Introduction to Flaviviruses

Flavivirus-Induced Neurological Diseases

At least eight flaviviruses may cause mild-to-severe neurologic syndromes in humans. Different viruses cause different disease neurological manifestations, which will be described in separate chapters. Some of these manifestations are encephalitis; meningitis; acute flaccid myelitis; microencephaly; Guillain-Barré syndrome; and sensory and motor disorders. Some of the neurological disorders are found primarily in adults (Guillain-Barré syndrome), while others are present in fetuses or infants of mothers infected during pregnancy (microencephaly). Most of these viruses pass into the central nervous system (CNS) from the blood by crossing the blood–brain barrier (BBB) using several host- or virus-induced mechanisms.

Flaviviruses can replicate in brain microvascular endothelial cells (BMECs) in vitro and, in the process, decrease the effectiveness of the tight junction proteins that are found between the cells of the BBB, thus increasing the barriers’ permeability. Infected BMECs also permit the basolateral release of infectious virions in the absence of a cytopathic effect. Some of these viruses infect glial cells of the CNS, such as astrocytes and microglia (small, phagocytic immune system cells in the brain). Activation of microglial triggers their production and secretion of inflammatory cytokines and chemokines that draw other leukocytes into the CNS. The infiltration of these inflammatory leukocytes can cause further disruption of the BBB as well as inducing neuropathology. Release and enzymatic activity of matrix metalloproteases by infected astrocytes damage the extracellular materials that hold the cells of the BBB tightly together. This allows for further flavivirus entry into the CNS.

Flavivirus-Induced Hemorrhagic Fever Diseases

At least five flaviviruses are known to produce mild-to-fatal hemorrhagic disease in humans. While most people develop only fever with or without severe joint or muscle pain, other people develop the classic symptoms of potentially life-threatening hemorrhagic fever. In the midst of the current DENV pandemic, the incidence of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) is increasing as different DENV serotypes invade new geological regions. This leads to rising numbers of people who have been infected with one DENV serotype, followed by infection with a different serotype. In such cases, antibodies produced in response to the first DENV serotype may cause a pathogenic cross-reaction that leads to enhanced, rather than decreased, disease manifestations, in a process named antibody-dependent enhancement (ADE).

General Information

Flaviviruses are spherical, enveloped viruses that are 40–50 nm in diameter ( Fig. 1.1 ).

Their genome consists of positive-sense, single- stranded RNA that is composed of approximately 11,000 nucleosides. This RNA lacks a poly-A tail, but contains 100 and 400–700 highly conserved nucleosides in its 5′ and 3′ untranslated regions (UTRs), respectively. The 5′ terminal region, containing many AG dimers, is formed of two domains, both involved in the replication. The first domain is completely found within the 5′ UTR and has a branched stem-loop, a structure that serves as a promoter for flavivirus replication. The second 5′ UTR domain extends in the genomes’ single open-reading frame. Its contents include the nucleosides that are 5′ of the AUG region that folds into the second stem-loop structure, the AUC start site, the nucleosides downstream of AUG region, the capsid protein’s coding region hairpin, the 5′ cyclization sequence, and a pseudoknot.

Flavivirus’ genomic RNA encodes a polyprotein that consists of 10 proteins, 7 of which are nonstructural (NS) genes. NS3 and its NS2B cofactor are found in the N-terminal serine protease domain. The NS3 C-terminal helicase domain performs three distinct activities, acting as an RNA helicase, a nucleoside triphosphatase, and a 5′ RNA triphosphatase. In its membrane-bound form, NS5 is the largest and most conserved flaviviral NS protein, consisting of four dimers that undergo intermolecular interactions. The N-terminal domain of NS5 contains a methyltransferase domain with three activities involved in RNA cap synthesis (guanylyltransferase, guanine-N7-ethyltransferase, and nucleoside-2′ O -methyltransferase), while the NS5’s C-terminal encodes the RNA-dependent RNA polymerase.

The 3′ terminus of flavivirus’ genome contains three domains within the 3′ UTR. Domain I is the most variable of the three. It contains two stem-loop structures in the form of pseudoknots and plays a role in the formation of subgenomic flavivirus RNAs (sfRNA1 and sfRNA2; RNA polymerase). The sfRNAs inhibit the host’s innate immune response and aid in adaptation to different host species. Some of the components of the variable region differ substantially between the genomes of high and low pathogenic viral isolates, signifying that this region contains virulence factors. Domain II of the 3′ UTR contains 1–2 conserved dumbbell-shaped structures required for replication and translation. Domain III is highly conserved among flaviviruses and contains sequences that interact with regions found at the 5′ end of the genomes that are necessary for genome circularization and genomic RNA replication. Domain III also contains a 3′ stem-loop structure that is flanked on its 5′ end by a short hairpin.

