**2.2.2 Nucleocapsid protein: N**

(MacNamara et al., 2005; 2008).

Structural proteins are transmembrane proteins located in the viral envelope, except for the N protein, which is associated with the viral RNA genome (Baric et al., 1988; Sturman et al., 1980). Replacement of the N gene of rA59 with that of JHM (NJHM) confers to the rA59 background a highly neurovirulent phenotype. The viral infectious dose inoculated into mouse brain to induce a 50% lethal dose (LD50) is decreased between 100- and 1000-fold. Inversely, the rJHM with NA59 is attenuated, but this is more evident on the attenuated phenotype (rA59/SJHM), indicating that replacing the MHV-JHM N gene with that of MHV-A59 is attenuating (Cowley et al., 2010). The rA59/NJHM induced a faster spread of virus in the CNS than rA59, whereas the rJHM/NA59 demonstrated a restrained viral spread as compare to rJHM. These data illustrated that the N background determined viral spread, which is a constituent of virulence. The NJHM is suspected to be associated to microtubules (Pasick et al., 1994) and then to be able to increase the transport of virus along axons to favor viral spread between neurons to other neurons. Unfortunately, the infection of primary hippocampal neuronal cultures by rA59/NJHM did not demonstrate an increased spread of virus as compared to rA59 (Cowley et al., 2010). Moreover, the N gene influences T-cell infiltration into the brain, as CD4+ and CD8+ T cell response is strong if the virus possesses NA59 and weak with the NJHM (Cowley et al., 2010).

#### **2.2.3 Hemagglutinin Esterase glycoprotein (HE)**

The hemagglutinin esterase (HE), is an envelope glycoprotein, which is present in some strains of the betacoronaviruses such as the human coronaviruses (HCoV-OC43 and HCoV-HKU1), as well as the bovine coronavirus (BCoV). The JHM strain of MHV expresses the HE glycoprotein, whereas MHV-A59 does not, due to a defective HE gene (Shieh et al.,

as rA59 and rA59/SJHM recombinant viruses target equally neurons and astrocytes (Phillips et al., 2002). The degree of neurovirulence is associated with the degree of spread through the brain. The faster the spread, the higher will be the neurovirulence. This increased viral spread and dissemination also induces a greater immune response to infection. The magnitude of lymphocytic infiltration in mouse brain following infection by rA59 with SJHM and rA59 with SA59 is different, as SJHM induced a more efficient CNS invasion of lymphocytes than SA59 and a significant increase in the percentage of CD8+ T cells (Phillips et al., 2002). Increased immune-mediated pathology was associated with rapid viral spread (Miura et al., 2008; Wodarz & Krakauer, 2000). The recombinant virus rA59, harboring the JHM S protein (SJHM) is not as virulent as wild-type recombinant rJHM, indicating that neurovirulence also depends on other background genes. The high neurovirulence of JHM is also associated with an increased innate immune response characterized by high numbers of infiltrating neutrophils and macrophages, demonstrated by comparing rJHM bearing SA59 to rJHM bearing SJHM during the acute phase of encephalitis (Iacono et al., 2006). Nevertheless, the viral strain background also plays a strong role, as viruses containing the rA59 genes demonstrated markedly increased levels of CD8+ and CD4+ T-cell infiltration compared to the rJHM background-containing viruses (Iacono et al., 2006). Virus-specific CD8+ T cells are critical for protection against neurotropic coronaviruses. On the other hand, the combination of a rapid and extensive spread through the CNS with the lack of induction of a significant T-cell response results in the high lethality of JHM-infected mice

Structural proteins are transmembrane proteins located in the viral envelope, except for the N protein, which is associated with the viral RNA genome (Baric et al., 1988; Sturman et al., 1980). Replacement of the N gene of rA59 with that of JHM (NJHM) confers to the rA59 background a highly neurovirulent phenotype. The viral infectious dose inoculated into mouse brain to induce a 50% lethal dose (LD50) is decreased between 100- and 1000-fold. Inversely, the rJHM with NA59 is attenuated, but this is more evident on the attenuated phenotype (rA59/SJHM), indicating that replacing the MHV-JHM N gene with that of MHV-A59 is attenuating (Cowley et al., 2010). The rA59/NJHM induced a faster spread of virus in the CNS than rA59, whereas the rJHM/NA59 demonstrated a restrained viral spread as compare to rJHM. These data illustrated that the N background determined viral spread, which is a constituent of virulence. The NJHM is suspected to be associated to microtubules (Pasick et al., 1994) and then to be able to increase the transport of virus along axons to favor viral spread between neurons to other neurons. Unfortunately, the infection of primary hippocampal neuronal cultures by rA59/NJHM did not demonstrate an increased spread of virus as compared to rA59 (Cowley et al., 2010). Moreover, the N gene influences T-cell infiltration into the brain, as CD4+ and CD8+ T cell response is strong if the virus possesses

The hemagglutinin esterase (HE), is an envelope glycoprotein, which is present in some strains of the betacoronaviruses such as the human coronaviruses (HCoV-OC43 and HCoV-HKU1), as well as the bovine coronavirus (BCoV). The JHM strain of MHV expresses the HE glycoprotein, whereas MHV-A59 does not, due to a defective HE gene (Shieh et al.,

(MacNamara et al., 2005; 2008).

