Lyssavirus Spp. – Rabies Viruses As A Still-Present Problem

The virus of this species constitutes the genotype 2 of the genus Lyssavirus and belongs to the phylogenetic group II. When mammals are infected, it causes a rabies-like illness in them. It occurs in central and southern Africa. In 1956, in Nigeria, LBV was first isolated from the megabats of the family Pteropodidae originating from the island of Lagos. It was also the first discovery of a virus related to RABV. To date no cases of human infection with this species have been documented [7,28].

Genotype 3 of the genus Lyssavirus includes MOKV, which at the same time belongs to the phylogenetic group II. It is isolated from mammals inhabiting the lands of sub-Saharan Africa [58], in which it causes symptoms similar to those induced by rabies. So far, the natural reservoir of this virus has not been identified, but it is assumed that small mammals, mainly cats, constitute it. So far, there have been only two recorded cases of human infection with a virus belonging to this species [14, 25].

This species was discovered for the first time in 1970 in southern Africa in a bat-bitten man who subsequently died of a disease manifesting itself with rabies-like symptoms [55]. DUVV constitutes genotype 4 classified within the phylogenetic group I. In 2006, also in South Africa, the second mortality was documented, when a man was scratched by a bat [40], while in 2007, in Amsterdam, the first recorded mortality case in Europe because of infection with DUVV was recorded. The bats from the suborder Microchiroptera are the probable carrier of Duvenhage virus from which it has been isolated only twice [56].

This virus, belonging to the genotype 5, was isolated in Spain from the bats of the species Eptesicus serotinus. The species is divided into EBLV-1a and EBLV-1b; it occurs throughout Europe – descriptions of infections from France, the Netherlands and Russia are available in the literature. Its carriers are insectivorous bats. By 2000, 630 cases of infections with this virus had been documented and by 2010 – 959 [2, 29, 48, 50, 51, 65].

The species was isolated in 1996 in the UK from a Myotis daubentonif bat. These viruses are found on the European continent, and the hosts, as in the case of European 1 bat lyssavirus, are insectivorous bats [3, 23, 29, 48].

The black fruit bats from the species Pteropus alecto constitute one of the five natural reservoirs of ABLV [5]. This virus belongs to the phylogenetic group I and constitutes genotype 7 of the genus Lyssavirus. It was first identified in 1995 in Australia [53], and a year later the first death resulting from ABLV infection was identified. So far, three deaths have been documented as being caused by the infection with ABLV. All these people developed rabies-like symptoms, after being scratched or bitten by the bats transmitting ABLV [16, 18].

The genomic material of the viruses of the genus Lyssavirus consists of a negative-sense single-stranded RNA molecule which encodes 5 viral proteins. These are: nucleoprotein N, phosphoprotein P, glycoprotein G, polymerase L and core protein M [1, 15]. 4 additional peptides are available via alternative initiation [52]. The leader sequence consists of about 50 nucleotides, preceded by genes encoding the proteins of the virus [65].

The virions are described as “approximately bullet-shaped” with an average length of 100–300 nm, and average diameter of 75 nm. In contrast, their infectious particles have an approximately cylindrical model [1, 15]. They are composed of two structural and functional units. The first of them is enclosed in a lipid envelope covered with approximately 400 G glycoprotein spike-like protrusions (constituting trimers) [65] of 10 nm in length, responsible for the recognition of specific viral receptors on cell membranes. The protrusions constitute the main surface antigens, determining the adsorption and inducing the formation of neutralizing antibodies [32]. The second subunit is formed of an internal, helical ribonucleocapsid (which is a ribonucleoprotein core), twisted symmetrically and composed of genomic RNA, which is closely related to the nucleoprotein (N protein), the L protein (RNA polymerase) and the RNA polymerase cofactor – the P protein.

The M, or core, protein located between the nucleocapsid and the lipoprotein envelope, which is responsible for the overall shape of the virus and is involved in budding – detachment of viral particles from host cells [65]. The N nucleoprotein is the main structural protein of the virus, protecting its RNA against ribonucleases, thus ensuring that it remains in the configuration allowing for transcription [32]. The virus structure is schematically shown in Fig. 1.

Fig. 1.

The genetic material of Lyssavirus is a single-stranded, linear, undivided negative-sense RNA, about 11–12 thousand nucleotides in size [37]. The expression of Lyssavirus genes occurs through RNA-dependent RNA polymerase, which binds the genome contained in the capsid in the leader region. Sequential transcription of each gene occurs through the recognition of start and stop signals contained in the viral genes. Next, the cap is attached to the mRNA and its polyadenylation by L proteins during synthesis takes place [52].

