**5.3 Disease manifestations in humans**

Humans present with a spectrum of diseases ranging from inapparent infections to lethal encephalitis. However, human infections are typically mild or asymptomatic, and severe encephalitis is less commonly seen. Fever, headache, convulsions, disorientation, ataxia, and mental depression appear in a subset of symptomatic cases, but primarily in patients under 15 years of age (de la Monte, et al., 1985; Rivas, et al., 1997; Weaver, et al., 1996). In the event neurological symptoms occur and the patient survives, sequelae are common (Weaver, et al., 2004). Virus has been isolated from throat swabs, serum and brains. Most of what is known of onset of disease has been determined based on accidental infection of laboratory workers where incubation period appears to be two to five days followed by sudden appearance of flu like symptoms though reports from natural disease outbreaks vary (CFSPH, 2008; Steele & Twenhafel, 2010). Acute disease typically lasts approximately four to six days. However, the disease can be biphasic with a secondary fever developing four to eight days after onset. During this second phase, neurological symptoms develop, though again a range is present from somnolence and mild confusion to seizures, ataxia, paralysis, and coma (Tsai, 1991). Following recovery from acute disease, patients develop generalized asthenia lasting one to two weeks. CSF protein and liver enzymes are typically elevated with cell counts in the CSF ranging from 12 to 900 WBC/mm3. An EEG tracing in one patient identified diffuse irregular slowing (Johnson, et al., 1968).

Gross findings in neurological cases include cerebral edema, but gross pathology of infections is poorly described in the literature (de la Monte, et al., 1985; Johnson, et al., 1968; Steele & Twenhafel, 2010). Fatal human cases are histopathologically characterized by edema, congestion, meningitis, and encephalitis in the brain. Vasculitis and hemorrhage are more rarely found (Johnson & Martin, 1974; Paessler & Weaver, 2009; Weaver, et al., 2004). Lymphocytes, mononuclear cells, and neutrophils infiltrate the meninges with cells extending into the Virchow Robins space and neuropil in a subset of cases. In the peripheral organs in the majority of cases, interstitial pneumonia associated with cellular infiltration, alveolar hemorrhage, congestion and edema present in the lung with diffuse hepatocellular degeneration and few infiltrates presenting in the liver (Walton & Grayson, 1988). In lymphoid tissues, lymphocyte degeneration, lymphoid depletion and follicular necrosis are accompanied by infiltrates of neutrophils as well as vasculitis (Steele & Twenhafel, 2010).

#### **5.4 Disease manifestations in experimental models**

Mouse and NHP models make the largest contribution to the understanding of VEE pathogenesis. However, hamsters and guinea pigs are also susceptible to infection, and early studies in these models initiated understanding of the host response to infection.

#### **5.4.1 Mouse model**

Mice are highly susceptible to VEEV infection (Holbrook & Gowen, 2008; Paessler & Weaver, 2009; Walton & Grayson, 1988; Zacks & Paessler, 2010). The mouse model mimics

Paessler & Weaver, 2009; Zacks & Paessler, 2010). Long thought to be a dead end host with levels of viremia below the limit able to reinfect mosquito populations, humans may be capable of acting as the primary vertebrate host for some strains of VEEV. Namely, VEEV ID strains circulate in an urban cycle in Peru with transmission occurring in a primary cycle between humans and mosquitoes. Restriction to geographic range is based primarily on

Humans present with a spectrum of diseases ranging from inapparent infections to lethal encephalitis. However, human infections are typically mild or asymptomatic, and severe encephalitis is less commonly seen. Fever, headache, convulsions, disorientation, ataxia, and mental depression appear in a subset of symptomatic cases, but primarily in patients under 15 years of age (de la Monte, et al., 1985; Rivas, et al., 1997; Weaver, et al., 1996). In the event neurological symptoms occur and the patient survives, sequelae are common (Weaver, et al., 2004). Virus has been isolated from throat swabs, serum and brains. Most of what is known of onset of disease has been determined based on accidental infection of laboratory workers where incubation period appears to be two to five days followed by sudden appearance of flu like symptoms though reports from natural disease outbreaks vary (CFSPH, 2008; Steele & Twenhafel, 2010). Acute disease typically lasts approximately four to six days. However, the disease can be biphasic with a secondary fever developing four to eight days after onset. During this second phase, neurological symptoms develop, though again a range is present from somnolence and mild confusion to seizures, ataxia, paralysis, and coma (Tsai, 1991). Following recovery from acute disease, patients develop generalized asthenia lasting one to two weeks. CSF protein and liver enzymes are typically elevated with cell counts in the CSF ranging from 12 to 900 WBC/mm3. An EEG tracing in one patient identified diffuse