**2.2.2 Nucleocapsid protein: N** 

NA59 and weak with the NJHM (Cowley et al., 2010).

**2.2.3 Hemagglutinin Esterase glycoprotein (HE)** 

1989). Nevertheless, MHV-A59 is able to induce an encephalitis in infected mice but the disease is less important as this strain is less neurovirulent than MHV-JHM. By using targeted RNA recombination to introduce the HE gene of MHV-JHM into the genome of MHV-A59, the role of the HE gene in neurovirulence was addressed. The expression of HE on the MHV-A59 background neither increased virulence in mice (as evaluated by the LD50), nor the production of infectious virus (in brain or liver tissue), or virus spread (revealed by the distribution of viral antigen) (Kazi et al., 2005). Whereas, Kazi and collaborators demonstrated that the expression of HE in combination with the MHV-JHM spike protein in rA59 enhances the disease outcome and increased viral spread in the brain, without changing viral replication, their results suggest that a structurally intact HE, in combination with the MHV-JHM spike protein, has a significant impact on the neurovirulence. In fact, even though the S protein is the main viral factor, which determines tissue tropism and infection of target cell, the HE viral protein appears to serve as a second receptor-binding protein, which increases infection and viral dissemination in the brain (Kazi et al., 2005).

#### **2.3 Human coronaviruses (HCoV)**

Human coronaviruses (HCoV) are recognized respiratory pathogens but, in rare cases, they may be associated with encephalitis. As previously mentioned, five different strains of human coronaviruses are currently described: HCoV-OC43, HCoV-229E, HCoV-NL-63, HCoV-HKU1 and SARS-CoV, and neuroinvasive properties were reported for at least three of these five strains. Indeed, our laboratory has demonstrated that HCoV-OC43 and HCoV-229E can infect human neural cells (neurons and glial cells) and does persist in human brains (Bonavia et al., 1997; Arbour et al., 1999a; 1999b; 2000). Others have reported the presence of HCoV-OC43 in a child with acute disseminated encephalomyelitis (Yeh et al., 2004). Moreover, the SARS-CoV epidemics reported in China in the fall of 2002, strongly illustrated the potentially fatal illness caused by a coronavirus (Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003). The severe acute respiratory syndrome described was usually transmitted by contact with droplets sprayed into the air by an infected person's coughing. Other symptoms can include high fever, headache, body aches, and pneumonia. A few years after the 2002-2003 epidemics, scientists found that SARS-CoV was able to also infiltrate brain tissue and cause significant CNS problems associated with edema, degeneration of neuronal cells and gliosis, and SARS-CoV RNA could be detected in the brain of patients who died of SARS (Gu et al., 2005; Xu et al., 2005). Moreover, the use of a transgenic mouse model expressing the recognized viral receptor, human angiotensinconverting enzyme 2 (hACE-2), showed that SARS-CoV possesses neuroinvasive properties, as the virus reached the brain by the olfactory bulbs, infecting neuronal cells and induced a lethal disease, with a restrained immune infiltration (Netland et al., 2008).

#### **2.3.1 Animal model to understand the human coronavirus infection**

We have characterized the neurotropic, neuroinvasive and neurovirulent properties of HCoV-OC43 through the development of an experimental animal model. We have reported that intranasal (IN) infection of mice with HCoV-OC43 led to acute encephalitis and to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence (Jacomy & Talbot, 2003). Damage to the CNS appeared to be mainly a consequence of direct virus-mediated neuronal injury. Indeed, as illustrated in Figure 1, this acute infection targeted neurons, which underwent vacuolation and degeneration. We were also able to demonstrate that caspase-related virus-induced apoptosis of neuronal cells both