The rabies virus penetrates, multiplies and spreads along neural pathways. It is thus qualified as a member of neurotrophic viruses [36]. The viral replication cycle begins with the attachment of viral G glycoproteins to the host’s specific nicotinic acetylcholine receptors or to the neural cell adhesion molecule. It initiates the infection process and allows clathrin-dependent endocytosis (pinocytosis) of the virus to host cells (internalization). Viruses accumulate in large cytoplasmic vesicles [32, 65]. Further, the virus’s lipid envelope becomes attached to the host cell membrane, which enables the release of the ribonucleocapsid into the cytoplasm [52]. This process is called uncoating [65]. It is promoted by the acidic environment of the vesicle [32]. Both the replication and transcription itself occur in the cytoplasm in Negri bodies [27].

The next stage of replication is the transcription of viral RNA. Then the viral mRNA is translated on free ribosomes in the cytoplasm leading to the synthesis of N, P, M and L proteins [65]. G protein is produced with the Golgi apparatus on ribosomes attached to the membrane, and then delivered, after incorporation into the membrane vesicles, to the cell surface [32]. The replication itself probably starts when there are enough nucleoproteins necessary to separate newly synthesized antigens from genomes [52]. The first stage is the synthesis of a full-length copy of the positive-sense viral genome. When it occurs, RNA transcription becomes continuous, thus ignoring the nucleotide sequences responsible for its possible interruption (STOP codons). Viral polymerase operates from the 3’ end of the genome and continues until a full copy of the genome has been formed. Duplicate strands with positive sense constitute then a matrix for the synthesis of a complete strand with negative sense, which constitutes the viral genome [65].

The final stage of the viral replication cycle consists in the binding of ribonucleocapsid (which consists of the genome associated with the L protein polymerase protein and N protein) with the protein core at the cytoplasmic membrane, which then – after folding it into a condensed form – induces the formation of the form referred to as “approximately bullet-shaped”. Subsequently, with the participation of the host’s ESCRT complexes in the plasma membrane, it is released by budding, thus leading to the disconnection of new virions after covering them with the ribonucleocapsid envelope [32, 52]. The replication cycle of the virus is shown in Fig. 2.

Fig. 2.

The infection occurs after the rabies virus enters a wound resulting from a bite, as well as after contact of contaminated saliva or infected brain of an animal with damaged skin, conjunctiva or mucous membranes. The virus does not penetrate undamaged skin. The most dangerous form of infection is the one effected through the nasal mucous membranes, as the olfactory nerve endings, located very close to the brain, occur therein, making its progress particularly fast. In rare cases, it is also possible to be infected by droplet-aerogenic [36] or iatrogenic routes of transmission [47]. There have also been isolated cases of infection through transplantation of various organs, i.e. lungs and liver, followed by the death of the recipient [48].

After entry, it replicates in tissues (in addition to the nervous tissue) or undergoes direct migration to peripheral nerves, thus penetrating into the central nervous system, to the dorsal root ganglia, through retrograde axioplasmatic transport. When the lyssavirus reaches the brain and spinal cord, a rapid progression of the infection follows, which includes the Purkini cells of the cerebellum, brain stem, hippocampus and the cells of the nuclei in the gyri of the pons. Sensory and motor fibres may be involved in this process, depending on the species of the animal [32]. In the case of bats these wounds are often very superficial (0.1–0.3 mm deep) and they only affect the epidermis, so that people exposed to contact very often do not associate infection with this bite. On the other hand, infected carnivorous animals cause infection through extensive deep wound bites [6]. The incubation period varies from 2 weeks to 6 years, but on average it is 2–3 months. It depends on the distance of the infection site from the brain, the extent of the wound, the dosage of the virus, age and the state of immunity. The estimated rate of virus migration, and thus the rate at which nerve infections progress, is 15–100 mm per day. The virus then travels through the afferent nerves of the peripheral nervous system from the central nervous system to highly-loosened areas (scalp, neck skin, nasal mucosa, renal parenchyma, pancreas lobules, retina, salivary gland, cornea, adrenocortical cortex), which also leads to infection of neighbouring tissues, i.e. secretory tissue of salivary glands [32].

