West Nile virus

What is West Nile virus (WNV)?

West Nile virus (WNV) belongs to the genus Yellow Fever Virus of the Flaviviridae family, which belongs to Japanese encephalitis, St. Louis encephalitis, yellow fever, dengue fever, Japan encephalitis, hepatitis C and other viruses. West Nile virus is an arbovirus that infects humans after being bitten by an infected mosquito. In more than 80% of cases, human infection is asymptomatic. In the remaining about 20% of cases, symptoms resemble those of pseudoflu syndrome. In 0.1% of all cases, viral infections cause neurological symptoms such as meningitis and meningoencephalitis.

West Nile virus origin and history

West Nile virus was first identified in 1937 when it was isolated from the blood of a febrile woman in the West Nile region of Uganda, hence the name West Nile virus. In 1950 Egypt described the ecological characteristics of the disease. In 1957, an epidemic occurred in Israel, and the virus was first noted to be associated with diseases of the central nervous system and is thought to be the cause of severe meningitis in the elderly. The virus was first noted in Egypt and France in 1960 as causing disease in horses. Since 1950, the virus has been endemic in Africa, the Middle East and countries bordering the Eastern Mediterranean without attracting attention. This neglected situation was quickly changed when the virus struck Bucharest, the capital of Romania, in 1996, causing encephalitis in about 400 people and nearly 40 deaths. A widespread epidemic occurred in southern Russia in September 1999, with nearly 1,000 people falling ill and at least 40 dying. In the past 5 years (1996~2000), the virus was isolated from 14 horses in the Czech Republic, and West Nile virus was isolated from 78 horses in Italy. In October 1999, an outbreak occurred among humans, horses, wild birds and zoo birds in New York and neighboring continents, ending the history of unmanned and animal infection reports in the Western Hemisphere, and was a milestone in the history of the development of the virus.

West Nile virus vectors

Birds are the reservoir host of the virus and the main source of WNV infection, and more than 70 species of birds have been identified to be involved in the transmission of the virus, some of which have high mortality rates, such as crows, great crows, magpies, blue jays, and gray birds, but the species of birds are not yet fully understood. Sick people and people with latent infection have also been suspected of being the source of the virus, but this has not been proven. Serologic tests suggest that in an epidemic, there are many people with latent infections who have only mild symptoms or no symptoms.

Mosquitoes are also reservoir hosts for a class of WNVs. The virus has been detected in many species of mosquitoes: Aedes mosquitoes, Anopheles mosquitoes, Anopheles vulgaris, and blue-banded mosquitoes. After the mosquito absorbs the blood containing the virus from the infected birds, the virus exists in the salivary gland of the mosquito after 10~14 days in the mosquito, and can spread the virus by biting other animals or humans, and the virus enters the blood of animals or people, and will enter the brain through the blood-brain barrier, causing encephalitis. There is no direct transmission between humans, poultry and birds. Researchers have found West Nile virus in overwintering mosquitoes. Based on the experience of Europe and the Middle East, it is suggested that the virus usually spreads to new places along the migration path of birds.

West Nile virus structure

The Viral structure of West Nile virus WNV is a sense strand single-stranded RNA virus. Its genome is about 11,000 nucleotides long. The coding region of the genome encodes three structural proteins and seven non-structural proteins. West Nile virus has three structural proteins, nucleocapsid protein (C), envelope protein (E), and membrane protein (prM/M). The WNV genome is first translated into polypeptides and then cleaved into individual proteins by the virus and host proteases. Under the electron microscope, the virus is medium in size, with a diameter of 21nm~60nm, round particles, sensitive to organic solvents and ultraviolet rays.

Fig.1 Structure of West Nile virus. (Lang Les.,2007)

The NS proteins of WNV virus (NS1 glycoprotein, NS2A, NS2B protease cofactor, NS3 protease and helicase, NS4A, NS4B, NS5 methyltransferase, MTase and RNA-dependent RNA polymerase, RdRp) form a replication complex in the endoplasmic reticulum membrane, mediate the processing of multiple proteins, and participate in defending against the host's innate antiviral response. Viral proteins that are critical to the viral cell cycle and have low mutation rates are the best candidate targets for antiviral drugs.

Fig.2 West Nile virus (WNV) genome and polyprotein organization. (Sinigaglia Alessandro, et al., 2020)

West Nile virus-related diseases

The virus usually spreads between June and October, and the current season is not over. Once infected with the virus, severe cases may cause fatal diseases such as encephalitis, West Nile virus fever, and rash.

West Nile virus usually breaks out in the summer, usually through mosquito bites, and the onset of illness usually occurs within 2 to 15 days after human infection. Infected people develop high fevers and headaches, and in severe cases, they die. The elderly, children and other groups are high-risk groups.