Coronaviruses as Encephalitis - Inducing Infectious Agents 191

and IL-6, released in the CNS, as well as chemokines and strong infiltration of immune cells (Jacomy et al., 2010). This is again of particular interest since accumulating evidence show that these types of factors may influence the degree of encephalitis. For instance, the mortality rate following JEV encephalitis is directly linked to the concentration of cytokine in serum and cerebrospinal fluid (CSF) in patients (Winter et al., 2004). Furthermore, neuronal cell death in a mice model of Japanese Encephalitis Virus (JEV) infection was demonstrated to be related to the activation of microglia and subsequent production of multiple proinflammatory mediators (Ghoshal et al., 2007). We also reported that apoptosis of neurons occurred in infected and non-infected cells, suggesting that it is independent of the presence of virus (Jacomy et al., 2006). Host adaptive immune response was demonstrated to contribute to HCoV-OC43-induced encephalitis (Butler et al., 2006). We also reported that mice surviving encephalitis developed motor dysfunctions as the result of viral persistence in the CNS (Jacomy et al., 2006). In addition to the direct effect of the viral pathogen, acute encephalopathy may be associated with viral infections and increased plasma concentrations of CXCL8/IL-8 and CCL2/MCP-1, CXCL10/IP-10 and IL-6, without viral neuroinvasion (hyperactivated cytokine/chemokine response) as demonstrated following infection by another respiratory virus, Influenza virus, which can cause encephalitis (Lee et al., 2010). JEV also induces a rapid inflammatory response, which is accompanied by an increased level of cytokines and chemokines in the serum and cerebrospinal fluid of infected patients. Moreover, levels of interferon (IFN)-alpha, TNF-alpha, and the cytokines IL-8 and IL-6 have been associated with a bad outcome (Burke

Excitotoxic neurotransmission, which is an excessive stimulation by the neurotransmitter glutamate on its specific receptors (AMPAr and NMDAr), seems to play an important role in neuronal damage during encephalitis as concentration of glutamate in CSF is significantly increased in acute encephalitis (Launes et al., 1998). Moreover, the concentration of glutamate was also correlated with a poor outcome during encephalitis in humans, as well as in an animal model of human encephalitis (Carmen et al., 2009; Launes et al., 1998). During the HCoV-OC43-induced encephalitis, we recently found that hippocampal neurons died in part by this pathological process (Brison et al., submitted for publication). Indeed, treatment of infected mice with the AMPA receptor antagonist (GYKI-52466) did not affect the survival rate of mice but histological analysis revealed that mice treated by the GYKI-52466 presented less neurodegeneration compared to infected mice without the AMPA antagonist, as determined by Neurosilver staining (Figure 2) (Brison et al., submitted for publication). Again, this is of particular interest since glutamate excitotoxicity was demonstrated to be involved in several viral infection such as West Nile virus, Sindbis virus, JEV, HIV and HSV (Blakely et al., 2009; Golembewski et al., 2007; Haughey et al., 2001; Mishra et al., 2007; Nargi-Aizenman et al., 2004). The loss of neuronal subpopulations in the brain during HIV dementia was also related to an indirect mechanism conjugating glial activation, cytokines released and excitotoxic transmission (Alirezaei et al., 2008; Masliah &

We have also demonstrated that the severity of the HCoV-OC43 induced-encephalitis depended on a number of factors such as the genetic background (Jacomy & Talbot, 2003) and the gender of the mice, as revealed by inoculation of male and female animals of different mouse strains, aged 21 days post-natal (DPN). At the equal infectious viral dose, male animals are more susceptible than female animals. For all strains of mice tested, male animals appeared more susceptible than female animals to encephalitis, as illustrated for

& Morill, 1987; Singh et al., 2000, Winter et al., 2004)

Mucke, 1996).

strains C57BL/6 and BALB/c in Figure 3.

*in vitro* and *in vivo* (Jacomy and Talbot, 2006). This type of virus-induced apoptosis is of particular interest as in the human brain, activation of caspase 3 is involved in pathologic pathway of influenza encephalopathy (Nakai et al., 2003), reovirus-induced encephalitis (Beckham et al., 2010) and Japanese-encephalitis virus-induced pathology (Yang et al., 2009).

Fig. 1. Infection of neuronal cells by HCoV-OC43. (A) Numerous cells positive for virus antigens at 7 days post-infection (DPI) in the CNS. Virus was present in hippocampal region (especially in pyramidal cell layers CA1, CA2 and CA3, as well as in dentate gyrus (DG)), in cortical area (Cortex), in Striatum as well as in Thalamus. (B) Magnification of pyramidal cell layers CA1, illustrating numerous infected cells stained in black. (C) Infected neurons in the Striatum, at higher magnification (D), numerous vacuolation were present in the cytoplasm of infected neurons (arrows). LV: Lateral ventricle; cc: corpus callosum; fi: fimbria of hippocampus.