In the pathogenesis of most viral infections, T CD8+ cells are involved, however, in the case of rabies, the virus enters the nerve cells which can lead to inflammation in the central nervous system and result in nerve damage and paralysis. The rabies virus has the features of a superantigen, which leads to the activation of a significant part of T lymphocytes. This indirectly leads to the body’s defensive response to other antigens unrelated to the infection [19].

The rabies virus leads to significant neurological damage after entering the central nervous system. As a result of the infection, an increased expression of chemokines belonging to the cytokine family occurs. In the case of mice infected in laboratory conditions, excessive activity of these proteins leads to the induction of apoptosis of infected cells, increased inflow of inflammatory cells into the central nervous system, and also to the sealing of the blood-brain barrier. The inflammatory process which occurred in mice infected with a low dosage of the virus under laboratory conditions led to the complete elimination of the virus from the brain. In the case of infecting rodents with the rabies virus which did not take place under laboratory conditions, invasion of the central nervous system occurred without initiation of the organism’s immune response [35].

At the time of the occurrence of the first clinical symptoms, whose bellwether example is, in most cases, neuropathic pain in the initial site of infection (caused by viral replication in the dorsal ganglion and ganglionitis), lyssavirus is already present throughout the organism. The main clinical symptoms are associated with the infection of the brain, cerebellum and spinal cord by the virus, and neuron degeneration [32]. Two main clinical pictures of rabies are distinguished: the furious form and the paralytic one [65], where it often happens that the furious form (agitated) proceeds into the paralytic form. After experiencing a period of agitation, in the case of surviving another fit of convulsions by the infected one, paralysis follows, which then leads to inevitable death. One of the forms of the occurrence of the disease is the so-called “silent rabies”, which runs without a form of craziness or is poorly expressed [36]. After approx. 7–10 days after the first symptoms of rabies, the patient enters a coma, after which they die from respiratory failure. After infection of the organism with the participation of Lyssavirus encephalitis occurs in about 80% of cases, which is a fatal agitated form of the disease. In the case of the development of mild paralytic rabies, paralysis is the dominant symptom [47].

In order to fully understand the way in which the organism is infected by the genus Lyssavirus spp., it is necessary to take into account the molecular mechanisms to which these viruses’ impact. The P protein of lyssaviruses may lead to the inhibition of RIG-I (retinoic acid-inducible gene I), belonging to membrane pattern recognising receptors (PRR) involved in the recognition of viruses by the non-specific immune response. The inhibition of RIG-I by the rabies virus P protein prevents the initiation of the anti-virus, apoptotic and pro-inflammatory response of the organism to infection [24, 26, 30, 31]. The P protein of the rabies viruses is also an antagonist of type I interferon (IFN). The P protein of lyssaviruses prevents the phosphorylation and dimerization of IRF3, i.e. the 3rd regulator factor of the interferon responsible for the expression of some genes with antiviral activity [12, 42]. Viral phosphoprotein may also inhibit signalling the STAT-I antiviral pathway, blocking the transport of STAT dimers to the nucleus [9, 63]. In this way, the P protein is presumably the key antagonist of interferons during the infection with the rabies virus, allowing viruses to replicate viruses in infected cells [8, 43].

Another element influenced by rabies viruses, more specifically Rabies virus (genotype 1.) and Mokola virus (genotype 3.), are the light chains of LC8 dynein, or the protein complex responsible for the movement of cell organelles along the microtubules. LC8 is expressed in various cell types, and the P protein of lyssaviruses, through interaction with this protein, probably allows the virus to propagate through the long axons of peripheral nerves from the primary site of infection, eventually enabling it to reach the CNS. LC8 is probably the key molecular link in the pathogenesis of rabies viruses and allows for the interaction of lyssaviruses with the cellular transport of the organism. However, it is not directly responsible for the final entry of viruses into the CNS [41, 54].

Rabies viruses can probably also alter ion channels and occupy cell receptors, and thus modify neurotransmission. In patients with rabies, a decrease of tetrahydrobiopterin (BH4) has been recorded, as well as a pathological decrease in serotoninergic and dopaminergic relays associated with the loss of BH4. BH4 deficiency ultimately leads to a decrease in the fluency of serotonin and dopamine turnover in the brain, and thus to the loss of these neurotransmitters and generalized slowdown of electrical activity in the cerebral cortex. The synthesis of neurotransmitters proceeds with the participation of phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, the essential cofactor of which is BH4. The decrease in BH4 concentration results in a shortage of, among others, adrenaline, noradrenaline, serotonin, dopamine and melatonin [62].

