**2. Coronavirus**

Coronaviruses are ubiquitous respiratory and enteric pathogens. They also represent one family of viruses that bear neurotropic and neuroinvasive properties in various hosts including humans, pigs, and rodents. Coronaviruses form a group of enveloped, positivesense, single-stranded polyadenylated RNA viruses that have the largest genome (~30 kb) among RNA viruses. They replicate in the cytoplasm of infected cells using a viral RNAdependent RNA polymerase that is translated from the genomic RNA very early after viral entry in the host cell. Coronaviruses first target respiratory and mucosal surfaces and then, depending of host and virus strain, may spread to other tissues (brain, eyes, liver, kidneys and spleen) and cause a range of pathologies such as pneumonia, encephalitis, neurodegenerative demyelination, hepatitis, enteritis, and nephritis among others (Resta et al., 1985; Riski & Hovi, 1980).

Human coronaviruses (HCoV) are represented by five different strains; HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and the causative agent of the severe acute respiratory syndrome (SARS), named SARS-CoV. Among these five strains, at least HCoV-229E and HCoV-OC43, as well as SARS-CoV possess neuroinvasive properties as viral RNA can be detected in human brains (Arbour et al., 2000; Gu et al., 2005; Xu et al., 2005).

However, these human coronaviruses are not well characterized as to their capacity to invade and infect the CNS. On the other hand, the murine counterpart of HCoV-OC43, which is designated Mouse Hepatitis Virus (MHV), represents one of the best-characterized models of this family. MHV infects mice and rats and some strains are neurotropic and

directly induce damage to CNS cells (virus-induced neuropathology). In acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the gray matter, as was described after herpes simplex virus (HSV), rabies, or some arbovirus infections. Rabies virus usually spreads to the CNS through retrograde peripheral nerve dissemination and one of the possible routes of HSV spread to the CNS is through the

Encephalitis caused by viruses generally can be classified into four different groups. (1) *Arboviruses* which appear to be the primary cause of acute encephalitis (Eastern Equine Encephalitis, Japanese Encephalitis, La Crosse Encephalitis, St. Louis Encephalitis, Western Equine Encephalitis, West Nile Virus Encephalitis). These viruses are transmitted to humans by the bite of infected mosquitoes and/or ticks. (2) *Enteroviruses*, such as coxsackievirus or polioviruses. These viruses spread through the fecal-oral route. Infection can result in a wide variety of symptoms ranging from mild respiratory illness (common cold), to, foot-and-mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis. (3) *Herpes viruses* constitute another major cause of encephalitis in North America. Members of this virus family include HSV, Epstein-Barr virus (EBV), cytomegalovirus (CMV), and varicella-zoster virus (VZV). They are highly contagious as they can be spread when an infected person is producing the virus. (4) Other viruses, following childhood viral diseases such as measles, mumps, and rubella can in rare cases develop secondary encephalitis. More recently, respiratory viruses were closely associated with encephalitis as reported for influenza virus (for reviews, see Maurizi, 2010 and Wang et al., 2010) or occasionally for

Coronaviruses are ubiquitous respiratory and enteric pathogens. They also represent one family of viruses that bear neurotropic and neuroinvasive properties in various hosts including humans, pigs, and rodents. Coronaviruses form a group of enveloped, positivesense, single-stranded polyadenylated RNA viruses that have the largest genome (~30 kb) among RNA viruses. They replicate in the cytoplasm of infected cells using a viral RNAdependent RNA polymerase that is translated from the genomic RNA very early after viral entry in the host cell. Coronaviruses first target respiratory and mucosal surfaces and then, depending of host and virus strain, may spread to other tissues (brain, eyes, liver, kidneys and spleen) and cause a range of pathologies such as pneumonia, encephalitis, neurodegenerative demyelination, hepatitis, enteritis, and nephritis among others (Resta et

