enable any definite conclusions to be drawn, it is possible in a patient such as the one with furious rabies presented in Figure 1,Figure 2 and panel 1, that once RABV has reached spinal motor neurons, it would take 3—4 days to infect the cervical dorsal root ganglia innervating the arm and skin of the neck,51, 52 a further 2 days before centrifugal transport in sensory axons begins, another 3 days for transport to neck hair follicles and skin,51, 52 and maybe 4 more days before reaching the regional (neck or axillary) lymph nodes. A further 4 days might then pass before T-cell activation in lymph nodes occurs66 and maybe another 3 days for ganglionopathy and neuropathic pain to begin (prodrome onset). The total estimated period (3 weeks) is well below the 7-week incubation in the patient described in figure 1 and panel 1, suggesting that he might have had low-level infection in his muscles for up to 4 weeks. The extensive centripetal and centrifugal viral propagation that had already occurred in this patient during incubation was shown by MRI changes (panel 1) and recovery of virus from his saliva at prodrome onset, which is mediated only by centrifugal spread to the salivary glands via their innervation.28, 67

The sequence of prodromal symptoms and signs closely parallels the proposed viral propagation sequence. Motor neurons and connected proprioceptive dorsal root ganglia supplying the bitten arm are the first nerve cells to be infected and the first to show progressive functional changes (Figure 2, Figure 3). Preferential entry via the motor route could explain why denervation potentials (detectable by day 3) precede sensory loss (day 5) and are initially localised to the bitten arm and ipsilateral neck paraspinal muscles (by day 3) but extend to the opposite arm and paraspinal muscles by day 6. Similarly, early and heavy infection of proprioceptive (Ia) dorsal root ganglia of agonist and antagonist muscles of the bitten arm (see above and figure 2), which mediate deep tendon reflexes, might explain the diminished deep tendon reflexes of the same arm by day 3 and their subsequent extinction (Figure 1, Figure 3). Late onset of infection of cutaneous (vs proprioceptive) dorsal root ganglia might explain why sensory function in the bitten arm is still intact on day 3, but affected by day 5 (figure 3). Finally, focal deficits (eg, weakness of left hand or wrist muscles, loss of deep tendon reflexes and position sense, pain attenuation) by day 6 might signify progressive neuronal dysfunction and ganglionopathy. The same logic might also apply to prodromal symptoms and signs in patients with paralytic rabies. An association between the location of the bite and the topography of prodromal symptoms and signs has been noted, which could be explained by RABV propagation in spinal pathways and dorsal root ganglia that are synaptically connected with motor neurons innervating the bitten limb. For example, in the patient with paralytic rabies described in panel 2, RABV propagation from motor neurons supplying the bitten right ankle to connected spinal interneurons and (lumbosacral) dorsal root ganglia might explain why pain, diminished deep tendon reflexes, and hypoaesthesia are initially localised to the right leg. Preferential infection via the motor route might also explain why Wallerian-like degeneration and inflammatory changes were much more severe in the ventral than in the dorsal spinal roots in a patient with paralytic rabies.68

Death in furious rabies: immune-mediated or virus-mediated mechanism?

The immune response to RABV has been hypothesised to contribute to the disease process, on the basis of experimental evidence that survival can be prolonged in immunosuppressed mice, whereas paralysis and death ensue with the return of immune responsiveness after passive transfer of immune serum or cells.19 The finding that patients who had cellular immunity to RABV antigen and raised serum cytokine concentrations tended to have furious rather than paralytic rabies19, 69 led to the belief that furious manifestations are immune-mediated (and associated with production of proinflammatory cytokines and nitric oxide),3 and that vaccination should be withheld in patients with symptomatic rabies because it might accelerate death.70, 71 However, accumulating evidence seems to prove otherwise. Attempts to prolong the clinical course of patients with rabies with high-dose steroids, antithymocyte globulin, or other immunosuppressive drugs have failed.14, 72Moreover, studies in dogs with early-stage furious or paralytic rabies did not show an exaggerated immune response in infected brains.8 On the contrary, cytokine and chemokine mRNA transcripts were barely detectable in the brain, particularly in dogs with furious rabies.8 A greater amount of rabies viral RNA was reported in the brains of furious dogs than in those of paralytic dogs, suggesting that furious manifestations are virus-mediated and associated with more extensive propagation of RABV to the brain than are paralytic manifestations.