Neuronal nitric oxide synthase is BH4-dependent, hence BH4 deficiency can also lead to potential brain ischemia as a result of impaired cerebral vascularity. In addition, in the case of the viral infection of macrophages, they may release pro-inflammatory factors, including chemokines, interleukins and cytokines as well as nitric oxide. The presence of the rabies virus in the cell also results in facilitating the production of nitric oxide. Increased synthesis of nitric oxide is probably part of the antiviral cell response to infection and is probably one of the factors of innate immune response contributing to the replication of viruses in the cell at an early stage of infection [33, 62].

In most documented cases of rabies in humans, specific antibodies against cells infected with the rabies virus were not detectable until the onset of acute symptoms of infection in the blood serum. Antibodies directed against viral glycoproteins were not detected in the cerebrospinal fluid either. On the other hand, in patients who were found to have antibodies in their serum, their concentrations were very low, described as almost undetectable. A case of a patient suffering from rabies and subjected to a pharmacologically induced coma is documented. This allowed for inducing the humoral response of the body and the generation of IgG antibodies detectable subsequently both in the blood serum and in the cerebrospinal fluid. This patient probably survived owing to the creation of specific antibodies by their body. The G glycoprotein of rabies viruses is the main antigen against which a protective immune system response is induced. There are also other elements, such as cytokines, which play a role in immunizing the organism against rabies. The induction of apoptosis probably correlates with the accumulation of G glycoprotein in infected cells. In turn, apoptosis and subsequent death of these cells results in a strong innate and acquired immune response of the body [13, 22].

Rabies viruses eventually lead to neuronal dysfunction which is probably caused by the destruction of the cytoskeleton integrity and synaptic neuron structures. Some pathogens of the genus Lyssavirus spp. are able to introduce significant changes in the expression of cytoskeletal network proteins of neuronal cells, including tubulin and beta-tropomyosin and vimentin isoforms, as well as cytoskeleton regulatory proteins. The disruption of the cytoskeleton integrity is probably one of the elements of the pathogenesis of rabies viruses. Rabies viruses require the integrity of the cytoskeleton actin network in order to penetrate neurons. Lyssaviruses are additionally able to introduce structural changes in organelles in neurons, and may, after penetration into a neuronal cell, affect their actin cytoskeleton, which physiologically fulfils key neuronal functions [11, 66].

The first symptoms of viral infection are similar to the common cold and include general weakness and fatigue, discomfort, fever, loss of appetite, nausea, vomiting and headache. They can last for up to several days. At the bite site prickling, tingling, pain or itching may also be felt. Other symptoms associated directly with the entry of the virus into the central nervous system may be brain dysfunction, elevated mood, anxiety and confusion. With the progression of infection, one may also experience insomnia, delirium, hallucinations and confusion. In the second phase of the disease, referred to as the paralytic form, acute neurological symptoms – dysarthria (speech disorders), dysphagia (swallowing difficulties), excessive production and salivation and nystagmus may also be observed.

The acute infection period usually ends after 2 to 10 days. At the time of the manifestation of the first symptoms of rabies, the disease will lead to death in most cases, and the symptomatic treatment is only intended to bring temporary relief to the patient. During the development of the full clinical picture of the disease, hydrophobia is also characteristic (20–50% of patients), which is manifested by the occurrence of swallowing muscle contractions and, in extreme cases, also by the whole-body convulsions at the sight or sound of pouring water, and aerophobia [48]. In addition, polyneuritis develops [32, 34, 36, 47].

The prevention of rabies infections aims to create specific immunity. The development of active immunity comes as a result of a series of vaccinations – specific active prevention. The vaccine contains an inactivated virus, which is also a specific antigen designed to generate immune memory by stimulating the immune system. Prevention may also include the generation of passive immunity by administering antibodies along with the injection of human immunoglobulin – specific active-passive prevention [17, 34]. The glycoprotein of the rabies virus (G protein) contains antigenic sites which are the target of both vaccine-induced antibodies and administered immunoglobulin [60].

The obligation to implement rabies vaccination applies to people exposed to infection as a result of contact with an animal infected or suspected of being infected, however, it is also recommended for people who plan to travel to the areas of the endemic occurrence of rabies [17].

