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Public Health Lab Scientists Recent Publication in the Journal of Virological Methods

Automated molecular detection of West Nile Virus in mosquito pools using the Panther Fusion system

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  • Automated platform successfully processed atypical mosquito sample matrix.

  • Panther Fusion provided equivalent performance to more manual workflow.

  • Higher throughput of automated assay creates ease for future testing expansion.


West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation. This study demonstrates the successful optimization of a WNV multiplex LDT (assay targets: ENV and NS1 genes) for high-throughput environmental surveillance testing of mosquito pool homogenates on the Panther Fusion System. Analytical sensitivity of the assay was 186 and 744 copies/PCR reaction for the ENV and NS1 targets, respectively. To assess the performance of this assay, a total of 80 mosquito pools were tested, including 60 previously tested pools and 20 spiked negative mosquito pools. Among the 60 previously tested specimens, the Panther Fusion WNV LDT demonstrated 100% positive and negative agreement with the CDC West Nile RT-qPCR assay. The Panther Fusion WNV LDT also detected all 20 spiked specimens. The Panther Fusion WNV LDT assay was successfully developed and optimized for high throughput testing with similar performance to the previously used CDC West Nile RT-qPCR assay.


West Nile Virus (WNV) is an arthropod-borne virus spread through mosquito vectors that causes the most domestically acquired arboviral disease in the US (Curren et al., 2018). WNV was first detected in the US in 1999 and it is the most prevalent flavivirus in the world (Kramer et al., 2019). The majority of WNV infections are asymptomatic. Typical symptomatic infections are self-limiting and cause nonspecific, febrile illness (Huntington and Allison, 2016). Less than 1% of cases result in neuroinvasive disease, which typically presents as meningitis, encephalitis, or acute flaccid paralysis (Emily McDonald et al., 2021). Since 1999, there have been over 48,000 reported cases of WNV infection, 24,000 reported neuroinvasive cases, and over 2300 deaths in the US (Hadfield et al., 2019). A 2019 collaborative report from the CDC, US Department of the Interior, and the US Department of Agriculture lists WNV as a top zoonotic disease of national concern (Centers for Disease Control and Prevention, 2019).

After the detection of the first case of WNV disease in the US, the CDC worked with state health departments to create a passive surveillance system called ArboNET in 2000. ArboNET continues to maintain surveillance data for nationally notifiable arboviral diseases from testing of mosquitos, dead birds, and sentinel animals, as well as reports of human or veterinary disease cases and presumptive viremic blood donors (Lindsey et al., 2012).

The District of Columbia has been conducting mosquito surveillance for WNV since 2000. The most recent CDC RT-qPCR method used was labor-intensive, involving an automated extraction, followed by 2 singleplex RT-qPCR reactions (screening and confirmation) (Lanciotti et al., 2000). This paper describes the adaptation of a modification of this CDC reference method to run as a multiplex assay on the Panther Fusion (Hologic), an automated extraction and RT-qPCR platform. The Panther Fusion’s Open Access capability allows users to implement laboratory-developed tests (LDTs) which include nucleic acid extraction, RT-qPCR, and result interpretation. This is the first report of an LDT validated on the Panther Fusion using mosquito homogenates, a surveillance specimen.

Section snippets

Primers and probes

The primer and probe sequences (Table 1) targeting the envelope (ENV) gene and non-structural (NS1) gene of the WNV RNA genome were previously published (Kauffman et al., 2003). Primers and probes were obtained from Integrated DNA Technologies and resuspended in TE buffer (pH 8.0). Aliquots were stored at −20℃ for the duration of the mosquito surveillance season.

Primer probe reconstitution (PPR) mix

The PPR mix was used to rehydrate lyophilized enzymes and nucleotides contained in the RNA/DNA cartridge to create master mix onboard

Analytical sensitivity and precision

In the initial LOD evaluation at 10-fold dilutions, 100% of replicates were detected for the ENV target at 10,000 copies/ml (149 copies/PCR reaction) and 90% of replicates were detected for the NS1 target at 100,000 copies/ml (1488 copies/PCR reaction) (data not shown). For the LOD evaluation at 2-fold serial dilutions, 100% of replicates were detected for the ENV target at 12,500 copies/ml (186 copies/PCR reaction) and 100% of replicates were detected for the NS1 target at 50,000 copies/ml


Arbovirus surveillance efforts from mosquito pools detect changes in arbovirus disease burden over time and location, providing an early warning system for the presence of arbovirus activity in mosquito populations before humans are affected. The surveillance results are notified to state and local mosquito control programs to initiate control measures to prevent potential outbreaks and to inform the public to take precautions to avoid contact with mosquitos. Surveillance for arboviruses is

Conflict of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Use of commercial instruments and materials are identified to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by DFS PHL.


This project was funded by the District of Columbia Department of Forensic Sciences to improve the Department of Health Arboviral Surveillance and Control Program to detect West Nile Virus in endemic mosquito species in the district.

CRediT authorship contribution statement

Patel Kajal M.: Data curation, Investigation, Project administration, Software, Validation, Visualization, Writing – original draft. Raj Pushker: Conceptualization, Resources, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (13)

  • Centers for Disease Control and Prevention. 8 Zoonotic Diseases Shared Between Animals and People of Most Concern in...
  • Emily J. Curren, Jennifer Lehman, Jonathan Kolsin, William L. Walker, Stacey W. Martin, J. Erin Stapes, Susan L. Hills,...
  • Emily McDonald, Sarabeth Mathis, Stacey W. Martin, J. Erin Staples, Marc Fischer, Nicole P. Lindsey Surveillance for...
  • James Hadfield et al.

    Twenty years of West Nile virus spread and evolution in the Americas visualized by Nextstrain

    PLOS Pathog.


  • James L. Hadler et al.

    Assessment of arbovirus surveillance 13 years after introduction of West Nile Virus, United States.

    Emerg. Infect. Dis.


  • Mark K. Huntington et al.

    Emerging vector-borne diseases

    Am. Fam. Physician


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