Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India

Abstract

In late 2009/early 2010, approximately 2000 people were affected by a mysterious viral outbreak in a southern district of Tamil Nadu; this particularly affected those living in coastal areas. Blood samples from affected patients were sent for clinical analysis to determine the actual cause of the illness, but reports were inconclusive. The present study describes the clinical observations and laboratory investigations involving molecular methods performed on 170 of the 2000 clinically suspected cases. These were patients who were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assays were used to detect West Nile virus (WNV) infection. Further investigation of the genetic diversity of the WNV implicated in ocular complications was undertaken by sequence phylogeny. Out of 170 samples, 25 (15%) were positive for chikungunya IgM antibody, 10 (6%) for chikungunya antigen, and 30 (18%) were positive for dengue IgM antibody. The remaining 105 seronegative samples were further processed for WNV detection by IgM capture ELISA and molecular methods. Out of the 105 samples, 35 (33%) were positive for WNV IgM antibody, 15 (14%) were positive for WNV by RT-PCR, and 27 (26%) were found to be positive for WNV by both real-time RT-PCR and RT-LAMP assays. Comparative evaluation with acute-phase patient serum samples revealed 100% concordance between the real-time RT-PCR and RT-LAMP assays. These assays had an overall higher sensitivity than the conventional RT-PCR as they picked up 12 additional samples with a low copy number of template. Further genotyping through sequence phylogeny revealed that all the WNV isolates were grouped in lineage I. The association of West Nile virus with ocular infection in South India during an epidemic of mysterious fever in the first half of 2010 was clearly established through molecular approaches employing envelope gene-specific real-time RT-PCR and RT-LAMP assays followed by nucleotide sequencing.