Rabies vaccination schedules are constantly updated and published by the WHO. Currently, modern, intramuscularly or subcutaneously administered CCEEV vaccines (Cell Culture Vaccines and Embryonated Egg-based Vaccines) containing inactivated RABV viruses are recommended. They are to be used both in preand post-exposure measures in the form of a series of injections carried out according to the instructions of the manufacturer of the vaccine. The most frequently recommended schedules are 3 doses for prophylactic vaccination and 5 (on days 0, 3, 7, 14 and 28) or 4 doses (on days 0, 3, 7 and 21) for post-exposure vaccination. CCEEV are among the most immunogenic vaccines. They are characterized by safety of use and good tolerance by patients [60].

Numerous anti-rabies programs have been implemented over the years, including the application of highly immunogenic safe vaccines produced in cell culture conditions to people from risk groups, as well as vaccination of foxes and pets – dogs and cats. They have permitted a significant reduction in the incidence of rabies in humans in Europe [48]. Immunoglobulins against the rabies virus can be administered to pregnant and breast-feeding women. All post-exposure prophylaxis measures should be applied to any who qualify according to WHO recommendations. HIV patients who meet certain conditions may also receive a vaccine against rabies. In the case of travellers, the risk of exposure to the rabies virus should be assessed individually, taking into account primarily the presence of rabies at the destination and planned activities, such as being in caves where presence and direct contact with bats is possible [64].

The Main Sanitary Inspectorate in Poland in the Protective Vaccination Program for 2018 differentiates the way the vaccine is administered after a bite due to the type of contact and the animal’s health condition [17].

The relatively long incubation period of the rabies virus in the human body creates the possibility of avoiding clinical consequences even after direct exposure [60]. The slow development of rabies allows for triggering an active immune response, thus providing protection against further infection [32]. This effect is possible only through the implementation of fast and effective post-exposure prophylaxis.

The first preventive step should be to wash the wound with soap and water or a substance which can inactivate the virus. Cleansing the wound leads to a reduction of the pathogen inoculum within it. Then administration of a series of vaccinations in order to provoke the organism to produce antibodies is recommended. Their presence reduces the risk of RABV entering the peripheral nerves. The next step in the preventive post-exposure measures is to administer immunoglobulin against the rabies virus in the wound area. It will provide necessary antibodies until the organism begins its production and will allow for neutralizing the virus at the site of its penetration [32, 60].

Until 2012, less than 10 cases of human recovery from rabies had been documented, and only in 2 of them previous pre-or post-exposure prophylaxis had been implemented [34]. In each case of contaminating the mucous membrane or open wound with saliva or brain tissue of an animal suspected of developing rabies, preventive measures should be applied [32]. When properly performed, immediate action after exposure to RABV shows 100% efficacy in the prevention of rabies. The disease and ultimately death therefrom mainly affects people who did not have access to fast, adequate post-exposure prophylaxis [60].

In any case of the suspicion of rabies, it must be confirmed by laboratory tests. Magnetic resonance imaging may also help in the diagnosis, during which moderate signal amplification in the T2 sequence is observed within particular areas of the brain – the stem, hypothalamus, hippocampus, as well as white and grey matters. In patients being in the coma stage, the signal is amplified after using gadolinium. In differential diagnosis of rabies, computed tomography does not play any role.

In laboratory tests, a lyssavirus infection can be ascertained by detecting an antigen of the virus, specific neutralizing antibodies in the serum or cerebrospinal fluid. Another method is the isolation of the virus itself or its nucleic acid. A viral antigen is detected by the direct immunofluorescence method, which is called a “gold standard”. For this purpose, saliva, corneal impression preparations (sensitivity 46%) or material prepared after skin biopsy collected from the neck are primarily used. In the last case, the sensitivity of the method is 50–94% and increases with the duration of the infection. A similar sensitivity is also found in the detection of lyssavirus nucleic acid in saliva using the PCR method with reverse transcriptase.

None of the tests is 100% effective, reliable and sensitive, so in order to confirm the infection performing several tests at the same time is recommended. In post-mortem diagnosis, the same methods are applied as in the intravital one, but the test material is mainly the brain tissue [48]. In order to confirm or exclude rabies, tests are also performed to detect intracytoplasmic inclusion bodies in infected neurons. They contain Negri bodies, or aggregates of viral ribonucleocapsids. They occur in up to 90% of the examined brain tissues of infected people [32].

