Latest developments and challenges in the diagnosis of human West Nile virus infection

Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus responsible for an increasing number of human outbreaks of neuroinvasive disease in Europe and in North America. Notwithstanding the improvements in the knowledge of virus epidemiology and clinical course of infection and the development of new laboratory tests, the diagnosis of WNV infection remains challenging and many cases still remain unrecognized. WNV genome diversity, transient viremia with low viral load and cross-reactivity with other flaviviruses of the antibodies induced by WNV infection are important hurdles that require the diagnosis to be performed by experienced laboratories. Herein, we present and discuss the novel findings on the molecular epidemiology and clinical features of WNV infection in humans with special focus on Europe, the performance of diagnostic tests and the novel methods that have been developed for the diagnosis of WNV infection. A view on how the field might evolve in the future is also presented.

Keywords:

• blood
• diagnosis
• human
• molecular test
• neutralization test
• serology test
• serum
• test performance
• urine
• West Nile virus

Financial & competing interests disclosure

This work was supported by the European Commission under FP7, project number 261426 (WINGS; Epidemiology, Diagnosis and Prevention of West Nile Virus in Europe). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• West Nile virus (WNV) is a mosquito-borne flavivirus which is maintained in nature in an enzootic cycle between birds and mosquitoes. Humans can be infected by the bite of an infected mosquito, but are not able to transmit the virus because of low viremia. Infection in humans is asymptomatic in most cases, while it presents as West Nile fever in 20% of cases and as West Nile neuroinvasive disease, a condition which includes meningitis, encephalitis and acute flaccid paralysis, in less than 1% of cases. The fatality rate is 10–30% in patients with encephalitis and a large proportion of patients who recover from WNV infection report on its sequelae.

• Phylogenetically, WNV is divided into lineages that differ from one another by 5–25%. Up to nine lineages have been proposed so far, but only lineage 1 and lineage 2 have been associated with human disease. In Europe, different WNV lineage 1a and lineage 2 strains are currently causing human outbreaks, while those circulating in North America are phylogenetically related since they are all derived from a single introduction of WNV lineage 1a.

• In patients with WNV infection, the viremic period starts 2–3 days post infection and lasts for 8–10 days. Since the virus is associated with red blood cells, viral load is higher in whole blood than in plasma, especially in the late phase of infection. In addition, the virus is excreted in urine where it can be detected at a higher titer and for a longer time than in blood. In the late phase of infection, the virus is sequestered in peripheral tissues and may be transmitted through organ donation.

• The direct diagnosis of acute WNV infection is based on the detection of WNV RNA by nucleic acid amplification methods. WNV RNA may be detected in peripheral blood from 2–3 days to 14–18 days post infection, although in approximately 70–80% of cases of infection, viral RNA is no longer detectable in blood at the time of symptom onset. Testing urine samples is more sensitive, since WNV RNA is detectable in 40–50% of patients with West Nile fever or West Nile neuroinvasive disease. Molecular methods include highly sensitive nucleic acid amplification tests for the screening of blood and organ donors and real-time reverse transcription-polymerase chain reaction (RT-PCR) or pan-flavivirus reverse transcription-polymerase chain reaction assay for the diagnosis in patients with symptoms. Low viral load at the time of symptom onset and the high diversity of WNV represent the major hurdles for molecular testing.

• WNV can be isolated from the serum samples of WNV-infected patients within the first 2–3 days post infection, before the appearance of neutralizing antibodies. The virus can be isolated also from the urine samples independently from the presence of antibodies in serum. WNV is a Biosafety Level-3 pathogen and must be handled within a class II biological safety cabinet in a Biosafety Level-3 facility.

• During acute infection, WNV nonstructural protein 1 is secreted and can be detected in serum. The application WNV nonstructural protein 1 antigen testing for the diagnosis of acute WNV infection should be further investigated.

• Detection of WNV antibodies in the serum and cerebrospinal fluid is one of the most widely used methods for the diagnosis of WNV infection. Several commercially available test systems exist, which are based on antigens such as recombinant proteins, virus-like particles or lysates of infected cells. Because of the sequence similarity of structural proteins of flaviviruses, mainly of the immunodominant envelope protein, antibodies against one flavivirus can cross-react with the structural proteins of another one, which can severely complicate specific diagnosis. An ELISA assay based on WNV envelope protein with mutations in cross-reacting epitopes showed higher specificity than with wild-type antigen. Improvement of the specificity of WNV serology assays is an important goal, since positive results obtained by ELISA or IFA require confirmation by a neutralization assay, such as plaque reduction neutralization test, which is considered the ‘gold standard’ for the differential diagnosis of flaviviruses.

• Due to the persistence of WNV IgM antibodies, their detection in patients with symptoms of WNV infection should not be used as a criterion for the diagnosis of a recent infection, especially in areas of endemic circulation of WNV. A fourfold or greater change in WNV-specific antibody titers between acute- and convalescent-phase serum specimens provides additional evidence of a recent infection. In addition, analysis of WNV IgG avidity has been proposed as a useful laboratory test to distinguish current-season from prior-season WNV infections.

• Next-generation sequencing-based shotgun metagenomics could be useful to detect WNV RNA and to sequence its full genome, when the presence of a novel WNV strain is suspected, but WNV RNA cannot be detected with the available PCR primer sets.

Notes