Heat Treatment as a Safe-Handling Procedure for Rift Valley Fever Virus

Abstract

Rift Valley Fever virus (RVFV) is a mosquito-borne virus with high pathogenic potential in ruminants and humans. Due to its high potential for spreading, it is considered a priority pathogen, and it is included in the Bluepoint list of the World Health Organization (WHO). Given the high pathogenic potential of the virus, it is crucial to develop a rapid heat-mediated inactivation protocol to create a safer working environment, particularly in medical facilities that lack a biosafety level 3 laboratory required for direct handling of RVFV. Our results reveal the broad tissue tropism of RVFV, showing the virus's capacity for replication in various cell lines. In terms of the thermal stability of RVFV, our findings showed that a 70 °C heat treatment did not fully inactivate the virus within 15 min. However, when exposed to 80 °C and 95 °C, the virus was completely inactivated after 15 min and 5 min, respectively. Additionally, our results indicated that heat-treatment only slightly decreased the integrity of the RVFV genome whether there is a high or low number of viral RNA copies. Overall, the study established a straightforward protocol for heat inactivation that may be beneficial in handling clinical and research samples of RVFV.

Keywords: RVFV; Rift Valley Fever virus; heat inactivation; safe-handling procedures; temperature; virus stability.

Figure 1

In vitro replication of RVFV and its cytopathic effect. The cell lines HTR-8, JEG-3, HEP-2, A549, and HUH-7 were exposed to RVFV with a multiplicity of infection (m.o.i) of 0.5 for a period of 48 h. The resulting infection displayed (A) pronounced cytopathic effects (CPE). CTRL = uninfected sample, RVFV = infected sample. The images represent one of three similar experiments. Viral replication at 48 h post-infection was verified in the infected cells using (B) TCID₅₀ assay and (C) quantitative RT-PCR. The histograms represent, respectively, the quantification of the viral infectious particles and the copies/mL of the viral RNA. Data are represented as the mean relative to the control plus S.D.

Figure 2

RVFV infectivity after heat treatment. To evaluate heat treatment on RVFV inactivation, the stocks derived from the different cell lines (HTR-8, JEG-3, HEP-2, A549 and HUH-7) were incubated at 70 °C, 80 °C, and 95 °C for 1, 5, 10, and 15 min. (A) The viral infectious particles were evaluated by TCID₅₀ assay in all the conditions tested. To find the quickest condition of inactivation, (B) the viral infectious particles, measured by TCID₅₀ assay, were also valued in samples treated at 95 °C for 1, 2, and 3 min. The histograms represent, respectively, the quantification of the viral infectious particles. Data are represented as the mean relative to the control plus S.D.

Figure 3

RVFV RNA detection after heat inactivation. To estimate the denaturation of viral RNA after heat treatment at different conditions RVFV RNA was measured by RT-PCR (measured as RNA copies/mL) in (A) stocks derived from the different cell lines incubated at 70 °C, 80 °C and 95 °C for 1, 5, 10, 15 min and (B) in diluted stocks to evaluate denaturation in the presence of few copies of viral RNA (mean RNA copies/mL: 10^1.2 copies/mL). The histograms represent the expression of viral RNA measured as copies/mL. Data are represented as the mean relative to the control plus S.D. ns = not significative.

Figure 4

RVFV heat inactivation in human samples. To evaluate the reliability of the method in human samples, urine samples were spiked with RVFV at different concentrations (respectively, 10^5.5, 10^4.5, 10^3.5, 10^2.5, 10^1.5 viral RNA copies/mL) and then heated at 60 °C for 1 h, 70 °C for 15 min, 80 °C for 10 min and 95 °C for 5 min. (A) RVFV RNA stability was determined by RT-PCR (measured as RNA copies/mL) NT = Not Treated Sample. (B) The images show the abolishment of the infectious ability of 95 °C inactivated samples on Vero E6 cell lines. CTRL = uninfected sample, RVFV = infected with spiked-sample, RVFVi = infected with 95 °C inactivated spiked-sample. The images are representative of multiple experiments.