Impaired neural tract integrity at brainstem and survival in paralytic rabies

In-vivo MRI examination showed greater signal abnormalities in the brainstem, hypothalamus, and temporal lobe of dogs with paralytic rabies than in the same parts of the brain in dogs with furious rabies, despite paralytic dogs having a lower viral titre overall (table and figure 4).8 No evidence of blood—brain barrier leakage was reported in dogs with either furious or paralytic rabies. MRI studies showed that abnormalities in the brainstem were prominent in paralytic dogs with early-stage rabies,7, 8 which might suggest that viral propagation is interrupted in paralytic dogs, resulting in less virus reaching the cerebral hemispheres. Post-mortem examination of brain (five furious and five paralytic cases) and spinal cord specimens (three of each clinical form) was done in ten naturally infected rabid dogs, including some that had MRI.9 To observe early pathological changes, all animals were killed shortly after developing signs of rabies. In both clinical forms, caudal—rostral distribution of RABV antigen, from greatest to least, was reported in the following order: spinal cord, brainstem, cerebellum, midline structures (caudate, thalamus), hippocampus, and cerebrum. By contrast, RABV RNA was most abundant in cerebral midline structures. More RABV antigen was detected in many more CNS regions in dogs with furious rabies than in those with paralytic rabies. Significantly higher RNA concentrations were noted in the cerebral cortex, thalamus, midbrain, and medulla of dogs with the furious subtype than in those with the paralytic subtype, whereas RNA concentrations in the spinal cord were similar in both clinical forms. Inflammation reported in the brainstem of dogs with paralytic rabies substantially correlated with increased MRI disturbances and increased extent of CNS immune response (table and figure 4).7—9 Thus, brainstem inflammation might impede viral propagation towards the cerebrum, resulting in longer survival in patients with paralytic rabies.

   

  

Figure 4 Full-size image (83K) Download to PowerPoint

MRI in canine rabies

Images are mid-sagittal fluid-attenuated inversion recovery (FLAIR) MRI images of the hypothalamus, midbrain, brainstem, cerebellum, and upper cervical cord of a dog with early-stage paralytic rabies (A and C) and a dog with early-stage furious rabies (B and D). Also shown are coronal FLAIR images of the temporal lobe, frontal lobe, thalamus/hypothalamus, and basal ganglion of a dog with early-stage paralytic rabies (E) and a dog with early-stage furious rabies (F). The dog with paralytic rabies has moderate-to-substantial abnormal hypersignal T2 change at the hypothalamus (green arrow in A and E), dorsal midbrain (white arrow in A), brainstem (asterisk in A), and upper cervical cord (double white arrows in C). Also noted is moderate hypersignal T2 change at bilateral temporal lobes (asterisks in E) sparing the frontal and parietal lobes. The dog with furious rabies has less well defined mild-to-moderate diffuse hypersignal T2 change involving the hypothalamus, midbrain, brainstem, and upper cervical cord (white arrows in B, D, and F) and cerebellum (asterisk in B). It has more diffuse but less well defined mild hypersignal T2 change involving the temporal lobes, frontal lobes, and parietal lobes (asterisks in F) than the paralytic dog. Reproduced from Laothamatas and colleagues,8 by permission of Informa Healthcare.

Diffusion tensor imaging (DTI) provides quantitative values compared with normal controls and has greater sensitivity than basic MRI for the detection of microstructural and macrostructural damage (neural tract integrity), as shown by diminished fractional anisotropy and increased fluid-attenuated inversion recovery (FLAIR) signals, respectively,7 and it is useful for the assessment of blood—brain barrier status. Our preliminary DTI data in rabid dogs suggest similar findings to those seen on MRI, with evidence of compromised neural tract integrity in the brainstems of paralytic dogs.7 Mean diffusivity or blood—brain barrier leakage was not increased in either furious or paralytic dogs with early-stage rabies. However, cytotoxic brain oedema (decreased mean diffusivity) was noted almost exclusively in paralytic dogs.7

Specific virus variants in furious and paralytic rabies

One hypothesis to explain clinical diversities in rabies would be the presence of furious and paralytic virus variants. However, a bite from the same dog was shown to have caused furious rabies in one patient and the paralytic form in another, which provides evidence against this hypothesis,19 although, theoretically, this finding would not rule out viral pathogenicity changes due to spontaneous point mutations after exposure. Mutations of the glycoprotein gene at amino acid position 333 (Arg) abolish virulence,73 whereas a substitution in the same gene at 194 (Asn) enhances pathogenicity.74 Other substitutions at positions 318 (Phe) and 352 (His), related to p75 neurotrophin receptor binding, might affect viral maturation and transport into the cell,35, 75 whereas substitutions at positions 273 (Glu) and 394 (Gln) of the RABV nucleoprotein gene enhance immune evasion and increase pathogenicity.76

