Molecular Determinants of West Nile Virus Virulence and Pathogenesis in Vertebrate and Invertebrate Hosts

Abstract

West Nile virus (WNV), like the dengue virus (DENV) and yellow fever virus (YFV), are major arboviruses belonging to the Flavivirus genus. WNV is emerging or endemic in many countries around the world, affecting humans and other vertebrates. Since 1999, it has been considered to be a major public and veterinary health problem, causing diverse pathologies, ranging from a mild febrile state to severe neurological damage and death. WNV is transmitted in a bird-mosquito-bird cycle, and can occasionally infect humans and horses, both highly susceptible to the virus but considered dead-end hosts. Many studies have investigated the molecular determinants of WNV virulence, mainly with the ultimate objective of guiding vaccine development. Several vaccines are used in horses in different parts of the world, but there are no licensed WNV vaccines for humans, suggesting the need for greater understanding of the molecular determinants of virulence and antigenicity in different hosts. Owing to technical and economic considerations, WNV virulence factors have essentially been studied in rodent models, and the results cannot always be transported to mosquito vectors or to avian hosts. In this review, the known molecular determinants of WNV virulence, according to invertebrate (mosquitoes) or vertebrate hosts (mammalian and avian), are presented and discussed. This overview will highlight the differences and similarities found between WNV hosts and models, to provide a foundation for the prediction and anticipation of WNV re-emergence and its risk of global spread.

Keywords: West Nile virus; molecular determinants; vertebrate and invertebrate hosts; virulence.

Figure 1

West Nile virus (WNV) transmission cycle. In nature, WNV transmission occurs through an enzootic cycle between birds and mosquitoes, the latter mainly of the genus Culex. Humans and mammals can be infected but are considered to be dead-end hosts.

Figure 2

Dissemination steps of WNV in the invertebrate vector. (1) Mosquito feeds on virus-infected blood. (2) WNV infects mosquito midgut: (A)—Virus binds to epithelial cells by a protein receptor. (B)—Viral replication in midgut cells. (C1)—Direct passage from basal lamina to hemolymph (1st way). (C2)—Direct paracellular passage through midgut cells (2nd way). (3) Dissemination of WNV to other organs like salivary glands: (a)—Infection of epithelial cells by direct passage from hemolymph to cells. (b)—Viral replication in epithelial cells. (c)—Release of virus from cells to salivary gland lumen, with or without apoptosis of epithelial cells (inspired by [68,69]).

Figure 3

Dissemination steps of WNV in vertebrate hosts such as humans. At the early phase of infection, WNV disseminates in the human skin after a WNV-infected mosquito bite. First, replication begins in the keratinocytes and dendritic cells (DCs) and is followed by a migration of the WNV into the dermis. After that, DCs containing the WNV migrate to the lymph nodes where the WNV is amplified. Systemic viremia gives rise to infection of peripheral organ infection, such as the spleen. More severe infections lead to neuroinvasion by two possible routes. The first (1) consists of an axonal retrograde transport along the spinal cord [70]. The second (2) involves blood vessel transport and crossing over the blood–brain barrier (BBB). Three steps are required to mediate the neurovirulence in the brain by the blood route: (A)—Crossing the BBB. (B)—The virus interacts with the brain cells. (C)—Infection of neurons. To cross the BBB, three methods are possible. (I)—The first is a “Trojan horse” mechanism, in which the WNV is internalized into lymphocytes that are able to cross the BBB. (II)—The second method is permeabilization of the BBB in response to TNF-α and MIF secretion. After MIF secretion by leucocytes, the MMPs are produced and increase the BBB permeability [51,52,53,54]. (III)—The third is enhancement of virus attachment, probably dependent on E protein glycosylation, which permits transcellular passage (inspired by [11,65]).

Figure 4

WNV evolution through genetic diversity balance between the avian hosts and mosquito vectors. Viral diversity increases in mosquitoes and decreases in birds.

Figure 5

Overview of the main reverse genetic methods. (a) First generic method for reverse genetics developed in 1989 for YFV. cDNA was produced from viral single-stranded RNA, and then inserted into a plasmid upstream of a pCMV promoter and downstream of the hepatitis delta ribozyme, followed by the simian virus 40 polyadenylation signal (HDR/SV40(pA)) sequence. After amplification and purification, the construction was introduced into permissive cells by transfection, and infectious clones were obtained. (b) Bacteria-free method used for TBEV in 1995. Two long PCR products were synthetized and joined by using restriction enzymes or fusion PCR. After in vitro transcription, infectious clones were generated after intracerebral inoculation of mice with the long PCR fragment. (c) Bacteria-free method using the CPEC (KUN) or Gibson reaction (DENV), 2013. Multiple PCR amplicons were inserted into a plasmid containing pCMV and HDR/Sv40(pA) sequences and joined by CPEC or Gibson reaction. Infectious clones were obtained after transfection of permissive cells. (d) New bacteria-free method for reverse genetics in flaviviruses. (A)—In the infectious subgenomic amplicons (ISA) method the genetic material consists of viral RNA, infectious clones, or de novo synthesis. PCR products that cover the entire genome are used for direct transfection of permissive cells. (B)—The new ISA-based reverse genetic method, called the subgenomic plasmids recombination method (SuPReMe), resembles the ISA method, but the genome fragments are cloned into plasmids at the restriction sites. After restriction enzyme digestion, genomic fragments are used for transfection of permissive cells, and infectious clones are produced (according to [79]).

Figure 6

Virulence determinants in the WNV genome found in mammalian hosts. Molecular virulence determinants found in vitro or in vivo in mammalian cells (Vero, A549) and mouse models are represented throughout the entire WNV genome. Green: mutations involved in the attenuated viral phenotype. Red: mutations involved in the more virulent viral phenotype (adapted from [88]).

Figure 7

Virulence determinants in the WNV genome found in avian hosts. Molecular virulence determinants validated in vitro in the DEF avian cell line or in vivo in American crows, house sparrows, red-legged partridges, and specific-pathogen-free (SPF) chickens. Green: mutations involved in attenuated viral phenotype. Red: mutations involved in more virulent viral phenotype.

Figure 8

Virulence determinants in the WNV genome found in mosquito vectors. Molecular virulence determinants validated in vitro in the C6/36 mosquito cell line or in vivo in Culex pipiens and Culex tarsalis. Green: mutations involved in attenuated viral phenotype. Red: mutations involved in more virulent viral phenotype.