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West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.
Key Words WNV replication, WNV proteins, cis-acting sequences, conserved RNA structures, RNA-protein interactions, virus-host interactions
West Nile virus (WNV) is a mosquito-borne virus that was first isolated in 1937 from the blood of a woman participating in a malaria study in the West Nile region of Uganda (136). Endemic in parts of Africa, the Middle East, and western Asia, particularly India, WNV has periodically been the causative agent of brief epizootics in France, Romania, Russia, Algeria, Madagascar, Senegal, and South Africa and of infrequent disease outbreaks in humans. Because a number of wild bird species develop high levels of virernia after WNV infection and sustain viremic levels of WNV of at least [10.sup.5] PFU/ml of serum (the minimum level estimated to be required to infect a feeding mosquito) for days to weeks, they are the main reservoir hosts in endemic regions and are also the usual source of the virus initiating epizootics outside endemic areas (11,86,113). For example, the sequence of the envelope gene of a WNV isolate (RO97-50) obtained from Culex mosquitoes in Bucharest in 1999 was identical to that of WNV isolates obtained in Kenya and Senegal from Culex mosquitoes (130). During the recent outbreaks in Israel and the United States, there has been a higher-than-normal death rate in birds (90).
Humans and horses are incidental hosts with low viremic levels and do not play a role in the transmission cycle. Although the incidence of clinical disease among WNV-infected humans is low, in recent outbreaks there has been an increase in the severity of disease among those that develop clinical symptoms (113). Fever is the most common symptom observed in humans. The course of the fever is sometimes biphasic, and a rash on the chest, back, and upper extremities often develops during or just after the fever (29). Symptoms also include headaches, muscle weakness, and disorientation. A few infected individuals develop encephalitis, meningoencephalitis, or hepatitis. The brainstem, particularly the medulla, is the primary CNS target (129). Humans age 60 and older have an increased risk of developing fatal disease (36a).
The largest known human epidemic of WNV occurred in 1974 in Cape Province, South Africa, with about 3000 human clinical cases (101). Recent outbreaks of WNV in humans and horses have been more frequent (Romania and Morocco in 1996; Tunisia in 1997; Italy in 1998; Russia and the United States in 1999; and Israel, France, and the United States in 2000) (113). Estimates from serological data obtained in Queens, New York, the epicenter of the U.S. outbreak in 1999, suggest that at least 1900 humans in Queens may have been infected with WNV. While the majority of the infected humans experienced either no symptoms or a low-grade fever, 62 developed clinical disease and of these 7 died as a result of the infection (77).
WNV has been isolated from Culex, Aedes, Anopheles, Minomyia, and Mansonia mosquitoes in Africa, Asia, and the United States, but Culex species are the most susceptible to infection with WNV (29,68). Also Culex mosquitoes feed on wild bird species that have high levels of viremia (145). Natural vertical transmission of WNV in Culex mosquitoes in Africa has been reported and is expected to enhance virus maintenance in nature (103). Mechanical transmission by ticks may also play a role in virus maintenance (29). The 1999 outbreak in New York represented the first introduction of WNV into the Western hemisphere (17). The mode of introduction of WNV into the United States is not known, but phylogenic analysis of the envelope gene of a WNV isolate from the New York outbreak indicated that it was most closely related to a goose isolate from Israel (WN-Israel 1998) (70,90). WNV transmission reoccurred in New York during the summers of 2000 and 2001, and the epizootic spread to other states, indicating that this viru s had become endemic in the United States. In future years, it is expected that viremic migratory birds will carry WNV to all parts of the United States as well as to Canada, the Carribean, and Central and South America (119). Mosquitoes capable of transmitting WNV to susceptible birds exist in all of these regions.
Environmental conditions such as heavy rains followed by flooding, irrigation, and high temperatures can cause an increase in mosquito populations and also in the incidence of mosquito-borne viruses such as WNV. However, in cities the lack of heavy rains, which periodically flush out the sewers, results in increased mosquito populations breeding in pools of stagnant water, as was the case for the 1999 outbreak in New York City. The convergence of birds at scarce pools of water also facilitates virus transmission.