Analysis of RABV nucleoprotein, phosphoprotein, and glycoprotein genes from samples obtained from patients with either furious or paralytic rabies did not show specific patterns.77 Mutations in the RABV samples did not differ between the two groups at positions 333 (Arg), 194 (Asn), 318 (Phe), or 352 (His) of the G gene, at positions 273 (Gln) and 394 (Glu) of the N protein gene, or at the carboxyl-terminal PDZ domain-binding motif, which is responsible for neuronal survival and apoptosis.78

However, this does not rule out the theory that mutations in the virus explain clinical diversity, because only the sequences of the glycoprotein, nucleoprotein, and phosphoprotein genes were analysed in brainstem samples, whereas other genes involved in pathogenicity (eg, the matrix protein gene) were not examined.79, 80

Poor immune response in rabies-infected CNS

The CNS cannot mount an adaptive immune response to RABV because it does not contain any primary immune organs (ie, no antigen presenting cells and classic lymphatic drainage); the adaptive immune response has to be triggered in the periphery. Yet, infected neurons and glial cells are able to mount innate antiviral type I interferon (α or β) and inflammatory cytokine responses after recognition of viral RNA by two classes of innate sensors: the endosomal transmembrane Toll-like receptors (TLRs) 3 and 7/8, and the cytoplasmic retinoic acid inducible gene 1-like helicases RIG-I (also known as DDX58) and MDA5 (also known as IFIH1).81—83Interferon α and interferon β exert antiviral functions via JAK/STAT signal transduction pathways,84 whereas the major transcription factor for proinflammatory cytokines, such as tumour necrosis factor α (TNFα) and interleukins, is nuclear factor κB (NF-κB), which also supports early interferon transcription (interferon β, interferon α4).83, 85

Despite being attacked by different defence mechanisms, RABV successfully invades its host and reaches the brain (figure 5). Although neurons have the machinery to sense RABV infection and trigger innate immune responses,86 the virus has evolved several strategies to escape or lower activation of innate sensors and the antiviral effects of interferons. These strategies might allow preservation of neuronal integrity so that the virus can propagate between neurons. Human brain neurons express the innate sensor TLR3,87 which can trigger cell death in the presence of interferons.88 However, the virus uses TLR3 as an evasive strategy,10 by sequestering it in Negri bodies where it plays a part in RABV multiplication (figure 5).89 The presence of RABV also activates the innate sensors RIG-I and MDA590, 91 and triggers type 1 interferon (via activation of interferon regulatory factor [IRF] 3 and 7 in association with activator protein 1 [AP-1] and NF-κB). However, the RABV phosphoprotein counteracts interferon antiviral effects, because it prevents transcription of interferon α/β genes by blocking phosphorylation of IRF3 and IRF7 by the kinases TBK1 and IKK-i (also known as IKBKE)92 and it also inhibits STAT signalling, the pathway by which interferon exerts antiviral activity,83,93—96whereas activation of NF-κB is not disturbed (figure 5). The virus nucleoprotein (amino acid positions 273 and 394) is also important for evasion of host RIG-I-mediated antiviral response.76 Thus, interferon induction in RABV-infected CNS is reduced97 and neuroinflammation is moderate.

   

  