While neurons are the primary target of HCoV-OC43, infected regions presented strong microglial reactivity and inflammatory reactions (neuroinflammation). Although initiation of immune responses by microglial cells is normally an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreversible brain damage. It is likely that strong microglial activation can trigger bystander damage, as they can release large amounts of cytokines and chemokines. In fact, we have recently shown that infection of susceptible mice by HCoV-OC43 induced the production of high level of numerous proinflammatory cytokines such as tumor necrosing factor (TNF)-alpha, interleukin (IL)-1 beta

*in vitro* and *in vivo* (Jacomy and Talbot, 2006). This type of virus-induced apoptosis is of particular interest as in the human brain, activation of caspase 3 is involved in pathologic pathway of influenza encephalopathy (Nakai et al., 2003), reovirus-induced encephalitis (Beckham et al., 2010) and Japanese-encephalitis virus-induced pathology (Yang et al., 2009).

Fig. 1. Infection of neuronal cells by HCoV-OC43. (A) Numerous cells positive for virus antigens at 7 days post-infection (DPI) in the CNS. Virus was present in hippocampal region (especially in pyramidal cell layers CA1, CA2 and CA3, as well as in dentate gyrus (DG)), in cortical area (Cortex), in Striatum as well as in Thalamus. (B) Magnification of pyramidal cell layers CA1, illustrating numerous infected cells stained in black. (C) Infected neurons in

the Striatum, at higher magnification (D), numerous vacuolation were present in the

of hippocampus.

cytoplasm of infected neurons (arrows). LV: Lateral ventricle; cc: corpus callosum; fi: fimbria

While neurons are the primary target of HCoV-OC43, infected regions presented strong microglial reactivity and inflammatory reactions (neuroinflammation). Although initiation of immune responses by microglial cells is normally an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreversible brain damage. It is likely that strong microglial activation can trigger bystander damage, as they can release large amounts of cytokines and chemokines. In fact, we have recently shown that infection of susceptible mice by HCoV-OC43 induced the production of high level of numerous proinflammatory cytokines such as tumor necrosing factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6, released in the CNS, as well as chemokines and strong infiltration of immune cells (Jacomy et al., 2010). This is again of particular interest since accumulating evidence show that these types of factors may influence the degree of encephalitis. For instance, the mortality rate following JEV encephalitis is directly linked to the concentration of cytokine in serum and cerebrospinal fluid (CSF) in patients (Winter et al., 2004). Furthermore, neuronal cell death in a mice model of Japanese Encephalitis Virus (JEV) infection was demonstrated to be related to the activation of microglia and subsequent production of multiple proinflammatory mediators (Ghoshal et al., 2007). We also reported that apoptosis of neurons occurred in infected and non-infected cells, suggesting that it is independent of the presence of virus (Jacomy et al., 2006). Host adaptive immune response was demonstrated to contribute to HCoV-OC43-induced encephalitis (Butler et al., 2006). We also reported that mice surviving encephalitis developed motor dysfunctions as the result of viral persistence in the CNS (Jacomy et al., 2006). In addition to the direct effect of the viral pathogen, acute encephalopathy may be associated with viral infections and increased plasma concentrations of CXCL8/IL-8 and CCL2/MCP-1, CXCL10/IP-10 and IL-6, without viral neuroinvasion (hyperactivated cytokine/chemokine response) as demonstrated following infection by another respiratory virus, Influenza virus, which can cause encephalitis (Lee et al., 2010). JEV also induces a rapid inflammatory response, which is accompanied by an increased level of cytokines and chemokines in the serum and cerebrospinal fluid of infected patients. Moreover, levels of interferon (IFN)-alpha, TNF-alpha, and the cytokines IL-8 and IL-6 have been associated with a bad outcome (Burke & Morill, 1987; Singh et al., 2000, Winter et al., 2004)

Excitotoxic neurotransmission, which is an excessive stimulation by the neurotransmitter glutamate on its specific receptors (AMPAr and NMDAr), seems to play an important role in neuronal damage during encephalitis as concentration of glutamate in CSF is significantly increased in acute encephalitis (Launes et al., 1998). Moreover, the concentration of glutamate was also correlated with a poor outcome during encephalitis in humans, as well as in an animal model of human encephalitis (Carmen et al., 2009; Launes et al., 1998). During the HCoV-OC43-induced encephalitis, we recently found that hippocampal neurons died in part by this pathological process (Brison et al., submitted for publication). Indeed, treatment of infected mice with the AMPA receptor antagonist (GYKI-52466) did not affect the survival rate of mice but histological analysis revealed that mice treated by the GYKI-52466 presented less neurodegeneration compared to infected mice without the AMPA antagonist, as determined by Neurosilver staining (Figure 2) (Brison et al., submitted for publication). Again, this is of particular interest since glutamate excitotoxicity was demonstrated to be involved in several viral infection such as West Nile virus, Sindbis virus, JEV, HIV and HSV (Blakely et al., 2009; Golembewski et al., 2007; Haughey et al., 2001; Mishra et al., 2007; Nargi-Aizenman et al., 2004). The loss of neuronal subpopulations in the brain during HIV dementia was also related to an indirect mechanism conjugating glial activation, cytokines released and excitotoxic transmission (Alirezaei et al., 2008; Masliah & Mucke, 1996).