Gross findings in neurological cases include cerebral edema, but gross pathology of infections is poorly described in the literature (de la Monte, et al., 1985; Johnson, et al., 1968; Steele & Twenhafel, 2010). Fatal human cases are histopathologically characterized by edema, congestion, meningitis, and encephalitis in the brain. Vasculitis and hemorrhage are more rarely found (Johnson & Martin, 1974; Paessler & Weaver, 2009; Weaver, et al., 2004). Lymphocytes, mononuclear cells, and neutrophils infiltrate the meninges with cells extending into the Virchow Robins space and neuropil in a subset of cases. In the peripheral organs in the majority of cases, interstitial pneumonia associated with cellular infiltration, alveolar hemorrhage, congestion and edema present in the lung with diffuse hepatocellular degeneration and few infiltrates presenting in the liver (Walton & Grayson, 1988). In lymphoid tissues, lymphocyte degeneration, lymphoid depletion and follicular necrosis are accompanied by infiltrates of neutrophils as well as vasculitis (Steele & Twenhafel, 2010).

Mouse and NHP models make the largest contribution to the understanding of VEE pathogenesis. However, hamsters and guinea pigs are also susceptible to infection, and early studies in these models initiated understanding of the host response to infection.

Mice are highly susceptible to VEEV infection (Holbrook & Gowen, 2008; Paessler & Weaver, 2009; Walton & Grayson, 1988; Zacks & Paessler, 2010). The mouse model mimics

habitat of the mosquito vector and primary vertebrate host (Watts, et al., 1998).

**5.3 Disease manifestations in humans** 

irregular slowing (Johnson, et al., 1968).

**5.4 Disease manifestations in experimental models** 

**5.4.1 Mouse model** 

both human and equine disease with mice developing neurotropic disease characterized by lethal encephalitis and lymphotropism following a biphasic disease course (Gardner, et al., 2008; Gleiser, et al., 1962; Steele, et al., 1998). Following peripheral routes of infection, mice display human like disease with progression from infection of the lymphoid tissue and ultimate destruction of CNS tissues. Clinical symptoms in mice include: lethargy, huddling, dehydration, weight loss, tremors, and paralysis or paresis with a minority of animals developing seizures (Davis, et al., 1994; Grieder, et al., 1995).

The lymphotropic nature of VEE results in severe myeloid depletion in rodents and lymphocyte destruction in lymph nodes and spleen (Davis, et al., 1994; Grieder, et al., 1995; Steele & Twenhafel, 2010; Zacks & Paessler, 2010). Peripheral infection is not present late in disease, but high levels of infectious virus are found in the CNS with death in immunocompetent mice occurring five to seven days after infection (Ludwig, et al., 2001; Vogel, et al., 1996).

Encephalitic pathologies vary based on background of mouse, route of inoculation and strain of virus (Ludwig, et al., 2001; Ryzhikov, et al., 1991; Steele, et al., 1998; Steele, et al., 2006; Stephenson, et al., 1988; Vogel, et al., 1996). Thus, based on study design, encephalitic pathologies range from mild neutrophilic infiltration to neuronal degeneration, necrotizing vascultis and Purkinje cell destruction. Lesions appear that are surrounded by necrotic cellular debris, perivascular cuffs composed of mononuclear cells, rarefaction of the neuropil and infiltration of neutrophils lymphocytes and macrophages. These lesions spread following the virus by an approximately 24 h delay from olfactory bulb to more caudal regions (Jackson, et al., 1991; Jensen & Jackson, 1966; Ludwig, et al., 2001; Ryzhikov, et al., 1991; Steele, et al., 1998; Steele, et al., 2006; Stephenson, et al., 1988; Vogel, et al., 1996). Once in the CNS viral tropism is primarily neuronal though CNS macrophages as well as astrocytes may become infected with VEEV, but do not appear to be a primary target (Jackson & Rossiter, 1997a; Schoneboom, et al., 2000b; Steele, et al., 1998; Steele, et al., 2006). As a result, neuronal damage is extensive and has been ascribed to both necrosis and apoptosis (Jackson & Rossiter, 1997b; Jensen & Jackson, 1966; Schoneboom, et al., 2000b; Schoneboom, et al., 2000d; Steele, et al., 2006). Neuropathology may also be attributed to bystander activation and death as damage to glial cells, lympohcytolysis and astrocytosis are reported in areas where virus is not detected (Davis, et al., 1994; Grieder, et al., 1995; MacDonald & Johnston, 2000b; Schoneboom, et al., 2000a; Schoneboom, et al., 2000c).

Routes of infection mimicking natural, mosquito borne infection utilize SC or footpad inoculation results in infection of DC, the primary cell type for VEE infection, at the site of inoculation. These cells then carry the virus to the DLN where VEE begins replication approximately four hours after infection (Aronson, et al., 2000; Davis, et al., 1994; Grieder, et al., 1997; Grieder, et al., 1995; Grieder & Nguyen, 1996b; MacDonald & Johnston, 2000a). The virus enters the blood stream by 12 hours and reaches high levels of serum viremia followed by infection of other tissues, particularly lymphoid. As such, VEEV presents in the spleen, gut and nasal-associated lymphoid tissues, thymus, bone marrow, and non-draining lymph nodes(Grieder & Nguyen, 1996a; Vogel, et al., 1996). Histological lesions found in peripherally infected animals are both neuronal and non-neuronal (Aronson, et al., 2000; Grieder, et al., 1997; Grieder, et al., 1995; Grieder & Nguyen, 1996a; Steele & Twenhafel, 2010).

