A reverse transcriptase-polymerase chain reaction assay for detecting Highlands J virus.

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Arbovirology

Background:

• Highlands J (HJ) virus is an arbovirus prevalent in Eastern US mosquitoes, posing a threat to domestic birds.
• Existing immunologic techniques for HJ virus detection are inefficient and prone to cross-reactivity.
• HJ virus shares an enzootic cycle with eastern equine encephalitis (EEE) virus, making it an important surveillance indicator.

Purpose of the Study:

• To develop a sensitive and specific molecular diagnostic assay for Highlands J virus.
• To improve the accuracy and efficiency of HJ virus detection in various sample types.
• To enhance arbovirus surveillance programs and avian disease monitoring.

Main Methods:

• Development of a reverse transcriptase (RT)-polymerase chain reaction (PCR) assay using primers targeting the E1 coding region of HJ virus.
• Testing the assay's sensitivity with serial dilutions of HJ virus-infected cell culture.
• Evaluating the assay's specificity against related arboviruses and direct detection in field samples.

Main Results:

• The developed RT-PCR assay demonstrated high sensitivity, detecting viral RNA at concentrations as low as 10 plaque-forming units/reaction.
• The assay showed high specificity, with no cross-reactivity observed with western equine encephalitis, EEE, LaCrosse, or Jamestown Canyon viruses.
• Successful detection of HJ virus RNA was achieved directly from infected bird brain tissue and field-collected mosquitoes.

Conclusions:

• The novel RT-PCR assay provides a rapid, sensitive, and specific method for HJ virus diagnosis.
• This molecular tool will significantly improve HJ virus surveillance, aiding EEE risk assessment.
• The assay facilitates accurate monitoring of HJ virus infections in domestic poultry and wild bird populations.