We have also demonstrated that the severity of the HCoV-OC43 induced-encephalitis depended on a number of factors such as the genetic background (Jacomy & Talbot, 2003) and the gender of the mice, as revealed by inoculation of male and female animals of different mouse strains, aged 21 days post-natal (DPN). At the equal infectious viral dose, male animals are more susceptible than female animals. For all strains of mice tested, male animals appeared more susceptible than female animals to encephalitis, as illustrated for strains C57BL/6 and BALB/c in Figure 3.

Coronaviruses as Encephalitis - Inducing Infectious Agents 193

How immunological, hormonal, and or genetic differences between males and females could affect gender differences in susceptibility to infection? Males and females are differentially susceptible to several pathogens including viruses (Fish, 2008; Klein, 2000). In fact, in the case of viral infection in general, this difference in susceptibility between males and females was shown for different viruses including Seoul Virus (Klein et al., 2000), and Influenza virus (Avitsur et al., 2011) and this is also true in the particular case of viral infection of the CNS, which leads to encephalitis, for other viruses such as HIV-1 (Wilson et al., 2006), and vesicular stomatitis virus (VSV) (Barna et al., 1996). The concept of sex-based (or genderbased) differences in host response to infection has been studied for many years and appears to be highly related to differences in immunological capacities between males and females (Purtilo & Sullivan, 1979; Fish, 2008). Furthermore, it appears now that even though the relative importance of different factors may vary with the type of infection, X-linked genes, hormones, immunity and, at least in humans, societal context are among the factors that explain this sex-based difference (Fish, 2008; Klein, 2000). Even though it remains difficult to clarify evidence on how the different factors make a difference between genders, studies aimed to addressing the question appear to more and more link specific hormones such as androgens in males and estrogens in females (Klein, 2000; Wilson et al., 2006), host innate immunity (expression of cytokines and of pattern recognition receptors such as toll-like receptors (TLR)) (Hannah et al., 2008; Hill et al., 1998) as well as acquired immunity involving

T and B lymphocytes (reviewed in Klein, 2000) to explain part of the puzzle.

are more susceptible (Schneider & Higgs, 2008; Wang et al., 2010).

**2.4 Other non human coronaviruses as encephalitis-inducing agents** 

described as agent able to induced encephalitis in certain conditions.

mice, as illustrated in Figure 5.

**2.4.1 Swine coronaviruses (HEV)** 

As already published in our model, C57BL/6 were more susceptible than BALB/c mice and CD1 mice were in between, being more resistant than C57BL/6 mice and more susceptible than BALB/c mice. As already described, susceptibility to intracellular pathogens depends on human genetics (for review, see Vannberg et al., 2011). Therefore, susceptibility to infectious diseases could be linked to loci or alleles that could favor clearance of virus, such as for hepatitis C virus (Ge et al., 2009; Thomas et al., 2009). In the contrary, genetics may determine persistence of infection, such as in the case of Hepatitis B virus (Kamatani et al., 2009) or restrict viral loads as shown in HIV-1 infections (Fellay et al., 2007; 2009) Furthermore, we also showed that susceptibility to intranasal infection diminished with age (Figure 4), as adult mice became resistant to intranasal infection. All infected mice (21 days post natal (DPN) and more) survived to intranasal infection, no infectious virus was detected in the brain even though viral RNA could be detected in the CNS, demonstrating neuroinvasive properties without evident signs of pathology. This is an interesting fact considering that this restrictive susceptibility to CNS infection and encephalitis appear to exist in humans, where mostly children, the elderly and immuno-compromised individuals

We also reported that mice became less susceptible to intracerebral infection with the age of

Over the years, some coronaviruses, which can infect cattle or domestic animals, were

Hemagglutinating encephalitis virus (HEV) was demonstrated to induce disease ranging from gastroenteritis to encephalomyelitis in piglets (Andries & Pensaert, 1980; Siddell et al.,

Fig. 2. Hippocampus of mice following HCoV-OC43 infection treated or not treated by the GYKI-52466 antagonist. Following infection, neurodegeneration was investigated with Silver staining in non-treated (A and higher magnification B) and AMPA receptor antagonist-treated mice (C and higher magnification D). Neurons that underwent degeneration appear in black. As illustrated at higher magnification, non-treated mice present more numerous dark CA3 neurons (B) as compared to mice treated with GYKI-52466 (D).