Figure 5 Full-size image (102K) Download to PowerPoint

RABV immune-evasion mechanisms

The diagram depicts rabies virus (RABV) inhibition of innate immune response (A) and evasion of adaptive immune response (B) in the CNS. In infected neurons, RABV prevents activation of innate sensor Toll-like receptor 3 (TLR3) by sequestering it in Negri bodies (A). In the neuronal cytoplasm, viral RNA is recognised by immune sensors RIG-I and MDA5 (A, centre-left), which normally triggers transcription of innate antiviral type I interferon α/β via formation of a protein complex (not shown, including adaptor protein IPS-1 together with TRAF3, TBKBP1, NAP1, TANK, TRADD, RIP1, and FADD) that triggers phosphorylation of IRF3/7 by TBK1/IKK-i. Phosphorylated IRF3/7 is transported to the nucleus to induce transcription of interferon α/β in conjunction with activator protein (AP-1) and nuclear factor κB (NF-κB); AP-1 is activated via mitogen-activated protein kinases and NF-κB by NEMO (not shown). The RABV phosphoprotein (P, in red circle) alters transcription of interferon α/β genes by blocking phosphorylation of IRF3/7 and its nuclear import (red crosses), while activation of AP-1 and NF-κB is not disturbed. The RABV phosphoprotein also inhibits the STAT signalling pathway. Interferon α/β binds to cell surface receptors (grey, IFNAR1/2) and induces STAT phosphorylation (p) by kinases JAK1/TYK2 (not shown); phosphorylated STAT/IRF9 complex is imported to the nucleus, where it activates interferon-stimulated genes (ISGs) that have antiviral activities. The RABV phosphoprotein prevents transcription of ISGs by binding to phosphorylated STAT in the cytoplasm, blocking its nuclear import, and by binding to the STAT/IRF9 complex in the nucleus (mediated by a shorter version of phosphoprotein).83Activated T cells and monocytes can cross the blood—brain barrier and invade the CNS (B), despite intactness of this barrier in patients with rabies. Surface expression of the immunosubversive molecules B7-H1, HLA-G, and Fas ligand (dark blue markers) by infected neurons leads to binding of T cells expressing the corresponding ligands, rapidly followed by death of migrating T cells (red cross).10

RABV is also able to evade the adaptive immune response in the CNS. Activation and entry into the CNS are not limiting factors for T cells and monocytes, which can infiltrate the CNS despite an intact blood—brain barrier (figure 5). After mice were infected with a virulent RABV strain, their brains were infiltrated with T cells expressing a marker of activation (Cd69) and Crmp2 (also known as Dpysl2), a marker of T-cell polarisation and migration.98 Invading T cells and monocytes, however, undergo apoptosis shortly after entry into the infected brain parenchyma (figure 5). Post-mortem immunohistochemical studies of the brains of human beings with rabies revealed that leucocytes were the only cells undergoing apoptosis.99 In post-mortem human brains infected with a vampire bat RABV variant, only infiltrating adaptive immune T cells (CD4+ and CD8+) were apoptotic, whereas natural killer cells, macrophages, astrocytes, or neurons were not.100 Paradoxically, RABV uses the innate immune response to induce apoptosis of infiltrating T cells. This strategy was demonstrated in a transgenic mouse model overexpressing Lgp2 (also known as Dhx58) to impair the RIG-I-mediated innate immune response; after RABV infection, lower morbidity and more viral clearance in the brain were noted in mice overexpressing Lgp2 than in a C57BL/6 strain of mice, with reduction of infiltrating CD4+ T cells but less disappearance of infiltrating CD8+ T cells,101 showing that host innate immune response favours the infiltration of T cells but simultaneously promotes CD8+ T-cell elimination. Elimination of infiltrating T cells is mediated by upregulation of immunosubversive molecules on the neuronal surface, such as B7-H1 (also known as CD274)102 and HLA-G,103, 104 which are interferon dependent, and Fas ligand, which is interferon independent.97, 105 Surface expression of B7-H1, HLA-G, and Fas ligand by infected neurons triggers death of migrating T cells expressing the corresponding receptors (figure 5).97

Although an intact blood—brain barrier in rabies is not an obstacle for migration of activated lymphocytes into the CNS, efficient entry, particularly of B cells, needs disruption of the blood—brain barrier.106—108 Failure to open the blood—brain barrier and deliver immune effectors was a crucial factor in the lethality of the virus in mice infected with a silver-haired bat RABV variant.107 Intrathecal production of rabies antibody by infiltrated B cells is needed for viral clearance from the CNS in mice infected with attenuated RABV.106

