**4. Infection and replication of viruses in brain cells**

#### **4.1 Primary receptors used by viruses to infect CNS cells**

The presence of cell membrane proteins that act as virus receptors determines whether a cell can be invaded by a virus or not. Dependent on the receptor(s) chosen, some viruses may infect nearly all mammalian cell types or only a small subset of cells from certain species. We herein have chosen to highlight only some of the receptors used by a few emerging or re-emerging viruses to infect specific cell types in the brain and to cause encephalitis. Several comprehensive reviews have discussed the role of different receptors for rabies (Acethylcholine nicotinic receptors, NCAM CD56 as well as the P75 neurotrophin receptor, NTR) (Lafon, 2005) and, hence, this aspect will not be discussed herein.

#### **4.1.1 Nectin-1 and 2 are cellular receptors for HSV**

Nectins are immunoglobulin (Ig)-like Cell Adhesion Molecules (CAMs) involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites (Takai et al., 2008). The nectin family which comprises 4 members,

encephalitis, capillary and endothelial inflammation of cortical vessels is a striking pathological finding, occurring primarily in the grey matter or grey white junction and this observation may facilitate virus entry. For example, HIV may gain access to CNS *in vivo* by this paracellular route as a result of endothelium activation by TNFα and other

Second, viruses may transmigrate across the BBB within virally infected leukocytes. For HIV, several studies suggest that virus shedding from infected CD4+ T cells, macrophages, and monocytes during migration through the BBB can instigate CNS replication into the parenchyma (Nottet et al., 1996; Persidsky et al., 1997; Schmidtmayerova et al., 1996). CMV can also be transferred to CNS by virus infected mononuclear phagocytes and bi-directional cell to cell transmission between infected monocytes and endothelial cells (Drevets and

Third, viruses can also penetrate the CNS by taking advantage of incomplete closure of the BBB. Despite the intercellular tight junctions between the capillary endothelial cells in most regions of the BBB, certain areas of the CNS such as the choroid plexus, posterior pituitary, and circumventricular organs are not completely protected by the BBB due to a fenestrated endothelial cell layer and sparse basement membrane (Zhang and Tuomanen, 1999). A number of blood-borne viruses including mumps (Herndon et al., 1974), HIV (Bagasra et al., 1996) and CHIKV (Couderc et al., 2008) have been suggested to penetrate across the choroid plexus micro- vessels and infect the epithelium. In the CSF space, viruses can subsequently

Finally, viruses can spread to the CNS through peripheral neuronal routes, like the motor neurons of the spinal cord ,olfactory neurons, retinal neurons, oculomotor neurons and trigeminal nerves , which are directly connected to the CNS, thus providing a convenient route for neurotropic viruses (Mori et al., 2005; Tirabassi et al., 1998). Viruses including HSV (Barnett et al., 1993), rabies virus (Jackson, 2003) and VEEV (Charles et al., 1995) are able to replicate within peripheral nerves and are transported into the CNS from the PNS along axons as the result of axonal transport of neurons. Certain enteroviruses can also spread to the CNS by infecting enteric neurons (Morrison and Fields, 1991). Virus spread within the

The presence of cell membrane proteins that act as virus receptors determines whether a cell can be invaded by a virus or not. Dependent on the receptor(s) chosen, some viruses may infect nearly all mammalian cell types or only a small subset of cells from certain species. We herein have chosen to highlight only some of the receptors used by a few emerging or re-emerging viruses to infect specific cell types in the brain and to cause encephalitis. Several comprehensive reviews have discussed the role of different receptors for rabies (Acethylcholine nicotinic receptors, NCAM CD56 as well as the P75 neurotrophin receptor,

Nectins are immunoglobulin (Ig)-like Cell Adhesion Molecules (CAMs) involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites (Takai et al., 2008). The nectin family which comprises 4 members,

proinflammatory cytokines (Fiala et al., 1997).

infect the ependymal cells and the surrounding brain tissue.

CNS by retrograde, anterograde or cell to cell diffusion mechanisms.

NTR) (Lafon, 2005) and, hence, this aspect will not be discussed herein.

**4.1.1 Nectin-1 and 2 are cellular receptors for HSV** 

**4. Infection and replication of viruses in brain cells 4.1 Primary receptors used by viruses to infect CNS cells** 

Leenen, 2000).