Fig. 3. Survival rate of female versus male mice to a same infectious dose. Mice were monitored for 30 days for mortality following intracerebral infection. C57BL/6 (A) and BALB/c (B) mice were infected, respectively, with 102.5 TCID50 or 100.5 TCID50 of virus. For the same infectious dose, male mice were more susceptible to viral infection than female.

Fig. 2. Hippocampus of mice following HCoV-OC43 infection treated or not treated by the GYKI-52466 antagonist. Following infection, neurodegeneration was investigated with Silver staining in non-treated (A and higher magnification B) and AMPA receptor antagonist-treated mice (C and higher magnification D). Neurons that underwent degeneration appear in black. As illustrated at higher magnification, non-treated mice present more numerous dark CA3 neurons (B) as compared to mice treated with

Fig. 3. Survival rate of female versus male mice to a same infectious dose. Mice were monitored for 30 days for mortality following intracerebral infection. C57BL/6 (A) and BALB/c (B) mice were infected, respectively, with 102.5 TCID50 or 100.5 TCID50 of virus. For the same infectious dose, male mice were more susceptible to viral infection than female.

GYKI-52466 (D).

How immunological, hormonal, and or genetic differences between males and females could affect gender differences in susceptibility to infection? Males and females are differentially susceptible to several pathogens including viruses (Fish, 2008; Klein, 2000). In fact, in the case of viral infection in general, this difference in susceptibility between males and females was shown for different viruses including Seoul Virus (Klein et al., 2000), and Influenza virus (Avitsur et al., 2011) and this is also true in the particular case of viral infection of the CNS, which leads to encephalitis, for other viruses such as HIV-1 (Wilson et al., 2006), and vesicular stomatitis virus (VSV) (Barna et al., 1996). The concept of sex-based (or genderbased) differences in host response to infection has been studied for many years and appears to be highly related to differences in immunological capacities between males and females (Purtilo & Sullivan, 1979; Fish, 2008). Furthermore, it appears now that even though the relative importance of different factors may vary with the type of infection, X-linked genes, hormones, immunity and, at least in humans, societal context are among the factors that explain this sex-based difference (Fish, 2008; Klein, 2000). Even though it remains difficult to clarify evidence on how the different factors make a difference between genders, studies aimed to addressing the question appear to more and more link specific hormones such as androgens in males and estrogens in females (Klein, 2000; Wilson et al., 2006), host innate immunity (expression of cytokines and of pattern recognition receptors such as toll-like receptors (TLR)) (Hannah et al., 2008; Hill et al., 1998) as well as acquired immunity involving T and B lymphocytes (reviewed in Klein, 2000) to explain part of the puzzle.

As already published in our model, C57BL/6 were more susceptible than BALB/c mice and CD1 mice were in between, being more resistant than C57BL/6 mice and more susceptible than BALB/c mice. As already described, susceptibility to intracellular pathogens depends on human genetics (for review, see Vannberg et al., 2011). Therefore, susceptibility to infectious diseases could be linked to loci or alleles that could favor clearance of virus, such as for hepatitis C virus (Ge et al., 2009; Thomas et al., 2009). In the contrary, genetics may determine persistence of infection, such as in the case of Hepatitis B virus (Kamatani et al., 2009) or restrict viral loads as shown in HIV-1 infections (Fellay et al., 2007; 2009) Furthermore, we also showed that susceptibility to intranasal infection diminished with age (Figure 4), as adult mice became resistant to intranasal infection. All infected mice (21 days post natal (DPN) and more) survived to intranasal infection, no infectious virus was detected in the brain even though viral RNA could be detected in the CNS, demonstrating neuroinvasive properties without evident signs of pathology. This is an interesting fact considering that this restrictive susceptibility to CNS infection and encephalitis appear to exist in humans, where mostly children, the elderly and immuno-compromised individuals are more susceptible (Schneider & Higgs, 2008; Wang et al., 2010).

We also reported that mice became less susceptible to intracerebral infection with the age of mice, as illustrated in Figure 5.

#### **2.4 Other non human coronaviruses as encephalitis-inducing agents**

Over the years, some coronaviruses, which can infect cattle or domestic animals, were described as agent able to induced encephalitis in certain conditions.

