Current Understanding of the Molecular Basis of Venezuelan Equine Encephalitis Virus Pathogenesis and Vaccine Development

Abstract

Venezuelan equine encephalitis virus (VEEV) is an alphavirus in the family Togaviridae. VEEV is highly infectious in aerosol form and a known bio-warfare agent that can cause severe encephalitis in humans. Periodic outbreaks of VEEV occur predominantly in Central and South America. Increased interest in VEEV has resulted in a more thorough understanding of the pathogenesis of this disease. Inflammation plays a paradoxical role of antiviral response as well as development of lethal encephalitis through an interplay between the host and viral factors that dictate virus replication. VEEV has efficient replication machinery that adapts to overcome deleterious mutations in the viral genome or improve interactions with host factors. In the last few decades there has been ongoing development of various VEEV vaccine candidates addressing the shortcomings of the current investigational new drugs or approved vaccines. We review the current understanding of the molecular basis of VEEV pathogenesis and discuss various types of vaccine candidates.

Keywords: Venezuelan equine encephalitis virus; alphavirus; blood brain barrier; encephalitis; inflammation; pathogenesis; vaccines; viral and host factors.

Figure 1

Venezuelan equine encephalitis virus (VEEV) transmission. VEEV is transmitted by a variety of mosquitoes. Enzootic strains are maintained in a cycle between small rodents and mosquitoes. Epizootic strains are transmitted by mosquitoes to equines, causing high titer viremia and high mortality. The virus is tangentially transmitted by mosquitoes from equines to humans that work in close contact with equines.

Figure 2

Biphasic replication of VEEV in mice. Mice infected via VEEV footpad injection mimics the natural mode of transmission by mosquito bites (1). After initial inoculation, regional dendritic cells take up the virus and transport it to the local draining lymph nodes, where VEEV replicates in lymphocytes (2) and is then released into the circulation resulting in viremia (3). VEEV replicates in various tissues, but lymphoid organs such as the spleen are primary replication sites (4). Around 24–36 h post infection, VEEV escapes into the olfactory tract infecting olfactory nerve endings initiating the first step of CNS phase of infection (5). Virus moves through the axons of the olfactory neurons and enters the olfactory lobe of the brain (large insert). In the brain, VEEV primarily infects the neurons, but glia and oligodendrocytes are also targets of VEEV infection. VEEV induced inflammation causes vascular cuffing and alteration in the BBB allowing mononuclear lymphocytes to enter the brain (6). VEEV mediated alteration of the BBB may allow the virus to enter the brain, but this route of entry is debated.

Figure 3

Organization of VEEV genome. VEEV genome is a single-stranded positive-sense RNA of 11.4kb length. VEEV encodes four nonstructural proteins (nsP1, nsP2, nsP3 and nsP4) and five structural proteins (capsid (C), envelope (E) 3, E2, 6k, and E1). VEEV genome has mRNA characteristics. 5’ untranslated region (UTR) is capped with methyl residue present on the 7-position of capping guanosine nucleotide. A short UTR is present after the nsp4 gene sequence that has a promoter sequence for a 26S subgenomic RNA. There are two open reading frames (ORF) in the genome. The first ORF in the 5’ region translate nonstructural proteins and the second ORF in sub genomic RNA translate structural proteins. 3’ end of the genome has a poly(A) tail.

Figure 4

Replication of VEEV in host cell. VEEV enters in the host cells by receptor mediated binding of the virus to the host cell membrane. Virus containing endosomes fuse with lysosomes resulting in formation of endolysosomes. Viral RNA is released in the cytoplasm following pH dependent conformational changes in the viral proteins, allowing fusion with the endolysosome membrane. VEEV is a single-stranded positive-sense RNA virus that replicates in the cytoplasm and does not have a nuclear phase of replication. In the second phase of infection, viral nsp are translated as P123 and P1234 polyproteins from the viral genomic RNA. Autolytic activity of nsp-2 cleaves the viral polyproteins in individual nsp-1, nsp-2, nsp-3, and nsp-4 proteins. Nsp-4 is a viral RdRp, which with methyltransferase activity of nsp-2, drives synthesis of negative-sense viral RNA. Negative-sense viral RNA is transcribed into smaller 26S subgenomic positive-sense RNA and a full-length positive-sense viral RNA. Subgenomic 26S RNA is translated into viral structural proteins namely capsid, a polyprotein of E3 and E2 called PE2, 6k, and E1. PE2 is processed into E3 and E2 proteins by cutting at the furin-cleavage site in PE2. Viral nsp-2 plays a role in capping of viral genomic RNA via its methyltransferase and guanylylation activity. Nsp-4 adds a poly-A tail to the viral genomic RNA via its terminal adenyltransferase activity. Full length positive- sense viral RNA is incorporated into a virus replication complex (VRC). Assembly of VRC is aided by interaction of viral non-structural proteins with host proteins such as nsp-3 with IKK-β and Fragile- X syndrome family proteins. Nsp-3 binds to other unknown host proteins during formation of VRC, the role of which is not yet understood. Nsp-1 conserved sequence element helps in the recognition of the core promoter element of the virus genome by VRC. In addition to direct involvement of viral proteins in replication and assembly, viral non-structural proteins interact with host factors to promote VEEV replication. Capsid proteins bind to components of the nuclear pore complex effectively blocking nuclear-cytoplasm-nuclear traffic and host protein translation. Nsp-2 interacts with karyopherin-alpha 1 for its nuclear localization function, role of which is not clearly understood. Nsp2 plays a role in loading of the viral RNA into nucleocapsid and maturation of virions. In the final step structural proteins E1 and E2 are embedded in the plasma membrane, and assembly and release of the mature virion particle occurs by encapsulating nucleocapsid and budding at plasma membrane [2].

Figure 5

Role of BBB in VEE. Endothelium of the BBB is actively involved in the inflammatory reaction during the CNS phase of VEEV infection. VEEV primarily enters the brain via the olfactory tract replicating first in the olfactory bulb of the brain. Presence of the virus in the brain is detected by the resident glia, the immune cells of the brain. Activation of glial cells release chemokines and cytokines that initiate the first steps of CNS inflammation. The initial inflammatory response probably initiates the alteration in the BBB. Chemokines such as MCP-1 have been shown to be directly involved in opening the BBB. Chemokines and cytokines activate brain microvascular endothelium, resulting in up-regulation of adhesion molecules such as ICAM-1. Ligands of ICAM-1 are expressed on peripheral circulating activated–leucocytes, which may or may not be infected with VEEV. Binding of circulating leucocytes with the brain microvascular endothelium elicits a cascade of events resulting in the transmigration of these cells across the BBB. In the third phase, inflammation and viral load in the brain is augmented by the transmigrating leucocytes. Infected leucocytes increase the viral load in the brain, which further activates the glia resulting in increased inflammatory reactions in the brain. Microglia are antigen presenting cells and may further contribute to an increase in inflammation by presenting the virus to peripheral leucocytes entering the brain and to the resident astrocytes. These events result in a continuous cycle of release of inflammatory cytokines and chemokines in the brain reaching a point where neurons may undergo inflammatory mediated cytotoxic damage in addition to apoptosis induced by direct virus infection.