The flaviviruses’ single open-reading frame, after cleavage by cellular and viral proteases, also produces three structural proteins: the envelope (E) protein, membrane (M) protein, and capsid (C) protein.

Flaviviruses’ Structural Proteins

The E protein is the major structural glycoprotein, with 180 copies on the surface of each virion. It is highly conserved among the members of the genus and is the primary protein in the viral envelope. The E protein is multifunctional and is involved in receptor-mediated endocytosis and fusion with endosomal membranes. It additionally contains hemagglutination activity, which allows the virus to bind to the host cells’ sialic acid moieties prior to membrane fusion. Due to its abundance and location on the viral envelope, the E protein induces the host’s immune system to produce protective hemagglutination-inhibiting (HI) and neutralizing antibodies. E proteins are oriented parallel to the membrane and are organized 30 “rafts,” each containing three E dimers. The dimers are situated in a head-to-tail manner and are transformed into active trimers after entering the target cell’s acidified endosomes.

The E protein is composed of several domains. Domain I (central domain) is approximately 120 amino acids long and can be N-glycosylated. Domain II is approximately 180 amino acids long and serves as the dimerization domain. It contains the fusion peptide as well as epitopes which cross-react with other flaviviruses and is important in diagnoses and vaccine development. The C-terminal region of Domain III has an immunoglobulin-like fold and is involved in binding to cellular receptors. Naturally occurring mutations on the upper lateral surface of the E protein’s Domain III are important determinants of neurovirulence and neuroinvasiveness. The presence or absence of a glycosylation site in this region plays a major role in whether or not the virus is able to escape neutralizing antibodies in mice. Immediately downstream of Domain III is an area important for anchoring the virus to membranes, interacting with the precursor of the membrane protein (prM) and for acid-induced conformational changes. Its two transmembrane regions anchor the virus to host cells and allow signal translocation of the viral NS1 into the lumen of the cell’s endoplasmic reticulum (ER).

In addition to the E protein, the membrane (M) protein is found on the viral surface. It is derived from its prM precursor after its cleavage by the host cell’s furin enzyme. prM prevents E protein from undergoing dimer-to-trimer transformation. The furin-induced cleavage allows activation of the E protein.

Over 500 copies of the capsid protein form antiparallel dimers. The capsid proteins act together with viral genomic RNA to form the electron-dense nucleocapsid. This provides an anchoring platform for prM and E proteins and viral assembly.

Flaviviruses’ NS Proteins

NS1 is multifunctional. It plays an important role in viral evasion of the host complement system of the immune response by decreasing the number of membrane attack complexes on its host cell’s plasma membrane. This protects the virally infected cell from the production of complement-induced pores, which would normally result in cell lysis. The escape from complement destruction of the host cell allows the virus to continue its life cycle. NS1 also protects extracellular viruses. Interactions between NS1 and NS4B regulate virus replication. NS1 also acts in concert with NS3, NS2B, NS4A, and NS4B to extensively modify host cell’s ER membranes. These alterations in the lipid structure of the ER are important for viral RNA replication, translation, and assembly.

NS2A, NS2B, and NS4B are integral membrane proteins, but have different functions. NS2A, NS3, and NS4B are involved in replication, viral assembly, and immunomodulation. The latter activity involves regulating the host cell’s ER unfolded protein response as well as the interferon-β (IFN-β) signaling pathway, a key antiviral immune system agent. NS2B also works in conjunction with NS3.

NS3 is the only known viral protein to serve as both a chymotrypsin-like serine protease in the N-terminal domain and a helicase/nucleotide triphosphatase (NTPase) that also includes ATPase activity. This activity is regulated by cytoplasmic NS4A and releases energy from ATP for use by the viruses as well as the host cells. The NS3 protease cleaves the newly transcribed viral polyprotein into its individual proteins, while the helicase is active during replication in strand unwinding prior to strand separation. It should be noted that flavivirus protease and helicase domains are arranged differently than those present in the other Flaviviridae member, hepatitis C virus, and are accordingly susceptible to different drugs.

NS5 has several functions. Its polymerase region incorporates ribonucleoside triphosphates into the nascent RNA during replication. Replication occurs in a close contact with the virally remodeled ER membrane. NS5’s capping functions include GTP binding and guanylyltransferase activity. The 5′ cap is vital to Flaviviridae members since it directs viral polyprotein translation, protects the 5′ end of the viral genome from cellular exonucleases, and is used by cells for self-nonself RNA discrimination. The NS5 polymerase contains four potential protein kinase C (PKC) family members’ phosphorylation sites. These sites are potential drug targets. Several antiviral NS5 drugs inhibit virus–cell membrane fusion during viral entry into host cells and dimer formation.