Prospective Investigation of Oxidative Stress in West Nile Virus Infection

Keywords

• Genome sequencing
• composite diagnosis

Lead sponsor

Institute of Tropical Medicine, Belgium is the lead sponsor of 104 studies on the registry; 21 are open to participants now.

Study population

Groups and cohorts

• WNND

  40 subject with West-Nile Neuroinvasive Disease will be recruited

• WNF

  40 subject with West-Nile Fever will be recruited

• controls

  20 control will be recruited. These controls will be aged matched to the cases.

Primary outcomes

1. Measure the redox status

   A multiparameter indexes of oxidative stress will be calculated to measure and summarize the redox status in cases and age matched controls. Association between the redox status and clinical, neuropsychological and radiological outcomes will be investigated. We will also examine the relative sensitivity of separate biomarkers of oxidative stress and autophagy as clinical predictors of WNV infection severity.

   Time frame: at recruitment, 10 days post-symptom onset and 20 days post symtom onset.

2. Assessment of Neurologic deficits

   As part of the descriptive analysis of biomarkers of disease severity and to study to neurologic sequelae of WNV infection. Will be assessed: specifically assessments of cranial nerves II- XII, motor strength in upper and lower extremities, sensory testing for pinprick and vibration, deep tendon reflexes, gait, coordination, and movement abnormalities

   Time frame: At recruitment, month 3 and month 12

3. Neuropsychologic performance

   Study the neuropsychologic sequelae of WNV infection during a 12-month followup period in following key domains: Attention, Memory, Executive Function, Emotion \& Social Cognition, Psychomotor Speed

   Time frame: 20 days post-symtom onset, month 3 and month 12

4. Longitudinal assessment of functional status Study the neurologic and neuropsychologic sequelae of WNV infection during a 12-month followup period.

   ECOG/WHO PS during a 12-month followup.

   Time frame: at recruitment, month 3 and month 12

5. MRI abnormalities

   Part of descriptive analysis of clinical markers of disease severity

   Time frame: at recruitment, month 3 and month 12

6. Brain iron content

   Part of the descriptive analysis of clinical markers of disease severity: Qualitative analysis per neuroanatomical region by iron-sensitive MRI sequence (SWI)

   Time frame: at recruitment, month 3 and month 12

7. Ophthalmological abnormalities

   As part of the descriptive analysis of clinical markers of disease severity ophthalmologic abnormalities will be assessed by slit lamp examination

   Time frame: at recruitment, with a follow-up of clinically indicated.

8. serum S100b concentration

   As part of the descriptive analysis of clinical markers of disease severity S100b concentration will be measured to asses the Blood-Brain barrier integrity

   Time frame: at recruitment, 10 days post symptom onset and 20 days post symptom onset

9. serum NSE concentration

   As part of the descriptive analysis of clinical markers of disease severity NSE concentration will be measured to asses the Blood-Brain barrier integrity

   Time frame: at recruitment, 10 days post symptom onset and 20 days post symptom onset

Secondary outcomes

1. Analysis of laboratory performance characteristics (e.g. sensitivity) of WNV-specific RT-PCR and viral isolation in clinical samples, compared to composite diagnosis of WNV infection

   Confirmed WNV infection is defined as one or more of following criteria: 1. Seroconversion of anti-WNV IgG antibodies (Indirect immunofluorescence testing (IIFT) Flavivirus 2, Euroimmun®, Lübeck, Germany) in convalescent serum (20 days after symptom onset) (all participants) 2. 4-fold increase of anti-WNV IgG antibodies in convalescent serum, compared to the screening sample (IIFT). 3. Anti-WNV IgM antibody detection (ELISA) in CSF\* (at VBH) (\*CSF sampled only when clinically indicated). 4. Positive WNV-specific RT-PCR result in serum, urine or CSF in samples obtained at screening. 5. Virus isolation by outgrowth assay in samples obtained at screening

   Time frame: 20 days

2. Description of molecular epidemiology of infecting WNV strain(s) and viral outgrowth diagnostic performance.

   The time frame for WNV detection after symptom onset by real-time reverse transcriptase polymerase chain reaction (RT-PCR) or virus isolation by outgrowth from blood or CSF is limited by the fast decline of circulating virus. Because the WNV RNA detection window in urine can be longer (up to 14 days), RT-PCR will be performed on all clinical samples at recruitment. The target sequences are a conserved region in the 5'UTR region and part of the capsid gene of WNV and detects both lineage 1 and 2 WNV. WNV has been successfully isolated from urine, in approx. 40% of samples obtained within 8 days of symptom onset. WNV isolation will be attempted in low-passage Vero E6 cells and BHK21 cells from urine samples with a high WNV RNA load (Cycle threshold (Ct)- values \<30).

   Time frame: 20 days

3. Identification of potential genetic signatures that correlate with virulence (neuro-invasion and morbidity) in our cohort.

   Whole genome sequencing (WGS) will be performed on a subset of RT-PCR positive serum, urine and CSF samples. This will allow us to investigate the temporal and spatial compartmentalization of the West Nile lineages in Romania as well as expand our understanding of genetic compartmentalization within hosts (urine, serum, CSF). In addition, the obtained isolates will be sequenced (from the viral outgrowth assay) to assess potential adaptations the virus undergoes during isolation. Finally, in a genome wide association study, the obtained viral sequences will be compared with the prospectively collected clinical data, to identify potential genetic signatures that correlate with virulence, neuroinvasion and morbidity. Our data will be compared to existing data on genetic determinants of virulence such as the presence of a glycosylation site in the E protein, substitutions in non-structural proteins 3, 4B or 5, or variation in the 3' noncoding region.

   Time frame: 20 days