Establishment of a one-step reverse-transcription qPCR to detect Getah virus in mosquitoes and feral swine in Alabama, USA

Abstract

Getah virus (GETV) is an emerging mosquito-borne alphavirus in the family Togaviridae, known to cause disease in a range of animal hosts including pigs, horses, and occasionally humans. The virus is endemic to several countries in Asia and the Western Pacific, where it has caused outbreaks characterized by febrile illness, reproductive failure in swine, and fever and rash in horses. Transmission occurs primarily via mosquitoes, particularly Culex, Aedes, and Anopheles species, some of which are also found in the United States. Although GETV has never been reported in North America, the presence of competent mosquito vectors and susceptible animal hosts raises concern over the potential for introduction and establishment. To date, no large-scale surveillance efforts have been conducted in the U.S. to assess the presence or absence of GETV in local vector or animal populations. To address this gap, we developed a highly sensitive and specific one-step reverse transcription PCR (RT-PCR) assay targeting a conserved region of the GETV genome. The assay was first optimized and validated using synthetic GETV RNA controls to ensure diagnostic accuracy. We then applied this molecular tool in a surveillance study involving both mosquito vectors and potential mammalian amplifying hosts. A total of 1,626 mosquitoes, collected across Alabama between 2019 and 2024, were pooled and tested. In parallel, 315 tissue and blood samples (kidney, spleen, and whole blood) were collected from feral swine, a species known to develop high viremia following GETV infection and serve as a major amplifying host in endemic regions. All samples were processed for total RNA extraction, followed by RT-PCR screening for GETV. All 1,626 mosquito pools and 315 swine samples tested negative for GETV RNA using the established RT-PCR assay. These findings suggest that detectable circulation of GETV were not identified in our mosquito and feral pig samples collected in Alabama. This is the first study to employ molecular techniques for large-scale GETV surveillance in the U.S. Our results provide the first molecular evidence supporting the absence of GETV in both mosquito and feral swine samples in the southeastern U.S., thereby offering valuable baseline data for future risk assessments. The significance of this work lies not only in the development of a reliable diagnostic tool but also in its application to proactive surveillance in an ecologically suitable region. Given the increasing global movement of animals and the documented expansion of arboviruses into new geographic regions, this work is timely and critical. While GETV is not currently present in the U.S., the detection of competent vectors and susceptible hosts reinforces the need for continued monitoring to enable early detection and rapid response should the virus be introduced. This study had several limitations. First, our surveillance relied solely on molecular detection of viral RNA, which identifies only active infections and may miss previous exposures. Serological assays, which could detect past infections through antibody presence, might offer a more comprehensive understanding of viral exposure. Unfortunately, no commercial ELISA kits are currently available for GETV antibody detection. Second, our host surveillance was limited to mosquitoes and feral pigs, despite the fact that horses and humans are also susceptible to GETV infection. Third, this study focused on a single state—Alabama—and cannot be generalized to the entire U.S. To build a more comprehensive understanding of the potential GETV threat, future studies should expand surveillance to include more geographic regions, additional host species, and incorporate both molecular and serological approaches. This study provides the first molecular evidence of the absence of Getah virus in mosquito and feral swine populations in Alabama. While the virus was not detected, the ecological presence of competent vectors and susceptible hosts in the U.S. warrants continued and expanded surveillance efforts to monitor for potential future introductions of this emerging pathogen.