The family Flaviviridae consists of three genera: Flavivirus, Pestivirus, and Hepacivirus. Members of the different genera are distantly related but share a similar gene order and conserved nonstructural protein motifs. The ~70 viruses currently classified in the genus Flavivirus are further subdivided into twelve antigenic serogroups (65). WNV is a member of the Japanese encephalitis virus (JEV) serogroup, which also includes Cacipacore, Koutango, JE, Murray Valley encephalitis, St. Louis encephalitis, Usutu, and Yaounde viruses (65, 115). Based on sequence homology, Kunjin virus, which is endemic to Australia and Asia, is now considered a WNV subtype (65, 131). WNV isolates have been grouped into two genetic lineages (1 and 2) on the basis of signature amino acid substitutions or deletions in their envelope proteins (12). All the WNV isolates associated thus far with outbreaks of human disease have been in lineage 1(70,90). Lineage 2 viruses are restricted to endemic enzootic infections in Africa. Because o f antigenic cross-reactivity between different flaviviruses, techniques such as in situ hybridization or sequence analysis of RT-PCR products are required to unequivocally identify WNV as the causative agent of an outbreak (17,90,139).
Because data are not available on all aspects of WNV and because it is likely that the properties of other flaviviruses will be similar to those of WNV, information obtained for other flaviviruses has been used when appropriate in this review. For additional information on flaviviruses, see (29,64,65,96).
The WNV genome is a single-stranded RNA of positive polarity (mRNA sense). A type 1 cap structure ([m.sup.7]GpppAmp) is present at the 5' end (40), but the 3' end terminates with [CU.sub.OH] (26, 151). Elavivirus genomes are the only mammalian plus-strand RNA virus genomes that do not have a 3' poly(A) tract. The 5' noncoding region (NCR) of the WNV genome RNA is 96 nts in length, while the 3' NCR is 631 nts. The WNV genome is 11,029 nt in length and contains a single open reading frame (ORF) of 10,301 nt (90). Ten mature viral proteins are produced via proteolytic processing of the single polyprotein by the viral seine protease (NS2B-NS3) and various cellular proteases (96, 108) (Figure 1). The three viral structural proteins, capsid (C), membrane (prM/M), and envelope (E), are encoded within the 5' portion of the genomic ORF, while the seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are encoded within the 3' portion (124).
VIRION MORPHOLOGY AND COMPOSITION
WN virions are small (~50 nm in diameter), spherical, enveloped, and have a buoyant density of ~1.2 g/[cm.sup.3] The spherical nucleocapsid is ~25 nm in diameter and is composed of multiple copies of the C protein. Cryo-electron microscopy data suggest that the virion envelope and capsid have icosahedral symmetry (65). Recent data indicate that the symmetry is conferred on the virus particle by interactions between E proteins rather than by interactions between capsid proteins (88a, 114). A precursor of C, designated anchored C, contains a hydrophobic region at its C terminus that is cleaved to generate the mature virion C protein. The encapsidation signal on the flavivirus genomic RNA recognized by the C protein has not yet been definitively mapped. Glutathione-S-transferase fusion proteins made from different fragments of the Kunjin virus C protein bound strongly to RNA probes from either the 3' or 5' noncoding regions of the genomic RNA (84). In infected cells, the C protein is located in the nucleus as we ll as in the cytoplasm late in the infection cycle (153a).