The Milwaukee protocol was developed by dr. Rodney Willoughby Jr. and constitutes a report on the experimental form of human rabies treatment in a 15-year-old girl from Wisconsin in the USA – Jeanny Giese, who developed rabies a month after having her left index finger bitten by a bat, which took place on 12 September 2004 [44]. Only 5 cases of people who did not die after being infected with the rabies virus had been known until this year. Each of these people underwent post-exposure prophylaxis in the form of administering a suitable vaccine. The girl from the USA, subjected to experimental therapy according to the Milwaukee protocol, was the first person who survived without immediate earlier post-exposure prophylaxis [4]. The wound was cleansed only with hydrogen peroxide. The first neurological symptoms were noticed after 37 days of the incident – the girl was admitted to a local hospital with high fever (39°C), speech disorders, left hand muscle contraction, double vision, nausea and vomiting, no other symptoms of flu or cold. The neurologist also established lack of mobility and bilateral paralysis of the abductor nerve in her. After diagnosing rabies by isolating antibodies against rabies (and not a live virus) during the second day of hospitalization, she was subjected to experimental treatment by being introduced into a coma and being kept in it for as long for as possible to protect the brain from further damage and thus enable the girl’s immune system to produce antibodies necessary to fight the virus. She was administered ketamine, ribavirin, amantadine and midazolam [44]. Ketamine is an NMDA receptor antagonist with neuroprotective effects on strokes and brain injuries, with efficacy proven in animals. In the therapy of rabies, it was used as an experimental drug, with no proven effect on human models. Theoretically, it was supposed to protect the brain against the excitotoxicity of glutamic acid, the main neurotransmitter in the central nervous system, however, in the case of rabies, no efficacy has been demonstrated even on an animal model [67]. Ribavirin, showing limited possibilities of penetrating the central nervous system, was administered in doses capable of increasing the level of proteins in the cerebrospinal fluid, and thus demonstrating the level of blood-brain barrier permeability [44]. Administering large doses of anaesthetics to the girl in order to introduce her into a pharmacological coma with a therapeutic effect was supposed to reduce the metabolic activity of her body and maintain the nerves in the best condition. This measure also had an experimental form, with efficacy confirmed only on animal models in the case of brain strokes and damage, as well as epileptic states. The coma was also supposed to prevent the instability of the autonomic nervous system occurring in the case of infection with the rabies virus [67].

After 8 days of hospitalization, signs of initiating a fight against the virus by the girl’s immune system were observed after performing a lumbar puncture. An increased level of anti-rabies antibodies was demonstrated, which allowed for commencing her recovery from coma. On the 12th day of hospitalization, a flu that did not respond to standard treatment revealed itself. On the 15th day of treatment, the room temperature at which the patient stayed was lowered by 5.5°C, which resulted in a decrease of the girl’s body temperature by 3.6°C. The patient was found to be free of the rabies virus after 31 days in the hospital, and there was no serious damage to her mental abilities after the illness and treatment. About 5 months after the hospitalization, the girl was aware and communicative, but she was diagnosed with dystonia (involuntary movements causing bending and twisting of individual parts of the body), dysarthria and ballism (sweeping, violent movements of the limbs, leading to unstable gait) [44].

The girl was probably infected with an extremely mild type of rabies virus transmitted by vampire bats of the subfamily Desmodontinae [61] or the bite occurred in a place far away from the central nervous system, which enabled her to survive rabies after the treatment planned according to the Milwaukee protocol. Consecutive 6 or more attempts at the treatment of symptomatic rabies with this type of treatment failed. On February 4, 2009 in Texas there was another successful therapy according to the Milwaukee protocol, also after a boy was bitten by a vampire bat [44].

The Milwaukee protocol has undergone many modifications since its creation. The last one was aimed at eliminating ribavirin and barbiturates from the pool of agents used to treat rabies with this method [61]. In addition, one of the side effects when using ribavirin may be a delay in the production of antibodies [4]. Only midazolam and ketamine have remained of the original medicines, which currently makes the Milwaukee protocol similar to a standard procedure in intensive care units around the world. In 2013, the World Health Organization officially announced that there is no confirmed, effective method of treating rabies [61]. One of the theories of eliminating infection is the induction of the organism’s inflammatory response with chemokines and IFN in the main role. It has found confirmation only in the case of mice infected with low doses of the virus under laboratory conditions, however, it was not confirmed for infections with Lyssavirus spp. under environmental conditions [35].