# Pasteurization temperatures effectively inactivate Rift Valley fever viruses in milk

Aurélie Pédarrieu, Camille Piro-Megy, Jordan Quellec, Catherine Cêtre-Sossah

<figure><img src="image_1.png" /></figure>

► To cite this version:

Aurélie Pédarrieu, Camille Piro-Megy, Jordan Quellec, Catherine Cêtre-Sossah. Pasteurization temperatures effectively inactivate Rift Valley fever viruses in milk. Journal of Virology, 2025, 99 (2), 10.1128/jvi.02026-24. hal-05084973

HAL Id: hal-05084973

https://hal.inrae.fr/hal-05084973v1

Submitted on 26 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
# Pasteurization temperatures effectively inactivate Rift Valley fever viruses in milk

Aurélie Pédarrieu,¹ Camille Piro-Megy,¹ Jordan Quellec,¹ Catherine Cêtre-Sossah¹

**AUTHOR AFFILIATION** See affiliation list on p. 2.

**KEYWORDS** Rift Valley fever virus, epidemiology, inactivation, milk

Rift Valley fever virus (RVFV) is a zoonotic arbovirus widely distributed across Africa, the Arabian Peninsula, and islands of the South West of the Indian Ocean, which causes recurrent epidemics affecting ruminant livestock as well as humans. RVFV is transmitted to humans by two main routes of infection, indirectly by infected vectors or directly by viral aerosolization from infected livestock tissues and body fluids (1).

The mode of transmission involved in recent RVF outbreaks in Kenya or Mayotte was not well identified for all cases, but the most commonly reported were exposure to animals or consumption of raw milk (2, 3). International agencies continue to seek RVF experts' recommendations and opinions on the best applicable control and prevention measures (4). To date, no studies have addressed the efficacy of temperature inactivation methods for infectious RVFV in milk. Pasteurization is a well-established method of heat inactivation, initially formalized by Pasteur for wine in 1864 (5).

To assess the effects of pasteurization on RVFs in milk and its usefulness in preventive and sanitary measures against RVF, we spiked whole cow's milk with two RVF viral strains, the Smithburn vaccine strain (6) or the MRU 25010-30 RVFV virulent field strain (7). Two temperature conditions representing the two common methods of pasteurizing milk were tested: (i) low-temperature long time (LTLT), which requires heating to 62.5°C for at least 30 minutes (8); and (ii) high-temperature short time (HTST), which requires heating to 72°C for at least 15 seconds (9). Vero cells grown at 37°C-5% CO₂ in minimal essential medium (MEM, Gibco, USA) supplemented with 2 mM L-glutamine and 10% decomplemented fetal bovine serum (FBS) (Corning, France) were infected in triplicate at a multiplicity of infection (MOI) of 0.1 in a 96-well plate format (1 × 10⁴ cells/well). Inocula, prepared by diluting the virus from the viral stock in whole cow's milk, were pasteurized using both mentioned methods and added to Vero cells at 37°C for 60 minutes. The medium was then replaced by complete MEM and incubated for 5 days. A mock control, consisting of the inoculum with medium only, was included.

We first evaluated RVFV infection by quantifying viral titers by tissue culture infectious dose 50% (TCID₅₀) calculated with Spearman-Kärber method (10) in infected Vero supernatants. We showed a significant decrease in viral titers to the limit of detection of the method for both strains in milk heated using both methods of pasteurization (Fig. 1A). We next confirmed these findings in milk-infected Vero cells with RVFV-specific labeling (Fig. 1B) (antibody anti-RVFV N nucleoprotein, red) that was only present in unpasteurized conditions and absent in pasteurized conditions.

These results demonstrate that commonly performed pasteurization techniques fully inactivate RVFV in milk, which is an important information to share considering the widespread public health risk that raw milk consumption represents in the RVF transmission cycle. This study provides baseline evidence to guide and strengthen future safety recommendations to avoid RVFV transmission through unpasteurized raw milk contributing to the spread of RVFV outbreaks.

**Editor** Stacey Schultz-Cherry, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Address correspondence to Catherine Cêtre-Sossah, catherine.cetre-sossah@cirad.fr.

Aurélie Pédarrieu and Camille Piro-Megy contributed equally to this article. Author order was determined alphabetically

The authors declare no conflict of interest.