Neuroinflammation might inhibit the neuroinvasiveness (ie, ability to invade the CNS) of RABV. Comparative analysis of cytokine and chemokine mRNA transcripts in 12 brain regions of dogs with naturally acquired, early-stage rabies revealed greater inflammatory cytokine transcription in the dogs with paralytic rabies than in those with the furious form (table).8 Interferon γ and interleukin 1β were detected exclusively in paralytic dogs. Granulocyte-macrophage colony-stimulating factor and interleukins 2, 4, 5, 8, and 10 were not detected in dogs with furious rabies. TNFα was detected in dogs with furious rabies and in those with the paralytic form, but was considered significantly increased only in those with paralytic rabies, whereas only CCL2 and VEGF were significantly increased in dogs with furious rabies. Interleukin 6, TNFα, interleukin 1α, interleukin 1β, and interferon γ cytokines have been reported to increase blood—brain barrier permeability in vitro and in vivo (by direct brain injection into piglets and rats) and to augment leucocyte CNS infiltration.109 In the dogs depicted infigure 4, the blood—brain barrier remained intact despite the presence of cytokines. Inflammatory cytokine transcription was associated with a lower viral load (in all CNS regions) in paralytic dogs than in furious dogs. Greater disturbances seen with MRI in dogs with paralytic rabies were associated with greater immune responses and less brain neuroinvasiveness (figure 4) than in furious dogs.8 During the late stage of disease, however, almost no cytokines were detected in the CNS in both rabies types, and similar viral quantities were detected in both forms.8

Proteomic profiling studies of the brains of dogs with late-stage rabies showed upregulation of immunoglobulin heavy chain in the brainstem of paralytic dogs and interferon α4 and SARM1 protein in the hippocampus of dogs with furious rabies.110 CRMP-2, a marker of activated infiltrating T cells, was downregulated in the spinal cord in both rabies forms, but was upregulated in the brainstem of dogs with paralytic rabies. Examination of 25 brains of patients with furious and paralytic rabies revealed no correlation between inflammation or viral antigen distribution and expression of interleukin 1β and TNFα (in microglia, macrophages, and lymphocytes).21 This might be explained by advanced stage of disease at examination.

A case study111 described a patient infected with a bat RABV variant who received treatment with a neuroprotective regimen of midazolam, ketamine, and propofol together with rabies immunoglobulin, ribavirin, and amantadine; the regimen was discontinued 48 days later because no neurological recovery occurred, and the patient died more than 2 months after disease onset.111 CSF analysis before death (3 weeks after cessation of the regimen, which is potentially immunosuppressive) and post-mortem brain examination showed robust inflammatory responses in the brain and CSF. The investigators propose that such a response at a very late stage might be related to immune reconstitution inflammatory syndrome.111

Preservation of neuronal integrity

RABV is transcribed and replicates in neuronal cell bodies and dendrites. Maintenance and preservation of these structures and the axon, where the complete enveloped particle is transported, might be as important for the virus as its ability to evade immune responses.10 RABV infection causes neuronal dysfunction rather than neuronal death.112, 113 Survival of infected neurons is ensured by the capacity of virulent RABV strains to prevent apoptosis by maintaining viral gene expression below threshold levels and by interfering with proapoptotic factors.73, 112, 114 Prevention of apoptosis is the hallmark of naturally acquired RABV infection; it depends on restricted expression of the glycoprotein protein and the glycoprotein gene sequence, including four amino acids at the carboxyl-terminal PDZ domain-binding motif that can bind cellular PDZ proteins, which control cell polarity and apoptosis.73, 78, 115 The matrix protein amino acid residues Arg 77 and Glu 81 might be associated with delayed apoptosis and increased pathogenicity of naturally acquired RABV.80 Thus, neurons that have been infected with RABV for several days (asymptomatic period) do not exhibit cytopathic changes and remain metabolically viable in vivo, expressing their neurotransmitters and transporting other markers.29—31,33,49,116 Yet, when severe clinical disease has developed, wild-type RABV infection in mice results in changes to host protein expression, particularly expression of proteins involved in ion homoeostasis and docking and fusion of synaptic vesicles to presynaptic membranes,117 which might lead to the defective neurotransmission recognised in rabies.113 Neuronal dysfunction might also be related to oxidative damage.118, 119 In moribund mice, mild structural damage involving exclusively neuronal processes (beading and fragmentation of axons and dendrites, with vacuoles that correspond with swollen mitochondria) was recognised;120 it was shown in vitro to be the result of oxidative stress, through virus-induced inhibition of NF-κB signalling, which plays a crucial role in axonal growth, neuronal survival, and antiviral responses.118, 119

Studies of human brains infected with naturally acquired RABV show that the virus is capable of preserving neuronal integrity to support its propagation. Apoptosis was evident in inflammatory cells but not in neurons.99,121 Cytochrome c leakage and evidence of mitochondrial membrane permeabilisation were absent in spinal cord and brainstem in patients with rabies, explaining the absence of anterior horn cell weakness and preservation of consciousness until the preterminal stage.122

