GENERAL INTRODUCTION: FLAVIVIRIDAE

Introduction

West Nile virus (WNV) infection is mosquito-borne and may cause severe and fatal neurological disease in birds, humans, horses, and, more sporadically, a wide variety of other animals. The virus was first isolated in the West Nile district of Uganda in 1937.¹⁸⁸ Prior to 1999, WNV caused only sporadic outbreaks of disease in humans and horses in Africa,^{77, 197} the Middle East^{69, 82} and Europe.⁷⁹ Kunjin virus (KUN), a variant of WNV found in Australia, caused an extensive outbreak in 2011 in horses. In the late 1990s and early 2000s, severe concurrent disease outbreaks occurred in the United States (US), France, and Israel.^{69, 79, 218} At present, WNV is endemic in all continents except Antarctica.⁴⁴ Following introduction to North America in 1999,^{64, 103} WNV was detected in Central and South America by 2005.^{46, 96, 134, 146} Expansion of all virus lineages with new subtypes has occurred in the US, Europe, and Eurasia.⁵¹ Finally, WNV was once considered a cause of subclinical or febrile disease in Africa, but reports of neurovirulent WNV have increased in Africa since 2007.²⁰⁶ The continued spread of WNV indicates that its associated disease will remain a threat to the health and well-being of livestock and humans worldwide.

Aetiology

West Nile virus is grouped within the genus Flavivirus of the family Flaviviridae (see General Introduction: Flaviviridae), an ever expanding group of viruses that as a family is one of the most  pathogenic to animals and humans.^{86, 219} Flaviviruses, including WNV, are positive sense, single-stranded RNA viruses measuring approximately 45-50 nm in diameter. The virions are spherical, enveloped and contain a nucleocapsid.¹³⁶ The genome is approximately 11kilobase (kb) and contains a single open reading frame (ORF) that is translated in its entirety and cleaved by both cell and viral proteases into 11 viral proteins, specifically three structural proteins, including capsid (C), premembrane (prM)/membrane (M), and envelope (E), and seven nonstructural (NS) proteins.^{57, 89, 102, 136, 157} The mature virion has 180 copies of the E protein arranged in a herringbone pattern covering a lipid bilayer.^{72, 99, 109, 132, 141, 145, 159, 193, 226} The E protein is dimerized, composed of two each of three domains (E-DI, E-DII, and E-DII) connected by hinges forming a type II viral fusion protein. E-DI is part of the central domain of the E protein and is an eight -barrel protein linked by two disulphide bonds with an N-link carbohydrate site. In vivo neurovirulence is altered with mutation of the amino acid residues 154-156, a N-Y-S glycosylation site of the E-DI.^{24, 76, 136, 157} A newly emerged neurovirulent strain of WNV in Australia (Kunjin virus [KUN]) is also glycosylated at this site whereas less virulent KUN viruses are not.⁷⁰ The domain E-DII mediates dimerization and has the glycine rich fusion loop protein that inserts into the membranes of target cells. Located in a hydrophobic pocket between E-DI and E-DIII, mutation of E-DII renders the virus unable to fuse with synthetic membranes.^{142, 145} E-DIII forms as an immunoglobulin-like structure at the carboxy-terminus and projects from the surface of the virus.^{50, 142, 159, 177, 178} Since monoclonal antibodies to this site result in neutralization of the virus, this domain contains the main binding epitopes for attachment and entry into cells.⁴² The prM protein is essential for E protein folding, trafficking through the Golgi, and prevents interaction of the virus with futher cell membranes during egress.^{108, 110, 122, 158, 224} Maturation of the virus is not considered complete until this protein is cleaved by a cellular furin-like serine protease.²²⁵ However, immature viral particles can be released independent of furin maturation and have demonstrated cellular infectivity.¹⁵⁸ 

The first step in viral infection occurs when domain III of the E protein binds to host cell receptors.¹¹¹ Receptor mediated endocytosis is clathrin-mediated, and the virus enters the host cell within a low pH vesicle.^{15, 27, 41} This low pH is the signal for E protein re-arrangements (switch from a dimer to a trimer form) to occur, exposing domain II of the E protein and allowing it to fuse to the vesicle membrane. The virus is then released from the vesicle into the cell cytoplasm. At this stage, a single strand of positive sense RNA is present in the host cell cytoplasm.¹¹¹ Translation occurs first, when host cell elongation initiation factors (eIF) bind to the 5’ untranslated region (UTR) of the viral genome. Host ribosomal subunits rRNA and tRNA with attached amino acids are then added in the traditional initiation, elongation, and termination steps of translation to form a polyprotein. Host cell proteases then cleave the polyprotein, allowing replication to occur. In subsequent translational events, the viral NS2B/NS3 protease carries out the cleavage of the polyprotein. Replication starts at the 3’ UTR of the viral RNA. The viral NS4B/NS5...