#### **2.4.1 Swine coronaviruses (HEV)**

Hemagglutinating encephalitis virus (HEV) was demonstrated to induce disease ranging from gastroenteritis to encephalomyelitis in piglets (Andries & Pensaert, 1980; Siddell et al.,

Coronaviruses as Encephalitis - Inducing Infectious Agents 195

Fig. 5. Susceptibility of mice to the same infectious dose depends of age. (A) C57BL/6 mice became totally unsusceptible to a 100.5 infectious dose at 28 days post-natal (DPN), whereas, 60% died following the same infectious dose when they were aged 21 DPN. (B) In the same manner, the susceptibility of CD1 mice to a 101.5 infectious doses depended on the age at the

1983). The virus was isolated from the brains of suckling pigs suffering from encephalomyelitis several years ago (Greig et al., 1962). The disease could be reproduced experimentally in piglets following intranasal inoculation (Alexander, 1962). A murine model demonstrated that this virus, as other coronaviruses including the HCoV-OC43, is neuroinvasive and neurotropic. In that latter case, neuroinvasion depends on a number of variables such as the route of inoculation and the age of the mice (Yagami et al., 1986; Hirano et al., 1990; 2004). As for the SARS-CoV in mice, HEV induced a poor inflammatory reaction in CNS (Hirano et al., 2004) and unlike HCoV-OC43-infected neurons (Jacomy &

Feline infectious peritonitis (FIP) is a common cause of death in cats, caused by a virulent feline coronavirus (FCoV). The virulent form of FCoV, called FIPV, occurs following mutations acquired during a persistent infection (Brown et al., 2009; Rottier et al., 2005; Vennema et al., 1998) and neurologic involvement could occur in FIP disease (Foley et al., 1998; Summers et al., 1995). The colon was identified as the major site of FCoV persistence; nevertheless the virus could also persist in tissue macrophages representing a source for viremia (Kipar et al., 2010). Infected macrophages disseminate systemically and trigger immunological responses resulting in microgranuloma formation, vasculitis, organ failure, and death (Pedersen and Boyle, 1980; Poland et al., 1996 and Vennema et al., 1998). The immunopathogenesis of the disease is poorly understood and often there is little coronavirus present in FIP-affected brain tissue (Foley et al 1998), even though infected phagocytes cells were recovered in CNS of cats (Poncelet et al., 2008), yet inflammatory cells are recruited to the brain and appear to contribute to disease, in part, through secretion of

Knowledge of mechanisms and consequences of virus interactions with the nervous system is essential to better understand potentially pathological relevant consequences and design intervention strategies that are highly appropriate to encephalitis. Therefore, collecting new

Talbot, 2003), HEV-infected cells showed no cytopathological changes.

time of infection.

**2.4.2 Feline coronaviruses (FCoV)** 

cytokines (Foley et al., 2003).

**3. Conclusion** 

Fig. 4. Neuroinvasive properties following intranasal (IN) inoculation of C57BL/6 aged from 7 to 14 DPN. After inhalation of virus (5000 TCID50), 5 mice of each age were sacrificed every 24 hours and the production of infectious virus (virus titers) measured in the CNS, lung, liver and spleen. A detectable level of HCoV-OC43 was found in the brain as early as 2 or 3 DPI (days post-infection), and reached its maximum level a few days later. Infectious virus was occasionally found in lung, spleen or liver (detection limit of the assay: 101.5 TCID50), especially at times when brain viral titers reached a maximum. Moreover, 7 and 10 DPN mice did not survive HCoV-OC43 inhalation and about 10% of 14 DPN mice did survive.

Fig. 4. Neuroinvasive properties following intranasal (IN) inoculation of C57BL/6 aged from 7 to 14 DPN. After inhalation of virus (5000 TCID50), 5 mice of each age were sacrificed every 24 hours and the production of infectious virus (virus titers) measured in the CNS, lung, liver and spleen. A detectable level of HCoV-OC43 was found in the brain as early as 2 or 3 DPI (days post-infection), and reached its maximum level a few days later. Infectious virus was occasionally found in lung, spleen or liver (detection limit of the assay: 101.5 TCID50), especially at times when brain viral titers reached a maximum. Moreover, 7 and 10 DPN mice did not survive HCoV-OC43 inhalation and about 10% of 14 DPN mice did

survive.

Fig. 5. Susceptibility of mice to the same infectious dose depends of age. (A) C57BL/6 mice became totally unsusceptible to a 100.5 infectious dose at 28 days post-natal (DPN), whereas, 60% died following the same infectious dose when they were aged 21 DPN. (B) In the same manner, the susceptibility of CD1 mice to a 101.5 infectious doses depended on the age at the time of infection.