Aerosol or IN infection results in a direct infection of the olfactory epithelium (Pratt, et al., 2003; Steele, et al., 1998; Vogel, et al., 1996). Other nasal epithelia tissues (respiratory or squamous) do not appear to become infected (Charles, et al., 1995; Davis, et al., 1995; Griffin, 2007; Pratt, et al., 2003; Steele, et al., 1998). VEEV is proposed to reach the brain via the

Encephalitic Development in Alphaviral Infection 225

Verlinde, 1968; Victor, et al., 1956). Thus, IN inoculation results in disease ranging from simply clinical signs of encephalitis and subsequent recovery to fulminant lethal

Of the three viruses, VEEV has the best characterized animal models, and therefore the most is understood regarding the specific host response to infection. However, significant obstacles due to the high biocontaiment level and the rapid death of the host animal following infection resulted in alternatives to typical infection models. Thus, the majority studies examining host response to VEE has been generated using models of attenuated virus or pre-existing immunity, so to date little is known about the primary host response to

Like other alphaviruses, VEEV is highly susceptible to the effects of type I IFN (Jordan, 1973). Attenuation in enzootic strains limits the virus' ability to interfere with type I IFN signaling pathways and partially explains the absence of typical disease symptoms following infection with enzootic strains (Grieder & Vogel, 1999; Jahrling, 1975; Jahrling, et al., 1976; Simmons, et al., 2009; Spotts, et al., 1998; White, et al., 2001). Conversely, epizootic and epidemic strains are able to limit host production of type I IFN through interference with signaling pathways, particularly STAT1 (Simmons, et al., 2009; White, et al., 2001a; Yin, et al., 2009). However, the susceptibility of enzootic and epizootic strains appears to vary significantly based on the virus strain(Anishchenko, et al., 2004). As with EEE, the ability to interfere with host transcription has been ascribed to properties of the capsid protein (Garmashova, et al., 2007a; Garmashova, et al., 2007b). Attenuating mutations in the 5' untranslated region of the genome are associated with susceptibility of the virus to the effects of type I IFN. Animals genetically modified with deficiencies in type I IFN signaling are highly susceptible to VEE infection and, even attenuated strains are uniformly lethal in these animals(White, et al., 2001). Additionally, artificial induction of type I IFN or prophylactic administration of type I IFN delays or prevents death in animal models (Julander, et al., 2008b). In the case of IN or aerosol infection, the rapid entry of virus to the CNS results in earlier neuroinvasion and may limit or alter the effectiveness of early innate

immune mechanisms such as IFN or other as yet other unexplored mechanisms.

Vaccination of immunocompromised mice genetically deficient in the IFN-γ receptor is only partially protective unlike complete protection observed in wild-type counterparts indicating type 2 IFN signaling is not required for complete protection unlike type I IFN

Current knowledge of the humoral response of the host is largely derived using models of pre-existing immunity (vaccination) or using attenuated strains of the virus. Early work identified high-level neutralizing serum antibody as essential following peripheral infection, and the production of neutralizing antibody is utilized as an endpoint in vaccine studies as well as marking efficacy of the IND vaccine, TC83, to vaccinate at risk laboratory personnel (Alevizatos, et al., 1967; Berge, et al., 1961; Eddy, et al., 1972; Engler, et al., 1992; Feigin, et al.,

encephalitis (Gleiser, et al., 1962; Steele & Twenhafel, 2010).

**5.5 Host response in experimental models** 

**5.5.1 Innate immune response to VEE** 

signaling (Paessler, et al., 2007).

**5.5.2.1 Antibody response** 

**5.5.2 Adaptive immune response to VEE** 

virulent infection.

olfactory nerve, but dental structure involvement has also been proposed (Charles, et al., 1995; Steele, et al., 1998; Steele & Twenhafel, 2010). Direct invasion of the brain via the bloodstream does not seem to be significant in VEE infection. Neuroinvasion results in caudal spread of the virus, and ultimately, overwhelming disseminated brain infection (Julander, et al., 2007; Julander, et al., 2008b). Thus, all routes of infection in mice with virulent forms of the virus result in neuroinvasion. However, infection of the nasal cavity, by either aerosol or IN infection, results in more rapid entry to the CNS depending on strain of virus used.

