Electron Microscopy Fixative Fails to Inactivate Rift Valley Fever Virus

Abstract

Rift Valley fever (RVF) virus, a member of family Bunyaviridae, genus Phlebovirus, is a mosquito‐borne zoonotic pathogen endemic to Sub‐Saharan Africa and the Arabian Peninsula that causes severe epizootics in ruminants. RVF outbreaks in ruminants are associated with mass abortions and high mortality in younger animals. Human infections at all ages are associated with acute febrile illness that may progress to more severe disease including encephalitis and severe hepatitis leading to fatal hemorrhagic fever. RVF virus (RVFV) is classified as an overlap select agent by the Center for Disease Control and Prevention and the US Department of Agriculture. In the US, work with virulent RVFV strains must be conducted in BSL‐3+ and requires RVFV specific select agent clearance.In conjunction with ruminant challenge model development for evaluation of new RVFV vaccines and therapies, we have been examining the pathology of RVFV in multiple organs. Our initial histopathology findings warranted examination of infected tissues by transmission electron microscopy (EM). Consequently, during early time‐point post‐infection calf necropsies, we collected 2–5 mm cube sized samples of calf kidney, liver and spleen in room temperature (RT) 2.5% glutaraldehyde in Sorenson's buffer (glut), a standard EM fixative that preserves morphology for EM. Glutaraldehyde shares fixation characteristics with 10% neutral buffered formalin that is already approved for use in our BSL‐3Ag facility. Post ~2 hours at RT, samples were stored at 4° C, as per advice from our EM experts, for a minimum of 7 days. Because glut was a new fixative for our facility, we conducted proof of inactivation tests on our samples. RVFV PCR‐positive, unfixed tissue from each organ of interest from one of the calves and PBS were used as positive and negative controls respectively. Samples were rinsed in PBS, homogenized and blind passaged 3 times using standard virus propagation protocols. The cell monolayers were checked post passage for cytopathic effect (CPE) and standard plaque assays were conducted after the third passage. Additionally, in a separate experiment, using full strength and serially diluted glut in a no virus simulated viral propagation protocol, we tested for a direct toxicity effect of glut on our Vero cells during the initial 1 hr incubation time‐period.Plaque formation was observed on cells incubated with glut samples containing RVFV‐positive tissues and positive control tissues but not on cells incubated with PBS or glut. This indicates that the glut treated tissue samples still contained viable RVFV. Currently, we have experiments planned to consider the role of temperature in glut viral inactivation.Support or Funding InformationThis work was funded by grants of the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.