Pathogenesis of paralytic rabies in man

Peripheral nerve dysfunction, axonopathy, or myelinopathy causes weakness in patients with paralytic rabies.2,68, 123 Axonopathy was reported in three patients with paralytic rabies (one Chinese, one Mexican, and one Thai, infected in their respective countries) who had an acute motor axonal Guillain-Barré syndrome variant.5,22, 68 Wallerian-like degeneration and inflammatory changes were more abundant in the ventral than in the dorsal nerve roots in the Chinese patient.68 Deposition of IgG and complement proteins on RABV-positive axons was also shown, suggesting an antibody-mediated complement attack. In support of this finding, one patient with furious rabies who received intravenous human rabies immunoglobulin developed weakness of the facial, limb, and neck flexor muscles 36 h after administration.124 However, the mechanism is unclear, since CSF rabies neutralising antibodies were not detected in this patient, or in another 30 Thai patients infected with dog RABV variants, 14 of whom had paralytic rabies.2 Antiglycolipid antibodies were not detected in one patient with axonal and two with demyelinating paralytic rabies;5 this finding is important because the exact pathogenic mechanism in paralytic rabies is unknown, and one hypothesis is that the humoral immune response plays a part.

Segmental demyelination and remyelination, or myelinated nerve fibre loss of the spinal roots and peripheral nerves, were characteristic findings in 11 patients with paralytic rabies.123 None of these patients had Wallerian-like degeneration as the only pathological feature. Such demyelination was absent in patients with furious rabies.125 Another two patients with electrophysiological evidence of demyelinating Guillain-Barré syndrome variants had inflammation of the dorsal and ventral spinal nerve roots that was more severe than in the spinal cord.5

MRI abnormalities provide clues for differential diagnosis with other encephalitides, in terms of preferential sites and extent of involvement (brain only or whole neuroaxis), presence of oedema (cytotoxic or vasogenic) or minute haemorrhage, and extent of signal intensity.7 MRI of patients with rabies can vary, since abnormalities can result from infection, host reaction, or complications (hypoxia, shock, bleeding, and metabolic derangements).3,5—7,126,127 Typically, MRI abnormalities are hypersignal T2 changes without contrast enhancement involving the spinal cord, brainstem, thalamus, limbic structures, and white matter during the non-comatose phase (figure 6). Both clinical forms of rabies in man have similar MRI features (table).6, 7 Lesions in the brachial plexus, spinal cord, and nerve roots are already seen at the prodromal stage as signal intensity abnormalities or enhancement.6 During the comatose phase, widespread T2 hyperintense lesions in the brainstem and forebrain can be seen; these are probably due to virus-induced neuronal injury and superimposed hypoxic insult.7 Neuronal injury might be related to oxidative stress, as shown in an animal model infected with fixed RABV (CVS strain).118 Blood—brain barrier breakdown is evident only during the late or comatose phase of the disease, as gadolinium-enhanced lesions along the brainstem and other midline structures.6, 7 MRI images are similar in patients with dog or bat RABV variants in terms of location and pattern of abnormal signal intensity.22, 128, 129 MRI might help with diagnosis when it is combined with clinical data and staging of disease severity and with knowledge of whether the patient is also affected with metabolic, electrolyte, or haematological disturbances or has a compromised cardiovascular status. Lesion characteristics at different stages have been reviewed in detail elsewhere.7

   

 

Figure 6 Full-size image (139K) Download to PowerPoint

MRI in patients with rabies

Images depict a conscious patient with furious rabies (A—C) and a comatose patient with paralytic rabies (D—F). (A) Coronal spin echo T2-weighted MRI of the brain showing diffuse ill-defined mild hypersignal T2 change involving the cerebral white matter and brainstem (asterisks), hippocampi, and temporal lobes (white arrows). (B) Ill-defined hypersignal T2 change seen in spinal cord (white arrow). (C) Enhancing left brachial plexus (white arrow) between the anterior and middle/posterior scalene muscles of the bitten limb. (D) Marked diffuse hypersignal T2 change involving the basal ganglia, hippocampi (asterisks), and cerebral white matter (green arrow). (E) Moderate ill-defined enhancing hypothalamus (green arrow), tectal plate and midbrain (white arrowhead), and the dorsal pons and medulla and anterior cervical cord (white arrows). (F) Moderate enhancement of the intrathecal ventral and dorsal nerve roots (white arrows). Reproduced from Laothamatas and colleagues,7 by permission of Elsevier.