Strain of mice also is an important determinant of outcome. C3H/HeN and BALB/C mice vaccinated dermally with TC-83, the live attenuated vaccine strain, survive. However, aerosol or IN infection with TC83 results in 90-100% mortality in C3H/HeN animals unlike inbred counterparts BALB/C that respond with no evidence of mortality (Julander, et al., 2007; Julander, et al., 2008c; Steele, et al., 2007; Steele, et al., 1998; Steele, et al., 2006). Similar experiences have shown that route of administration can alter neuroinvasion with nonpathogenic viruses failing to enter the CNS by peripheral routes, but exhibiting the ability to enter the CNS by IC, IN, or aerosol administration. However, neuroinvasion, regardless of route, does not always correlate with mortality and virus strains avirulent in the periphery may remain so once in the CNS (Grieder, et al., 1995; MacDonald & Johnston, 2000a; Steele, et al., 1998; Steele, et al., 2006). This creates an interesting dichotomy between neuroinvasiveness and neurovirulence of the virus and indicates that outcome depends on factors beyond neuroinvasion or viral replication to generate mortality.

#### **5.4.2 Hamster and guinea pig models**

In guinea pigs and hamsters, VEEV causes acute, fulminant disease associated with extensive necrosis of lymphoid tissues, and death typically occurs prior to development of CNS disease making these models limited for studies of human infections and encephalitis (Gorelkin & Jahrling, 1975; Jackson, et al., 1991; Jahrling & Scherer, 1973a; Jahrling & Scherer, 1973b; 1973c; Walker, et al., 1976)

#### **5.4.3 Nonhuman primate model**

Like the majority of symptomatic human infections, infection in NHP represents an acute biphasic, nonspecific febrile disease with infection of lymphoid organs (Danes, et al., 1973; Gleiser, et al., 1962; Monath, et al., 1974a; Nalca, et al., 2003b; Pratt, et al., 1998; Reed, et al., 2007; Verlinde, 1968; Victor, et al., 1956) . In a comprehensive study with rhesus macaques infected IP, animals developed a transient viremia and biphasic fever, but otherwise displayed no clinical signs of disease with complete resolution of pathologies by five weeks PI. Lymphoid depletion occurred rapidly by two days post-infection with extensive lymphoid necrosis, later followed by lymphoid hyperplasia as the animals recovered from infection. Lesions in the brain characterized by perivascular cuffs associated with lymphocytes and gliosis were also apparent and developed around six days post-infection starting at the olfactory bulb and spread caudally throughout the brain (Danes, et al., 1973; Gleiser, et al., 1961; 1962; Monath, et al., 1974b; Pratt, et al., 1998; Reed, et al., 2007; Verlinde, 1968; Victor, et al., 1956). A range of CNS involvement presents depending on strain of virus utilized and route of inoculation with IN and IC inoculation being particularly severe. In cynomolgus macaques similar findings were reported except in the case of IN or aerosol infection where CNS damage is more severe and, in the case of IC infection, lethal (Danes, et al., 1973; Monath, et al., 1974b; Pratt, et al., 1998; Reed, et al., 2007; Steele & Twenhafel, 2010;

olfactory nerve, but dental structure involvement has also been proposed (Charles, et al., 1995; Steele, et al., 1998; Steele & Twenhafel, 2010). Direct invasion of the brain via the bloodstream does not seem to be significant in VEE infection. Neuroinvasion results in caudal spread of the virus, and ultimately, overwhelming disseminated brain infection (Julander, et al., 2007; Julander, et al., 2008b). Thus, all routes of infection in mice with virulent forms of the virus result in neuroinvasion. However, infection of the nasal cavity, by either aerosol or IN infection, results in more rapid entry to the CNS depending on strain

Strain of mice also is an important determinant of outcome. C3H/HeN and BALB/C mice vaccinated dermally with TC-83, the live attenuated vaccine strain, survive. However, aerosol or IN infection with TC83 results in 90-100% mortality in C3H/HeN animals unlike inbred counterparts BALB/C that respond with no evidence of mortality (Julander, et al., 2007; Julander, et al., 2008c; Steele, et al., 2007; Steele, et al., 1998; Steele, et al., 2006). Similar experiences have shown that route of administration can alter neuroinvasion with nonpathogenic viruses failing to enter the CNS by peripheral routes, but exhibiting the ability to enter the CNS by IC, IN, or aerosol administration. However, neuroinvasion, regardless of route, does not always correlate with mortality and virus strains avirulent in the periphery may remain so once in the CNS (Grieder, et al., 1995; MacDonald & Johnston, 2000a; Steele, et al., 1998; Steele, et al., 2006). This creates an interesting dichotomy between neuroinvasiveness and neurovirulence of the virus and indicates that outcome depends on

In guinea pigs and hamsters, VEEV causes acute, fulminant disease associated with extensive necrosis of lymphoid tissues, and death typically occurs prior to development of CNS disease making these models limited for studies of human infections and encephalitis (Gorelkin & Jahrling, 1975; Jackson, et al., 1991; Jahrling & Scherer, 1973a; Jahrling &