Human coronaviruses (HCoV) are represented by five different strains; HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and the causative agent of the severe acute respiratory syndrome (SARS), named SARS-CoV. Among these five strains, at least HCoV-229E and HCoV-OC43, as well as SARS-CoV possess neuroinvasive properties as viral RNA can be detected in human brains (Arbour et al., 2000; Gu et al., 2005; Xu et al.,

However, these human coronaviruses are not well characterized as to their capacity to invade and infect the CNS. On the other hand, the murine counterpart of HCoV-OC43, which is designated Mouse Hepatitis Virus (MHV), represents one of the best-characterized models of this family. MHV infects mice and rats and some strains are neurotropic and

olfactory tracts.

coronaviruses (Yeh et al., 2004).

al., 1985; Riski & Hovi, 1980).

2005).

**2. Coronavirus** 

neuroinvasive, causing a large spectrum of diseases from hepatitis to encephalitis and chronic demyelination (Stohlman & Hinton, 2001; for reviews, see also Bender & Weiss, 2010 and Lane & Hosking, 2010).

#### **2.1 Murine Hepatitis Virus: An agent of encephalitis**

MHV exhibits various organ tropisms as well as pathogenic potentials. The most common strains used for pathogenesis studies are the neurotropic MHV-JHM (previously called MHV-4), the hepatotropic/neurotropic MHV-A59 and the hepatotropic MHV-3. Experimental infections of rodents with these strains provide animal models for human diseases such as hepatitis, encephalitis, and demyelinating diseases such as multiple sclerosis. Infection of mice by the intranasal or intracerebral route with MHV-JHM or MHV-A59 serves as a model for studying encephalitis and determinants of neurovirulence. MHV is part of the family *Coronaviridae* and the genus betacoronavirus. Its genome is 32-

kilobases long and comprises different open reading frames (ORFs), which encode four structural proteins: spike (S), envelope (E), membrane (M), nucleocapsid (N), with some strains also expressing a gene encoding two other structural proteins: hemagglutininesterase (HE) and internal protein (I). The genome also encodes three nonstructural proteins, which functions remain poorly understood. The assembly of coronavirus virions needs a concerted action of three structural proteins: the membrane protein (M), the small envelope protein (E), and the nucleocapsid protein (N) (de Haan & Rottier, 2005; Masters 2006). Among these three proteins, no role in pathogenesis has been reported for M and E.

#### **2.2 Viral molecular determinants of encephalitis**

While the molecular determinants of pathogenesis remain poorly understood, there is evidence that both host and viral factors play a role in coronavirus-induced disease. Experiments completed during the last decade have used infectious cDNA clones to produce viruses of high and low virulence to investigate, by the mean of reciprocal chimeric viruses, the molecular determinants of neurovirulence. Viral genes responsible for high MHV neuropathogenesis contribute to viral spread, replication and activation of innate/adaptive immunity.

#### **2.2.1 Spike glycoprotein: S**

The S protein mediates attachment of the virus to its receptor on the target cell and viral fusion with the cell membrane, as well as viral entry and cell to cell spread (Collins et al., 1982; Williams et al., 1991). Based on previous studies, which used numerous variant viruses selected for resistance to neutralizing monoclonal antibodies, an association was made between various mutations or deletions in the S gene and neuroattenuation of the different strains of MHV (Gallagher et al., 1990; Wege et al., 1988). More recently, the use of recombinant MHV viruses with a modified spike (S) glycoprotein has definitively identified the S protein as a major determinant of neurovirulence. The recombinant A59 (rA59) virus which contains the S gene of JHM (SJHM) confers a highly neurovirulent phenotype. The viral infectious dose inoculated into mouse brain to induce a 50% lethal dose (LD50) is decreased by more than 1000-fold, demonstrating the role of S in neurovirulence (Phillips et al., 1999). Neuronal infection has long been proposed to be a major determinant of MHV neurovirulence (Dubois-Dalcq et al., 1982), and the recent use of recombinant viruses demonstrated that even if neurovirulence is increased, cellular tropism remained the same,

Coronaviruses as Encephalitis - Inducing Infectious Agents 189

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).

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

**2.3 Human coronaviruses (HCoV)** 

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