**Published** 30 January 2025

Copyright © 2025 Pédarrieu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<figure><img src="image_3.png" /><figcaption>FIG 1 Pasteurization temperatures inactivate RVFVs in milk. (A) Viral quantification was measured using TCID₅₀ method in mock and RVFV-infected supernatants (Smithburn and MRU 25010–30, MOI 0.1) harvested 5 days post-infection (dpi). Two-way analysis of variance (ANOVA) (****P < 0.0001). (B) RVFV-infected cells (Smithburn and MRU 25010–30, MOI 0.1) were fixed at 5 dpi using a 4% paraformaldehyde (PFA) solution for 15 minutes at room temperature (RT) and were labeled with a mouse monoclonal IgG1 anti-nucleoprotein N RVFV antibody (anti-N RVFV, 1:50 [Cirad, 15G6-4B8]) and a secondary antibody (polyclonal donkey IgG anti-mouse IgG Alexa Fluor 555 [1:500 {Invitrogen A31570}], red), an actin probe (Actin Green [Ready Probes, Alexa Fluor 488, 1:50 {Thermo Fisher, 37110}], green), and DAPI (1:1,000 [Sigma-Aldrich, MBD0015], blue) for nuclear labeling (scale bar, 100 µm). Labeled wells were imaged by inverted microscope AXIOVERT A1 (Zeiss, France) using Archimed 6.1.4 software and were analyzed with Image J Software bundled with 64-bit Java 1.8.0_172. Similar data were obtained for both strains, only Smithburn-infected conditions are presented.</figcaption></figure>

## Biosafety statement

All experiments were conducted in biosafety level 3 (BSL-3) laboratories while dealing with infectious materials or biosafety level 2 (BSL-2) laboratories when working on inactivated materials.

## ACKNOWLEDGMENTS

We are grateful to ONARDEP (Dr Bezeid Ould El Mamy) for the Mauritanian field work and sample collection.

## AUTHOR AFFILIATION

¹ASTRE, CIRAD, INRAE, University of Montpellier, Montpellier, France

## AUTHOR ORCIDs

Catherine Cêtre-Sossah http://orcid.org/0000-0002-0869-1681

## AUTHOR CONTRIBUTIONS

Aurélie Pédarrieu, Formal analysis, Methodology, Validation, Writing – review and editing | Camille Piro-Megy, Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review and editing | Jordan Quellec, Data curation, Software, Writing – review and editing | Catherine Cêtre-Sossah, Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing

## REFERENCES

1. Wright D, Kortekaas J, Bowden TA, Warimwe GM. 2019. Rift Valley fever: biology and epidemiology. J Gen Virol 100:1187–1199. https://doi.org/10.1099/jgv.0.001296

2. Gerken KN, Mutuku FM, Ndenga BA, Agola GA, Migliore E, Fabre EP, Malumbo S, Shaita KN, Rezende IM, LaBeaud AD. 2022. Urban risk factors
for human Rift Valley fever virus exposure in Kenya. PLOS Glob Public Health 2:e0000505. https://doi.org/10.1371/journal.pgph.0000505

3. Youssouf H, Subiros M, Dennetiere G, Collet L, Dommergues L, Pauvert A, Rabarison P, Vauloup-Fellous C, Le Godais G, Jaffar-Bandjee MC, Jean M, Paty MC, Noel H, Oliver S, Filleul L, Larsen C. 2020. Rift Valley fever outbreak. Em Inf Dis:769-772. https://doi.org/10.3201/eid2604.191147

4. Nielsen SS, Alvarez J, Bicout DJ, Calistri P, Depner K, Drewe JA, Garin - Bastuji B, Gonzales Rojas JL, Gortázar Schmidt C, Michel V, et al, EFSA AHAW Panel (EFSA Panel on Animal Health and Welfare). 2020. Rift Valley Fever: risk of persistence, spread and impact in Mayotte (France). EFS2 18:6093-6154. https://doi.org/10.2903/j.efsa.2020.6093

5. Currier R. 2023. Pasteurisation: Pasteur's greatest contribution to health. Lancet Microbe 4:e129-e130. https://doi.org/10.1016/S2666-5247(22)00324-X

6. Smithburn KC. 1949. Rift Valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine. Br J Exp Pathol 30:1-16.

7. El Mamy AB, Lo MM, Thiongane Y, Diop M, Isselmou K, Doumbia B, Baba MO, El Arbi AS, Lancelot R, Kane Y, Albina E, Cêtre-Sossah C. 2014. Comprehensive phylogenetic reconstructions of Rift Valley fever virus: the 2010 northern Mauritania outbreak in the *Camelus dromedarius* species. Vector Borne Zoonotic Dis 14:856-861. https://doi.org/10.1089/vbz.2014.1605

8. Peila C, Moro GE, Bertino E, Cavallarin L, Giribaldi M, Giuliani F, Cresi F, Coscia A. 2016. The effect of holder pasteurization on nutrients and biologically-active components in donor human milk: a review. Nutrients 8:477. https://doi.org/10.3390/nu8080477

9. Ranieri ML, Huck JR, Sonnen M, Barbano DM, Boor KJ. 2009. High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk. J Dairy Sci 92:4823-4832. https://doi.org/10.3168/jds.2009-2144

10. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493-497. https://doi.org/10.1093/oxfor-djournals.aje.a118408