nectin-1 or poliovirus receptor related 1 (PRR1 also called HveC, CD111, HIgR), nectin-2 (also termed PRR2, HveB, CD112), nectin-3 and nectin-4. Each of them contains an extracellular region with three Ig-like domains, a single transmembrane region, and a cytoplasmic tail region. Nectin-1, -2 and -3 are expressed ubiquitously in multiple cell types such as epithelia, fibroblasts and neurons, whereas nectin-4 is mainly expressed in the human placenta. Nectin-1 and -2 have been identified as HSV entry mediators (Hve) **(see Table 1)**. Nectin-1 can serve as entry receptors for both HSV-1 and HSV-2 (Geraghty et al., 1998). In contrast, nectin-2 has more limited entry activity. Indeed, human nectin-2 is only a weak entry receptor for HSV-2 and certain strains of HSV-1 carrying out mutations in the glycoprotein D (Lopez et al., 2000). A more recent study also assessed the roles of the two known entry receptors, HVEM and nectin-1, in neuronal infection in the CNS and the development of encephalitis in a mouse model (Kopp et al., 2009). Intracranial injection of HSV was performed directly into the hippocampus of Wild-type (WT), HVEM KO, nectin-1


(See text for abbreviation)

Table 1. Encephalitis due to emerging and re-emerging viruses

Virus-Induced Encephalitis and

binding with NiV glycoprotein G.

**4.1.4 Scavenger receptor class B member 2 (SCARB2) and EV71** 

lysosomes and late endosomes (reviewed in (Eskelinen et al., 2003).

**4.1.5 CD4 as the primary receptor for HIV** 

Innate Immune Responses – A Focus on Emerging or Re-Emerging Viruses 67

Two independent studies identified Ephrin B2 (EFNB2) as the receptor for HeV and NiV, which are emergent paramyxovirus. Of the ten potential receptor-encoding plasmids they used to transfect non-permissive HeLa cells, only the human EFNB2 plasmid confers HeV fusion permissiveness. They further demonstrated that soluble EFNB2 blocks both viruses fusion in human EFNB2-transfected HeLa cells. They also showed that both HeV and NiV glycoprotein G and EFNB2 soluble protein could be specifically and reciprocally captured by ELISA. In addition, both viruses glycoprotein G were efficiently coprecipitated by EFNB2/Fc. Another study identified EFNB2 as an entry receptor for NiV (Negrete et al., 2005). The authors also reported that EFNB2 binds to the NiV glycoprotein G. They observed that soluble Fc-fusion proteins of EFNB2 effectively blocked NiV fusion and entry into permissive cell types. Moreover, transfection of EFNB2 into non-permissive cells renders them permissive for NiV fusion and entry. Interestingly they found that soluble EFNB2 inhibited NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons. EFNB3 was later discovered as an additional receptor for NiV using the Chinese hamster ovary cell line (CHO-pgsA745) that does not express ephrins endogenously (Negrete et al., 2006). Interestingly, the authors characterized the important conserved Leu-Trp residues in the G-H loop of EFNB2 and B3 that are critical for their

The concept of scavenger receptors (SCAR) was first described by their ability to bind modified low-density lipoproteins (Goldstein et al., 1979). SCAR can bind a variety of natural/endogenous, modified host, microbial (bacterial, viral, fungal, and parasitic), environmental, soluble or particulates ligands by endocytosis and phagocytosis (reviewed in (Mukhopadhyay and Gordon, 2004)). SCARB2 (also known as lysosomal integral membrane protein II or CD36b like-2) belongs to the scavenger receptor class B subfamily, which also includes SCARB1 and CD36 (Calvo et al., 1995; Eskelinen et al., 2003). SCARB2 is a type III double-transmembrane protein with N- as well as C-terminal cytoplasmic tails and is located primarily in lysosomes and endosomes. SCARB2 participates in membrane transportation and the reorganization of the endosomal-lysosomal compartment (Kuronita et al., 2002). Deficiency of SCARB2 in mice causes ureteric pelvic junction obstruction, deafness and peripheral neuropathy (Gamp et al., 2003). SCARB2 is widely expressed in

Since its identification in California in 1969 (Schmidt et al., 1974), enterovirus 71 (EV71) has been recognized as a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) associated with severe neurological sequelae in some cases (Blomberg et al., 1974). EV71 infections can progress to aseptic meningitis, acute flaccid paralysis and fatal encephalitis (McMinn, 2002). A recent paper demonstrated that SCARB2 is a receptor for EV71 (Yamayoshi et al., 2009). The authors showed that EV71 binds soluble SCARB2 or cells expressing SCARB2, and the binding is inhibited by an antibody to SCARB2. Furthermore, expression of human SCARB2 enables normally unsusceptible cell lines to support EV71 propagation and develop cytopathic effects. Yet, the detail of the interaction between EV71

and SCARB2 at the cellular level, resulting in CNS infection has not been identified.

Human CD4 exhibits a poly-Ig-like domain structure which is homologous to an increasingly large number of recognition molecules, and consists in four tandem variable-

KO and double KO mice. The results indicated that nectin-1 deficient mice showed no signs of disease after intracranial inoculation, and no HSV antigens were detectable in the brain parenchyma. However, HSV antigens were detected in non-parenchymal cells lining the ventricules. In the double KO mice, the results showed an absence of disease and no detectable expression of viral antigens even in non-parenchymal cells, indicating that infection of these cells in the nectin-1 KO mice was dependent on the expression of HveM.
