Development of an indirect ELISA for the detection of venezuelan equine encephalitis virus specific antibodies in horses.

Overview

• This research developed a sensitive and specific indirect ELISA test to detect antibodies against Venezuelan equine encephalitis virus (VEEV) in horses.
• The new assay provides a fast and easy-to-use diagnostic alternative, useful for surveillance and monitoring of VEEV, especially considering the risk of virus introduction into new regions such as Europe.

Background

• VEEV Characteristics: Venezuelan equine encephalitis virus is a zoonotic pathogen primarily affecting equines (horses and related species), with high mortality rates between 19% and 83%.
• Transmission Cycle: The virus circulates between vertebrate hosts (equines) and mosquito vectors, particularly species from the Aedes and Culex genera.
• Geographical Relevance: VEEV causes sporadic outbreaks in Central and South America affecting both equids and humans.
• Concerns for Europe: Although VEEV is not currently found in Europe, factors such as global climate change, increased international trade, and presence of competent mosquito vectors increase the risk of VEEV introduction.
• Regulatory Response: The European Commission tasked the European Union Reference Laboratory (EURL) for equine diseases with developing a rapid, easy-to-implement serological test to detect VEEV infection in horses for early diagnosis and monitoring.

Objective

• To develop an indirect enzyme-linked immunosorbent assay (ELISA) targeting the VEEV E2 glycoprotein for the detection of VEEV-specific antibodies in horses.
• The assay was intended to be both sensitive and specific while being straightforward to implement in diagnostic and surveillance laboratories.

Methods

• Antigen Selection: The ELISA was based on the E2 glycoprotein of VEEV, a key viral surface protein that induces an antibody response.
• Sample Collection: A total of 469 equine serum samples were collected, including:
  • Non-infected horses (negative controls)
  • Horses naturally infected with VEEV, Eastern equine encephalitis virus (EEEV), and/or Western equine encephalitis virus (WEEV)
  • Horses vaccinated against EEEV/WEEV (to test potential cross-reactivity)
• Assay Evaluation: The indirect ELISA’s performance was assessed by calculating sensitivity (ability to correctly identify positive cases) and specificity (ability to correctly identify negative cases).

Results

• The developed indirect ELISA demonstrated:
  • Sensitivity: 97.3% — indicating a very high accuracy in detecting horses with VEEV-specific antibodies.
  • Specificity: 93.8% — demonstrating good capacity to avoid false positives, even in horses vaccinated against related encephalitis viruses.
• The assay showed minimal cross-reactivity with antibodies from EEEV/WEEV infections or vaccinations.
• The test was simple to perform and adaptable to routine laboratory workflows.

Significance and Applications

• This ELISA serves as an alternative to the more complex and time-consuming virus neutralization tests, facilitating easier large-scale testing.
• It enables accurate sero-epidemiological surveys to:
  • Map the current distribution of VEEV in endemic regions (Americas).
  • Monitor potential introduction or emergence of VEEV in new regions such as Europe.
• By providing a quick and reliable diagnostic tool, veterinary health authorities can better manage risks and respond proactively to outbreaks.
• The approach supports global health security in the face of factors driving the geographic spread of mosquito-borne zoonotic diseases.