Like the majority of symptomatic human infections, infection in NHP represents an acute biphasic, nonspecific febrile disease with infection of lymphoid organs (Danes, et al., 1973; Gleiser, et al., 1962; Monath, et al., 1974a; Nalca, et al., 2003b; Pratt, et al., 1998; Reed, et al., 2007; Verlinde, 1968; Victor, et al., 1956) . In a comprehensive study with rhesus macaques infected IP, animals developed a transient viremia and biphasic fever, but otherwise displayed no clinical signs of disease with complete resolution of pathologies by five weeks PI. Lymphoid depletion occurred rapidly by two days post-infection with extensive lymphoid necrosis, later followed by lymphoid hyperplasia as the animals recovered from infection. Lesions in the brain characterized by perivascular cuffs associated with lymphocytes and gliosis were also apparent and developed around six days post-infection starting at the olfactory bulb and spread caudally throughout the brain (Danes, et al., 1973; Gleiser, et al., 1961; 1962; Monath, et al., 1974b; Pratt, et al., 1998; Reed, et al., 2007; Verlinde, 1968; Victor, et al., 1956). A range of CNS involvement presents depending on strain of virus utilized and route of inoculation with IN and IC inoculation being particularly severe. In cynomolgus macaques similar findings were reported except in the case of IN or aerosol infection where CNS damage is more severe and, in the case of IC infection, lethal (Danes, et al., 1973; Monath, et al., 1974b; Pratt, et al., 1998; Reed, et al., 2007; Steele & Twenhafel, 2010;

factors beyond neuroinvasion or viral replication to generate mortality.

**5.4.2 Hamster and guinea pig models** 

Scherer, 1973b; 1973c; Walker, et al., 1976)

**5.4.3 Nonhuman primate model** 

of virus used.

Verlinde, 1968; Victor, et al., 1956). Thus, IN inoculation results in disease ranging from simply clinical signs of encephalitis and subsequent recovery to fulminant lethal encephalitis (Gleiser, et al., 1962; Steele & Twenhafel, 2010).

### **5.5 Host response in experimental models**

Of the three viruses, VEEV has the best characterized animal models, and therefore the most is understood regarding the specific host response to infection. However, significant obstacles due to the high biocontaiment level and the rapid death of the host animal following infection resulted in alternatives to typical infection models. Thus, the majority studies examining host response to VEE has been generated using models of attenuated virus or pre-existing immunity, so to date little is known about the primary host response to virulent infection.

### **5.5.1 Innate immune response to VEE**

Like other alphaviruses, VEEV is highly susceptible to the effects of type I IFN (Jordan, 1973). Attenuation in enzootic strains limits the virus' ability to interfere with type I IFN signaling pathways and partially explains the absence of typical disease symptoms following infection with enzootic strains (Grieder & Vogel, 1999; Jahrling, 1975; Jahrling, et al., 1976; Simmons, et al., 2009; Spotts, et al., 1998; White, et al., 2001). Conversely, epizootic and epidemic strains are able to limit host production of type I IFN through interference with signaling pathways, particularly STAT1 (Simmons, et al., 2009; White, et al., 2001a; Yin, et al., 2009). However, the susceptibility of enzootic and epizootic strains appears to vary significantly based on the virus strain(Anishchenko, et al., 2004). As with EEE, the ability to interfere with host transcription has been ascribed to properties of the capsid protein (Garmashova, et al., 2007a; Garmashova, et al., 2007b). Attenuating mutations in the 5' untranslated region of the genome are associated with susceptibility of the virus to the effects of type I IFN. Animals genetically modified with deficiencies in type I IFN signaling are highly susceptible to VEE infection and, even attenuated strains are uniformly lethal in these animals(White, et al., 2001). Additionally, artificial induction of type I IFN or prophylactic administration of type I IFN delays or prevents death in animal models (Julander, et al., 2008b). In the case of IN or aerosol infection, the rapid entry of virus to the CNS results in earlier neuroinvasion and may limit or alter the effectiveness of early innate immune mechanisms such as IFN or other as yet other unexplored mechanisms.

Vaccination of immunocompromised mice genetically deficient in the IFN-γ receptor is only partially protective unlike complete protection observed in wild-type counterparts indicating type 2 IFN signaling is not required for complete protection unlike type I IFN signaling (Paessler, et al., 2007).

#### **5.5.2 Adaptive immune response to VEE**

#### **5.5.2.1 Antibody response**

Current knowledge of the humoral response of the host is largely derived using models of pre-existing immunity (vaccination) or using attenuated strains of the virus. Early work identified high-level neutralizing serum antibody as essential following peripheral infection, and the production of neutralizing antibody is utilized as an endpoint in vaccine studies as well as marking efficacy of the IND vaccine, TC83, to vaccinate at risk laboratory personnel (Alevizatos, et al., 1967; Berge, et al., 1961; Eddy, et al., 1972; Engler, et al., 1992; Feigin, et al.,