Antibody assay might not be useful in the diagnosis of rabies associated with dog RABV variants. RABV neutralising antibody in serum (and not in CSF) was detected in only six of 31 Thai patients and in none of 43 unvaccinated patients with dog RABV variants from Cambodia, Madagascar, and Senegal.3, 130 Immune recognition of rabies nucleoprotein and neutralising antibody titre development is inadequate in these patients.131 21 of 43 patients in the USA (1960—2009) had neutralising serum antibody; 18 were infected with bat RABV variants, two with dog variants, and one with a bobcat variant.13 Only recently have antibody assays been considered in dog RABV variant endemic countries because of the potential to assess chance of recovery, particularly when viral RNA is not detected.15—18 However, patients with neutralising antibody in their serum or CSF but no detected viral RNA have died despite intensive care support.13

RABV RNA can be detected in saliva, extracted hair follicles or a biopsy sample of skin tissue from the nape of the neck that contains hair follicles, CSF, and urine.48,130,132—134 Descriptions of the biological samples and molecular methods for detection have been detailed elsewhere.135 Frequently used techniques, such as hemi-nested reverse transcription (RT) PCR (targeting the large polymerase [L] gene) and TaqMan real-time RT-PCR (nucleoprotein gene), had similar degrees of sensitivity when serial dilutions of purified RNA from RABV-infected dog brain tissue were used.136 When previously positive clinical samples (saliva) from ten patients with rabies, and RABV-infected dog and human brain tissue, were tested, results with each technique were similar.136However, the volume of tissue might be crucial for adequate sensitivity.136 Hemi-nested RT-PCR assay of nuchal skin biopsy specimens containing hair follicles (diameter roughly 4 mm; total volume 20 mm3) is almost 100% sensitive.130 Similar sensitivity was obtained when at least three serial saliva samples were examined or when three types of specimen (saliva, CSF, urine, or hair follicles) were assayed simultaneously.134, 136 This result, however, might apply with certainty only to patients with furious rabies, since results were negative in half (three of six) of patients with the paralytic form.134 Negative results were higher in patients with paralytic rabies than in those with furious rabies: saliva samples (seven of nine (78%) vs eight of 53 [15%]), CSF (three of five [60%] vs 14 of 25 [56%]), urine (five of five (100%) vs 20 of 36 [56%]).134 Hair follicle tests showed negative results in 12 of 25 (48%) patients with the furious form compared with one of one with the paralytic form.134

Recovery after rabies has been reported in four patients.15—18 Prediction of which patients are likely to recover is not possible, although most survivors with good functional recovery had bat RABV variant rabies.15—18Patients infected with bat RABV variants (who died or survived) had clinical manifestations that differed in many respects from those who have survived classic forms of dog RABV variant rabies.3, 4, 13, 23 Differences in cellular tropism37 or in the routes of spread, or both, might account for these discrepancies.28 Because only a few experimental studies are available of the propagation of bat RABV variants in vivo,37, 137 it is unclear whether higher chances of survival in patients with bat RABV variant rabies might be related to differences in the modalities of propagation of bat versus dog RABV variants. Importantly, all four rabies survivors described in the scientific literature with good recovery, with or without treatment, had a vigorous and early immune response, with autosterilisation (ie, no detected virus or RNA in tissue or biological fluids) and rabies antibodies detected in serum and CSF.13—18 Two received coma induction therapy,16, 18 one had standard intensive care support,15 and another had presumptive abortive infection17 and did not receive any intensive support. Two patients, one of whom did not receive coma induction therapy, had non-neutralising antibodies, suggesting that other mechanisms played a part in eradication of the virus.17, 18

The suffering that patients with furious rabies go through cannot be readily explained by pain in the throat or phobic spasms. We have routinely used benzodiazepines, barbiturates, ketamine, or even intravenous morphine. However, deepening of consciousness by the use of sedatives to the extent that ventilatory support is needed should be avoided.

So far, no proven standard treatment for rabies exists. Combination therapy with rabies immunoglobulin (polyclonal or monoclonal) plus vaccination, ribavirin (antiviral drug), ketamine (with some NMDA receptor antagonistic effect), and interferon α has been advocated.138 Large doses of intravenous human rabies immunoglobulin (25 g for 4 consecutive days) were given to a patient with furious rabies. Anti-rabies antibody was not detected in the CSF after treatment, confirming the intactness of the blood—brain barrier;125 however, this treatment was able to attenuate the autonomic symptoms.

