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Int J Mol Sci. 2019 Apr 19;20(8). pii: E1934. doi: 10.3390/ijms20081934.

Optimizing PCR Detection of West Nile Virus from Body Fluid Specimens to Delineate Natural History in an Infected Human Cohort.

Author information

1
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. rodion.gorchakov@gmail.com.
2
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. Bonnie.Gulas-Wroblewski@bcm.edu.
3
Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA. Bonnie.Gulas-Wroblewski@bcm.edu.
4
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. ronca@bcm.edu.
5
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. jruff@bcm.edu.
6
University of South Carolina, Arnold School of Public Health, Columbia, SC 29208, USA. MSNOLAN@mailbox.sc.edu.
7
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. rberry@bcm.edu.
8
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. Rojelio.Alvarado@bcm.edu.
9
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. sm22@bcm.edu.
10
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children's Hospital, Houston, TX 77030, USA. kmurray@bcm.edu.

Abstract

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.

KEYWORDS:

Houston West Nile cohort; PCR; West Nile virus; prolonged detection; saliva; semen; urine; virus shedding; whole blood

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