Encephalitic Development in Alphaviral Infection 227

survival, encephalitis, and the repair of neural damage and homeostasis in the brain(Paessler & Weaver, 2009; Paessler, et al., 2007; Yun, et al., 2009; Zacks & Paessler, 2010). The role of CD8+ T-cells explored previously by Jones et al. and arrived at much the same conclusion that CD8+ T-cells were not cytolytic or immunoprotective in VEE encephalitis (Jones, et al., 2003). However, given the pleiotropic roles of CD8+ T-cells and their significance in other viral infections the elimination of a role in VEE infection is

Functional antibody production is not required for recovery from infection with an genetically modified, attenuated VEE virus while T-cells were critical to complete protection and survival of the animals. Persistence, as seen previously in γδ deficient mice, was present in mice deficient in functional B-cell response (μMT knock out) and infection was less controlled in animals depleted of CD3, CD4 or CD8 T-cells with CD4 cells appearing to

Additionally data indicate that IFN-y secretion from these cell populations contributes to survival. However, their role in primary infection has not been well defined. The ability of animals to sustain high levels of virus in the CNS calls into question the importance of viral replication in host pathogenesis. Vaccinated animals maintain equivalent levels of virus to uninfected counterparts with differential outcome indicated that viral load is not the best discriminator for mortality(Paessler, et al., 2007; Yun, et al., 2009). Additionally, such evidence indicates that the efficacy of antivirals once the virus invades the CNS may be limited and therapeutic efforts may be better focused on limiting or altering the host response to generate a non-pathogenic response. However, further understanding of the host response and pathogenic and protective mechanisms of resolution of infection are

Adams, A.P., Aronson, J.F., Tardif, S.D., Patterson, J.L., Brasky, K.M., Geiger, R., de la Garza,

Adler, W.H. & Rabinowitz, S.G. (1973). Host defenses during primary Venezuelan equine

Aguilar, P.V., Paessler, S., Carrara, A.-S., Baron, S., Poast, J., Wang, E., Moncayo, A.C.,

Aguilar, P.V., Weaver, S.C. & Basler, C.F. (2007). Capsid protein of eastern equine encephalitis virus inhibits host cell gene expression. *J Virol*, 81, 8, 3866-3876, Aguilar, P.V., Adams, A.P., Wang, E., Kang, W., Carrara, A.-S., Anishchenko, M., Frolov, I.

and qualitation of the immune response. *J Immunol*, 110, 5, 1354-1362, Aguilar, M.J. (1970). Pathological changes in brain and other target organs of infant and

M., Carrion, R., Jr. & Weaver, S.C. (2008). Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis

encephalomyelitis virus infection in mice. II. In vitro methods for the measurement

weanling mice after infection with non-neuroadapted Western equine encephalitis

Anishchenko, M., Watts, D., Tesh, R.B. & Weaver, S.C. (2005). Variation in Interferon Sensitivity and Induction among Strains of Eastern Equine Encephalitis

& Weaver, S.C. (2008a). Structural and Nonstructural Protein Genome Regions of

contribute the most significantly in viral control (Brooke, et al., 2010).

integral to effective therapeutics development.

virus strains. *J Virol*, 82, 18, 9035-9042,

virus. *Infect Immun*, 2, 5, 533-542,

Virus. *J. Virol.*, 79, 17, 11300-11310,

doubtful.

**6. References** 

1967; Jochim & Barber, 1974; Pittman, et al., 1996; Walton, et al., 1972). However, more recent research efforts indicate that circulating antibody may be irrelevant once virus reaches, invades, and begins replicating in the CNS and the role of antibody produced in the endogenous micro-environment of the CNS has been poorly explored (Paessler, et al., 2007; Yun, et al., 2009). Research from Sindbis models using neuroadapted strains of this particular alphavirus indicate that neutralizing antibody is capable of non-cytolytic clearance of virus from neurons (Burdeinick-Kerr, et al., 2009; Griffin, et al., 1997; Griffin, 2010). Primary tropism of VEE in the periphery is for dendritic cells; however, in the CNS it replicates in neurons where non-cytolytic clearance may be of great importance(MacDonald & Johnston, 2000a; Schoneboom, et al., 1999b).

Monoclonal antibodies against specific surface glycoproteins, particularly E1 and E2, may act as useful therapeutic agents and may provide passive immunity to infected animals (Hart, et al., 2000; Hart, et al., 2001; Hart, et al., 1997; Mathews & Roehrig, 1982; Roehrig, et al., 1988; Roehrig & Mathews, 1985). Alterations in the E2 protein are capable of altering the pathogenesis of the virus and preventing neuroinvasion (Davis, et al., 1995). Since aerosol and IN infection bypasses the need to develop viremia to be neuroinvasive, the IgG peripheral neutralizing antibodies may not afford protection against nasal routes of infection. Studies of the antibody response indicate that not all species are equally protected by the same vaccines and specific IgA production at the nasal mucosal surfaces may play a critical role in prevention from aerosol infection (Hart, et al., 1997). More recent studies indicate that administration of hyperimmune sera is only effective against peripheral infection, and has little effect once virus invades the CNS though transfer is able to prolong survival probably due to depression of peripheral infection (Hart, et al., 1997). However, the role of plasma cells or locally produced antibody in the brain by memory B-cells remains to be determined for this infection (Paessler, et al., 2007; Yun, et al., 2009). Research efforts in the 1970's indicated that Fc-dependent clearance of the virus does not rely on complement (Mathews, et al., 1985). Vaccinated mice with non-functional B-cells (μMT deficient) are highly susceptible to intranasal infection, and when infected with attenuated strains develop persistent viral infection (Brooke, et al., 2010; Paessler, et al., 2007; Yun, et al., 2009).