1983). The virus was isolated from the brains of suckling pigs suffering from encephalomyelitis several years ago (Greig et al., 1962). The disease could be reproduced experimentally in piglets following intranasal inoculation (Alexander, 1962). A murine model demonstrated that this virus, as other coronaviruses including the HCoV-OC43, is neuroinvasive and neurotropic. In that latter case, neuroinvasion depends on a number of variables such as the route of inoculation and the age of the mice (Yagami et al., 1986; Hirano et al., 1990; 2004). As for the SARS-CoV in mice, HEV induced a poor inflammatory reaction in CNS (Hirano et al., 2004) and unlike HCoV-OC43-infected neurons (Jacomy & Talbot, 2003), HEV-infected cells showed no cytopathological changes.

#### **2.4.2 Feline coronaviruses (FCoV)**

Feline infectious peritonitis (FIP) is a common cause of death in cats, caused by a virulent feline coronavirus (FCoV). The virulent form of FCoV, called FIPV, occurs following mutations acquired during a persistent infection (Brown et al., 2009; Rottier et al., 2005; Vennema et al., 1998) and neurologic involvement could occur in FIP disease (Foley et al., 1998; Summers et al., 1995). The colon was identified as the major site of FCoV persistence; nevertheless the virus could also persist in tissue macrophages representing a source for viremia (Kipar et al., 2010). Infected macrophages disseminate systemically and trigger immunological responses resulting in microgranuloma formation, vasculitis, organ failure, and death (Pedersen and Boyle, 1980; Poland et al., 1996 and Vennema et al., 1998). The immunopathogenesis of the disease is poorly understood and often there is little coronavirus present in FIP-affected brain tissue (Foley et al 1998), even though infected phagocytes cells were recovered in CNS of cats (Poncelet et al., 2008), yet inflammatory cells are recruited to the brain and appear to contribute to disease, in part, through secretion of cytokines (Foley et al., 2003).

#### **3. Conclusion**

Knowledge of mechanisms and consequences of virus interactions with the nervous system is essential to better understand potentially pathological relevant consequences and design intervention strategies that are highly appropriate to encephalitis. Therefore, collecting new

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#### **4. Acknowledgment**

Work from the Talbot laboratory was funded by operating grants MT-9203 from the Institute of Infection and Immunity, Canadian Institutes of Health Research, and 42619-2009 from the National Sciences and Engineering Research Council of Canada, a Tier-1 Canada Research Chair to P.J.T., as well as a studentship from the Canadian Multiple Sclerosis Society to E.B..

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**1. Introduction** 

successful resolution of infection.

(Hawley & Eitzen, 2001; Weaver, 2005).

**10** 

**Encephalitic Development** 

Slobodan Paessler and Katherine Taylor

Viruses are currently the most common cause of encephalitis. With more than 20 viruses known to cause human encephalitis, arboviruses, arthropod-borne viruses, represent a significant number of emerging infectious diseases both in the United States and worldwide. The *Alphavirus* genus in the family *Togaviridae* contains three viruses capable of causing human encephalitis: Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV). WEEV and EEEV are endemic to the United States and South America while VEEV circulates in Central and South America; however, spread of epidemic outbreaks has resulted in disease in North America. No specific therapy or vaccine is currently available against these viruses. In this review, we summarize the development and progression of alphavirus encephalitis in both human populations and murine models of alphavirus infection. We concentrate on the host response that characterizes the development of central nervous system (CNS) disease following alphaviral infection. In addition, we focus on immune factors that influence

Alphaviruses represent a significant public health threat as both emerging infectious diseases and possible agents of bioterrorism. Natural, endemic infections of alphavirus disease are currently absent in certain areas of the globe where the mosquito vector capable of transmitting alphaviruses exists making potential virus introduction into such unaffected areas a significant public health concern (Calisher, 1994; Hawley & Eitzen, 2001; Weaver & Barrett, 2004; Zacks & Paessler, 2010). Additionally, many of the encephalitic alphaviruses are highly stable as an aerosol, cause significant mortality or incapacitating disease, and grow rapidly in easily utilized, readily available cell culture systems. This combination of factors makes this group of viruses of significant interest for biodefense in the United States

One of three of the original serological groups of arboviruses, the group A complex became the *Alphavirus* genus within the *Togaviridae* family representing enveloped, plus-strand RNA viruses (Porterfield, 1975; Porterfield, 1986). Pathogenic alphaviruses can be roughly grouped further by a combination of phylogenetics, geographical circulation, and disease manifestation into two groups. Viruses circulating in the Old World (Europe, Asia, and Africa) typically cause arthralgia, malaise or a rash. (Griffin, 2007; Zacks & Paessler, 2010). New World alphaviruses' infections result in a flu-like syndrome that may progress to neurological involvement. New World encephalitic alphaviruses include EEE, WEE, and

**in Alphaviral Infection** 

*University of Texas Medical Branch* 

*United States of America* 

brain: potential role of the chemokine mig in pathogenesis. Clin. Infect. Dis. 41, 1089-1096.