The Milwaukee protocol initially aimed to induce coma with an electroencephalographic stage of burst suppression.16 Various sedatives (midazolam, barbiturates, ketamine), amantadine, which is supposed to reduce brain excitotoxicity,139 and ribavirin were given to a patient who then recovered with minimal sequelae.16 However, following the protocol did not save more than two dozen fully alert, previously healthy, young, or middle-aged patients with symptomatic rabies.12,14,129,140—142 The role of excitotoxic mechanisms and the benefit of ketamine in in-vitro and in-vivo experiments are not supported by the data and scientific evidence.12, 14, 140, 143, 144 The current Milwaukee protocol consists of ketamine and midazolam, similar to what is used by physicians in dog RABV variant endemic countries to relieve suffering and dysautonomia. Coma induction is no longer recommended in the protocol. Nimodipine has also been added to the protocol to relieve vasospasm.145, 146 However, data have been conflicting; neuroimaging and post-mortem examination did not reveal findings compatible with territorial spasm in patients with dog RABV variant rabies.6,7, 147 Whether territorial spasm might be specifically related to bat RABV variants is not known. In view of autonomic disturbances in patients with rabies, nimodipine should be administered with extreme caution because it potentially poses the risk of severe hypotension and shock.3

Rabies is unique because it carries the highest fatality rate among all viral encephalitides. The mechanisms that allow this virus to invade and partly hide from the hosts immune defences, sometimes for extensive periods of time, before overwhelming the host are fascinating for scientists and clinicians.

Improved understanding of the mechanisms underlying rabies neuropathogenesis in man and animal models is necessary for the development of new therapeutic approaches. Temporary blood—brain barrier disruption by the use of ultrasound and microbubbles, and therapeutic miRNAs or nanoengineered molecules, together with generalised or regional brain cooling methods are prospective treatment options.14, 45, 148, 149 A live-attenuated triple-glycoprotein RABV variant is a promising vaccine candidate for both pre-exposure and postexposure prophylaxis of rabies because it induces immune mechanisms capable of containing experimental CNS infection with pathogenic RABV.150, 151 Any new drugs or treatment protocol should be proven not to pose any potentially harmful risk to already seriously ill patients.

Physicians and neurologists need to promote awareness of the absolute requirement of prompt administration of evidence-based prophylactic treatment to all individuals exposed to RABV. This approach is still deficient or even non-existent in many parts of the world. Equally, if not more importantly, is the need to control this disease in its main vector, the dog. The knowledge and means to do so are available, but the will and appropriate support from societies and governments are often not.

We searched PubMed for English language articles published from 1967, to Dec 1, 2012, containing the terms “rabies” in conjunction with other key terms, including “encephalitis”, “human”, “virus”, “pathophysiology”, “pathology”, “treatment”, “propagation”, and “transneuronal”. Data for this review also came from references on neural connectivity and those contained within older relevant review articles. Most of the review articles that we have cited contain clinical data of many individual cases or case series, as well as many original references of basic science studies and experiments in animal models.

Contributors

All authors contributed to the scientific literature search and the writing and revision of the paper.

Conflicts of interest

We declare that we have no conflicts of interest.

Acknowledgments

Research by the authors described in this Review article was sponsored by the Thailand Research Fund (DBG5180026, RDG5420089), the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (HR1160A-55), the Thai Red Cross Society, the US Naval Health Research Center BAA-10-93 under cooperative agreement number W911NF-11-2-004, the Centre National de la Recherche Scientifique (CNRS), France, and the European Union (QLRT-2001-00151, EUROKINESIS, and BIO4-CT98-0546, TransVirus). The views and conclusions contained in this document are those of the authors.

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a WHO Collaborating Centre for Research and Training on Viral Zoonoses, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand

b Neurobiology and Development (UPR3294), Institute of Neurobiology Alfred Fessard, Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France

c Advanced Diagnostic Imaging (AIMC) and Department of Radiology, Ramathibodi Hospital, Faculty of Medicine, Mahidol University, Bangkok, Thailand

Correspondence to: Prof Thiravat Hemachudha, WHO Collaborating Centre for Research and Training on Viral Zoonoses, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok 10330, Thailand