#### **5.5.2.2 Other cell-mediated immune responses**

Recent research indicates that T-cells are crucial in recovery from VEE. Specifically, CD4+ Tcells contribute to resolution of infection. While adoptive transfer of primed CD3+ and CD4+ T-cells generated via vaccination ameliorates encephalitis in vaccinated αβ T-cell receptor deficient animals, CD8+ T-cells fail to generate protection. Vaccination of γδ T-cell receptor deficient animals is partially protective, but animals develop a persistent viral infection to 28 days. Thus, while αβ T-cell subsets appear to be required for protection, γδ Tcells do not and viral persistence in these animals may be an indirect effect of a deficit in Tcell help for B cells in these animals (Paessler, et al., 2007; Yun, et al., 2009). Earlier studies of the immune response to the attenuated, live vaccine strain TC83 identified a Th 1 mediate immune response with local activation of CD4+ and CD8+ T-cells (Bennett, et al., 2000; Jahrling & Stephenson, 1984; Phillpotts, 1999; Phillpotts, et al., 2003; Phillpotts & Wright, 1999). Later data examining transcriptional profiles in the brain and sera corroborate an overwhelming proinflammatory response and support the Th1 bias in response to infection(Davis, et al., 1994; Grieder, et al., 1997; Grieder & Vogel, 1999; Koterski, et al., 2007). These studies indicated that T-cells are critical to the host defense against infection,

1967; Jochim & Barber, 1974; Pittman, et al., 1996; Walton, et al., 1972). However, more recent research efforts indicate that circulating antibody may be irrelevant once virus reaches, invades, and begins replicating in the CNS and the role of antibody produced in the endogenous micro-environment of the CNS has been poorly explored (Paessler, et al., 2007; Yun, et al., 2009). Research from Sindbis models using neuroadapted strains of this particular alphavirus indicate that neutralizing antibody is capable of non-cytolytic clearance of virus from neurons (Burdeinick-Kerr, et al., 2009; Griffin, et al., 1997; Griffin, 2010). Primary tropism of VEE in the periphery is for dendritic cells; however, in the CNS it replicates in neurons where non-cytolytic clearance may be of great importance(MacDonald

Monoclonal antibodies against specific surface glycoproteins, particularly E1 and E2, may act as useful therapeutic agents and may provide passive immunity to infected animals (Hart, et al., 2000; Hart, et al., 2001; Hart, et al., 1997; Mathews & Roehrig, 1982; Roehrig, et al., 1988; Roehrig & Mathews, 1985). Alterations in the E2 protein are capable of altering the pathogenesis of the virus and preventing neuroinvasion (Davis, et al., 1995). Since aerosol and IN infection bypasses the need to develop viremia to be neuroinvasive, the IgG peripheral neutralizing antibodies may not afford protection against nasal routes of infection. Studies of the antibody response indicate that not all species are equally protected by the same vaccines and specific IgA production at the nasal mucosal surfaces may play a critical role in prevention from aerosol infection (Hart, et al., 1997). More recent studies indicate that administration of hyperimmune sera is only effective against peripheral infection, and has little effect once virus invades the CNS though transfer is able to prolong survival probably due to depression of peripheral infection (Hart, et al., 1997). However, the role of plasma cells or locally produced antibody in the brain by memory B-cells remains to be determined for this infection (Paessler, et al., 2007; Yun, et al., 2009). Research efforts in the 1970's indicated that Fc-dependent clearance of the virus does not rely on complement (Mathews, et al., 1985). Vaccinated mice with non-functional B-cells (μMT deficient) are highly susceptible to intranasal infection, and when infected with attenuated strains develop persistent viral infection (Brooke, et al., 2010; Paessler, et

Recent research indicates that T-cells are crucial in recovery from VEE. Specifically, CD4+ Tcells contribute to resolution of infection. While adoptive transfer of primed CD3+ and CD4+ T-cells generated via vaccination ameliorates encephalitis in vaccinated αβ T-cell receptor deficient animals, CD8+ T-cells fail to generate protection. Vaccination of γδ T-cell receptor deficient animals is partially protective, but animals develop a persistent viral infection to 28 days. Thus, while αβ T-cell subsets appear to be required for protection, γδ Tcells do not and viral persistence in these animals may be an indirect effect of a deficit in Tcell help for B cells in these animals (Paessler, et al., 2007; Yun, et al., 2009). Earlier studies of the immune response to the attenuated, live vaccine strain TC83 identified a Th 1 mediate immune response with local activation of CD4+ and CD8+ T-cells (Bennett, et al., 2000; Jahrling & Stephenson, 1984; Phillpotts, 1999; Phillpotts, et al., 2003; Phillpotts & Wright, 1999). Later data examining transcriptional profiles in the brain and sera corroborate an overwhelming proinflammatory response and support the Th1 bias in response to infection(Davis, et al., 1994; Grieder, et al., 1997; Grieder & Vogel, 1999; Koterski, et al., 2007). These studies indicated that T-cells are critical to the host defense against infection,

& Johnston, 2000a; Schoneboom, et al., 1999b).

al., 2007; Yun, et al., 2009).