Less than 1% of people infected with WNV develop severe neurological disease, including encephalitis, meningitis, or myelitis. The mortality rate is 10% in patients with neuroinvasive WNV disease, and about 50% of survivors suffer long-term neurological effects, including movement impairment or cognitive sequelae. After systemic infection, WNV crosses the blood-brain barrier (BBB) and eventually enters the CNS. After invading the CNS, WNV directly infects nerve cells, leading to nerve cell death through caspase 3 apoptosis. CNS tissue damage due to neuroinvasive WNV infection may result from nerve cell death and nerve inflammation. Nerve inflammation is characterized by activation of microglia and astrocytes, production of inflammatory cytokines, breakdown of BBB, and infiltration of peripheral immune cells.

West Nile virus test

Antibody detection and nucleic acid detection are two commonly used West Nile virus detection methods. When the West Nile virus enters the body, the immune system produces antibodies specific to it, which can be detected in the blood of the infected person, as well as the corresponding viral nucleic acid. Detection of West Nile virus-specific antibodies can be performed by immunofluorescence and enzyme immunoassays.

In the setting of suspected West Nile encephalitis disease (WNED), diagnosis is usually made by real-time RT-PCR detection of viral RNA in serum or cerebrospinal fluid (CSF) samples. Identification of the West Nile virus genome in cerebrospinal fluid or serum during the acute phase of neurological involvement is generally considered a confirmatory diagnostic parameter. When viral RNA is not detectable in CSF, detection of a specific IgM immune response in CSF or serum is considered a reliable diagnostic indicator in the early stages of infection, which can then be confirmed by detection of IgG seroconversion (manifested by a fourfold or greater increase in serum IgG levels) during the recovery phase of infection.

West Nile virus treatment

West Nile virus vaccines

Although several veterinary vaccines have been licensed, WNV vaccines for humans are still in clinical trials, including two live attenuated chimeric vaccines (WN/EN4-3δ30, ChimeriVax-WN02), a DNA vaccine (VRC-WNVDNA017-00-VP), and a recombinant subunit vaccine (WN-80E) and two inactivated whole-virus vaccines (HydroVax-001, formalin-inactivated whole-virus vaccine). Although inactivated virus vaccines elicit only moderate immune responses, all vaccines have been associated with minor adverse events, and most vaccines have shown good immunogenicity. In the phase 2 trial, the recombinant live attenuated yellow fever virus (ChimeriVax-WN02) strain expressing the WNV pre-membrane and envelope (prM-E) gene was found to have good safety and immunogenicity.

Anti-WNV inhibitors

WNV NS2B-NS3 protease inhibitors

Flaviviruses NS2B-NS3 are serine proteases consisting of an N-terminal domain of NS3 and a short cofactor of the hydrophilic core sequence of NS2B. Favivirus proteases are well conserved and can be used as potential targets for pan-flavivirus inhibitors. Palmatine chloride is an orally active non-competitive West Nile virus (WNV) NS2B-NS3 protease.

WNV NS3 helicase domain inhibitors

A library of compounds reported to inhibit other helicases, mainly HCV helicases, was screened and found that benzothiazole and pyrrolidone were candidates with good activity for DENV and WNV ATPase/helicase. In addition, the antihelminth drug ivermectin was identified as a potent in vivo inhibitor of helicase activity of flaviviruses NS3, including West Nile virus.

WNV NS4B inhibitors

As a possible target of antiviral drugs, the NS4B protein has been the subject of several studies. Among the candidate compounds against NS4B, the natural plant alkaloid lycorine and its derivatives have been shown to have broad-spectrum activity against a variety of viruses, including WNV, by inhibiting viral RNA replication.

WNV NS5 MTase/GTase inhibitors

Nucleoside analogues have also been tested as inhibitors of the MTase domain of WNV NS5. Compound NSC 12155 was identified as a broad-spectrum inhibitor with strong activity against WNV by virtually screening putative compounds that bind to the SAM binding site of flavivirus MTase.

WNV NS5 RdrP inhibitors

The RdRp domain of the yellow fever virus NS5 protein has been considered a drug target by many researchers, in particular the discovery that the guanosine analog ribavirin has a wide range of antiviral effects, including activity against HCV, can inhibit WNVNS5, and reduce WNV replication in cell lines. Nucleoside analogues and other small molecules with competitive inhibitory activity have been extensively developed and tested in vitro and animal models.

References

• Lang, Les. The Food and Drug Administration approves second West Nile virus screening test for donated blood and organs. Gastroenterology 133.5 (2007): 1402.
• Sinigaglia, Alessandro, et al., New avenues for therapeutic discovery against West Nile virus. Expert Opinion on Drug Discovery 15.3 (2020): 333-348.

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