The two viral envelope proteins, E and M, are both type I integral membrane proteins with C-terminal membrane anchors. The E proteins of some strains of WNV have no N-linked glycosylation sites, whereas others have a single glycosylation site (2, 12, 156). The acquisition of a carbohydrate residue on the WNV E protein after passage in mosquito cells was not linked to attenuation of the virus (33). Cysteine residues in the E protein ectodomain are strictly conserved and all of them form intramolecular disulfide bonds (109). Crystallographic analysis of the three-dimensional structure of a soluble fragment of a flavivirus E protein revealed dimers composed mostly of [beta]-sheets arranged in a head-to-tail orientation and suggested that the distal ends of each monomer were anchored in the membrane (64, 122). The E protein dimers in mature virions lie fairly flat against the lipid bilayer. The Eprotein has both receptor-binding and pH-dependent fusion activities and is composed of three domains (64). Domain m co ntains a fold typical of an immunoglobulin constant domain and has been postulated to contain the receptor-binding region; the lateral surfaces of the Domain III structures of mosquito- and tick-borne flavivirus E proteins differ significantly from each other and mutations in Domain III alter virulence (64, 91). A study on yellow fever virus suggested that a region of Domain II may also be involved in the binding of virus to cells in monkey brains (106).
WNV REPLICATION CYCLE
WNV replicates in a wide variety of cell cultures, including primary chicken, duck, and mouse embryo cells and continuous cell lines from monkeys, humans, pigs, rodents, amphibians, and insects, but does not cause obvious cytopathology in many cell lines (22). Glycosaminoglycans play a role in flavivirus entry (36, 91). However, Murray Valley virus mutants with substitutions in the hydrophilic region (FG loop) of the E protein that resulted in an increased dependence on glycosaminoglycans during entry of cultured cells showed decreased neurovirulence in mice (91). The identification of two glycoproteins as putative receptors of dengue 4 virus (128) suggest that additional host cell surface molecules are necessary for fiavivirus entry. Because flaviviruses are transmitted between insect and vertebrate hosts during their natural transmission cycle, it is likely that the cell receptor(s) they utilize is a highly conserved protein.
After binding to an unknown cell receptor(s), virions enter cells via receptor-mediated endocytosis followed by low-pH fusion of the viral membrane with the endosomal vesicle membrane releasing the nucleocapsid into the cytoplasm (64) (Figure 2A). The genome RNA is released and translated into a single polyprotein (Figure 2B). The viral serine protease, NS2B-NS3, and several cell proteases then cleave the polyprotein at multiple sites to generate the mature viral proteins (Figure 2C). The viral RNA-dependent RNA polymerase (RdRp), NS5, inconjunction with other viral nonstructural proteins and possibly cell proteins, copies complementary minus strands from the genomic RNA template (Figure 2D), and these minus-strand RNAs in turn serve as templates for the synthesis of new genomic RNAs (Figure 2E). Flaviviral RNA synthesis is semiconservative and asymmetric. Genome RNA synthesis is about 10 times more efficient than minus-strand RNA synthesis (38, 41). Data obtained by Chu & Westaway (38) with a Kunjin virus su ggested that only a single, nascent (-) RNA is copied from a plus-strand template at a time (replicative form, RF), while the minus-strand template is efficiently reinitiated so that multiple, nascent plus stands are simultaneously copied from a single minus-strand template (replicative intermediate, RI). Once established, both plus- and minus-stand viral RNA synthesis can continue even in the absence of protein synthesis, indicating that transient viral polyprotein precursors are not required (38, 41, 154). Extensive reorganization and proliferation of cytoplasmic, perinuclear endoplasmic reticular (ER) membranes are observed in infected cells (96). Nascent genome RNAs could function as templates for translation and transcription and as substrates for encapsidation. Data obtained with a Kunjin replicon suggest that translation is a prerequisite for replication of nascent RNAs (81) and that replication is a prerequisite of encapsidation (83a). At the beginning of the replication cycle, nascent genome RNAs may alternate between replication and translation because a sufficient pool of structural proteins has not yet accumulated.
Virion assembly occurs in association with rough ER membranes (Figure 2F, G). Little is known about this process because budding intermediates and free nucleocapsids in the cytoplasm have rarely been observed by electron microscopy (EM), but they may have been observed by cryo-EM (107). …