**5.5.2.2 Other cell-mediated immune responses** 

survival, encephalitis, and the repair of neural damage and homeostasis in the brain(Paessler & Weaver, 2009; Paessler, et al., 2007; Yun, et al., 2009; Zacks & Paessler, 2010). The role of CD8+ T-cells explored previously by Jones et al. and arrived at much the same conclusion that CD8+ T-cells were not cytolytic or immunoprotective in VEE encephalitis (Jones, et al., 2003). However, given the pleiotropic roles of CD8+ T-cells and their significance in other viral infections the elimination of a role in VEE infection is doubtful.

Functional antibody production is not required for recovery from infection with an genetically modified, attenuated VEE virus while T-cells were critical to complete protection and survival of the animals. Persistence, as seen previously in γδ deficient mice, was present in mice deficient in functional B-cell response (μMT knock out) and infection was less controlled in animals depleted of CD3, CD4 or CD8 T-cells with CD4 cells appearing to contribute the most significantly in viral control (Brooke, et al., 2010).

Additionally data indicate that IFN-y secretion from these cell populations contributes to survival. However, their role in primary infection has not been well defined. The ability of animals to sustain high levels of virus in the CNS calls into question the importance of viral replication in host pathogenesis. Vaccinated animals maintain equivalent levels of virus to uninfected counterparts with differential outcome indicated that viral load is not the best discriminator for mortality(Paessler, et al., 2007; Yun, et al., 2009). Additionally, such evidence indicates that the efficacy of antivirals once the virus invades the CNS may be limited and therapeutic efforts may be better focused on limiting or altering the host response to generate a non-pathogenic response. However, further understanding of the host response and pathogenic and protective mechanisms of resolution of infection are integral to effective therapeutics development.

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**1. Introduction** 

**11** 

*USA*

**and Diagnosis** 

**Human Rabies Epidemiology** 

Brett W. Petersen and Charles E. Rupprecht *Centers for Disease Control and Prevention, Atlanta*, *GA* 

Rabies is a fatal viral infection that is most commonly spread to humans through the bite of an infected animal. The disease is an acute progressive encephalitis caused by highly neurotropic zoonotic viruses belonging to the *Lyssavirus* genus in the *Rhabdoviridae* family (Kuzmin, 2009). Of the twelve species of lyssaviruses, rabies virus (RABV) is the most important with respect to its impact on public health. RABV is distributed globally and found on all continents except Australia and Antarctica. In the United States, multiple RABV variants circulate in wild mammalian reservoir populations including raccoons, skunks, foxes, and bats. Rabies has the highest case fatality rate of any infectious disease and kills an estimated 55,000 people annually, primarily in developing countries within Africa and Asia (Knobel, 2005). However, rabies is a preventable disease. Postexposure prophylaxis (PEP) consisting of rabies immune globulin and rabies vaccine is successful in preventing the disease when administered promptly after an exposure to the virus has occurred. Additionally, vaccination of domestic animals against rabies and stray animal control programs greatly reduce the risk of RABV transmission to humans. Implementation of these measures in developed countries such as the United States has led to drastic declines in the incidence of human rabies. Despite this success, rabies remains a significant public health issue. Each year approximately 7,000 rabid animals are reported in the United States (Blanton, 2010). Up to 35,000 people annually are estimated to receive PEP due to exposures to suspect rabid animals (Christian, 2009). Given the high cost of rabies PEP, this represents a substantial economic burden as well. A clear understanding of the epidemiology of human rabies in the United States can help to manage these human exposures using the best available evidence. In this way, the risk of infection can be assessed more precisely and ensure rabies PEP is administered more judiciously. The identification of epidemiologic patterns can also be used to focus educational messages for human rabies prevention and thereby increase public awareness of rabies and the importance of seeking medical care after a potential exposure occurs. Furthermore, providing accurate descriptions of the clinical presentation of human rabies is essential in recognizing and diagnosing the disease in a timely fashion. Delayed or missed diagnoses place others at risk of exposure if appropriate infection control precautions are not instituted, exposures are not treated appropriately, or organs or tissues from an infected individual are used for transplantation (Houff, 1979; Javadi, 1996; Hellenbrand, 2005; Kusne, 2005; Srinivasan, 2005). An early diagnosis also

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