# In vivo risk assessment of yellow fever virus transmission through breastfeeding, and mechanistic insights

Jeanne Pascard, Sophie Desgraupes, Aurélie Chiche, Patricia Jeannin, Rebecca Kanaan, Antoine Gessain, Han Li, Pierre-Emmanuel Ceccaldi, Aurore Vidy

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Jeanne Pascard, Sophie Desgraupes, Aurélie Chiche, Patricia Jeannin, Rebecca Kanaan, et al.. In vivo risk assessment of yellow fever virus transmission through breastfeeding, and mechanistic insights. Journal of Infectious Diseases, In press, pp.jiaf637. 10.1093/infdis/jiaf637. pasteur-05438224

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The Journal of Infectious Diseases

MAJOR ARTICLE

*In vivo risk assessment of yellow fever virus transmission
through breastfeeding, and mechanistic insights*

Jeanne Pascard ¹#*, Sophie Desgraupes ¹#§, Aurélie Chiche ², Patricia Jeannin ¹, Rebecca Kanaan ¹, Antoine Gessain ¹, Han Li ², Pierre-Emmanuel Ceccaldi ¹, Aurore Vidy ¹*

¹Epidemiology and Pathophysiology of Oncogenic Viruses Unit, Virology Department, Institut Pasteur & Université Paris-Cité, 28 rue du Docteur Roux, 75724 Paris cedex, France; ²Cellular Plasticity and in Age-related Pathologies Group, Developmental and Stem Cell Biology Department, CNRS UMR3738, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex, France.

**BACKGROUND:** Yellow fever virus (YFV), a mosquito-borne Orthoflavivirus, remains a significant public health concern, especially in regions with low vaccine coverage. Since 2010, yellow fever vaccination is not recommended for breastfeeding women due to reported cases of vaccine strain transmission through breast milk causing neonatal meningoencephalitis. However, breastfeeding transmission efficiency of the vaccine strains remains unknown, and wild-type strains transmission has been suggested following viral RNA detection in milk. Obtaining direct evidence of breastfeeding-related transmission in humans is challenging as vector-borne exposure confounds analyses, making animal models essential for assessing this risk.

**METHODS:** We used A129 mouse model to investigate YFV transmission via breastfeeding for wild-type and vaccine strains, and human epithelial *in vitro* models to explore mechanisms of mammary and intestinal barrier crossing. **RESULTS:** Wild-type and vaccine strains spread to mammary glands, targeting mainly stromal and immune cells, and are excreted into milk as free and cell-associated virus. *In vitro*, mammary epithelial cells also support infection, suggesting two mechanisms of epithelial crossing. Neonates are susceptible to oral infection, showing

© The Author(s) 2025. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

<footnote>*: Co-corresponding Authors</footnote>

<footnote>#: These Authors contributed equally to this work</footnote>

<footnote>§: Present Address: IRIM-CNRS, 1919 route de Mende, 34090 Montpellier, France</footnote>

<footnote>Email addresses of co-corresponding authors: aurore.vidy@pasteur.fr and jeanne.pascard@pasteur.fr</footnote>
higher infection rates for wild-type virus but evidence of neuroinvasion for both strains. These strains infect and cross an *in vitro* human intestinal barrier model, suggesting this epithelium as a potential viral entry site for neonates. Finally, the virus can be transmitted from infected dams to suckling pups via breastfeeding, though rarely. **CONCLUSION:** This study demonstrates YFV transmission through breastfeeding in an animal model and supports the biological plausibility of this route, highlighting its potential among YFV transmission risks.

**KEY WORDS:** Yellow Fever Virus, A129 Mouse Model, Mother-to-Child Transmission, Breastfeeding, Mammary Gland, Breast Milk, Intestinal Epithelium, Epithelial Crossing.

INTRODUCTION

Yellow fever virus (YFV) is a mosquito-borne Orthoflavivirus that causes a potentially fatal haemorrhagic disease, with ~200,000 cases and 30,000 deaths annually worldwide [1]. Clinical manifestations range from mild symptoms to severe jaundice and haemorrhagic fever, with fatality rates up to 60% in severe cases [2]. Despite the availability of a safe and effective live-attenuated vaccine (17D), YFV remains endemic in 44 countries, and outbreaks continue due to insufficient immunization coverage [3]. Currently, three vaccine substrains are used worldwide: 17D-204, 17DD and 17D-213 [4].

In addition to mosquito-borne transmission, YFV has been implicated in non-vectorial routes such as blood transfusion [5,6], organ transplantation [6], and vertical transmission [7–12]. Notably, several cases of breastfeeding transmission involving YFV vaccine strains have been reported. In each instance, exclusively breastfed infants developed meningoencephalitis shortly after maternal vaccination [9–12]. Detection of vaccine RNA or YFV-specific IgM in the infants’ serum and/or cerebrospinal fluid provided compelling evidence of transmission via breastfeeding [9–11]. Consequently, since 2010, the U.S. Centers for Disease Control and Prevention (CDC) recommend against vaccinating breastfeeding women, except for epidemics or unavoidable travel to endemic areas [13].

Although less documented, wild-type YFV transmission via breastfeeding remains plausible. During Brazil’s 2016–2018 outbreak, YFV RNA was detected in breast milk of an infected mother whose infant also developed symptoms [14] — raising concern about this potential, but often underestimated, transmission route. However, establishing breastfeeding transmission of mosquito-borne viruses is difficult in endemic areas, where the omnipresence of vectors makes it nearly impossible to exclude mosquito-borne transmission.

To address this challenge, criteria adapted from Koch’s postulates have been proposed [15]. These include epidemiological evidence of breastfed infant infection, detection of infectious virus in breast milk, exclusion of alternative transmission routes, and reproduction of transmission via oral or breastfeeding routes in animal models. While epidemiological data are
informative, animal models are essential to confirm causality under controlled conditions. Importantly, transmission of the YFV 17D vaccine strain through breastfeeding is considered proven, as this attenuated strain cannot be transmitted by mosquitoes [16] — thereby excluding vectorial transmission and strengthening the causal link.

Using these criteria, breastfeeding transmission has been confirmed for three human viruses: human T-cell lymphotropic virus type 1 (HTLV-1), human immunodeficiency virus (HIV), and human cytomegalovirus (CMV) [15], and is suspected for several arboviruses. Infant infections have been reported following maternal infection with chikungunya virus (CHIKV), dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), with viral genomes from all four — and infectious DENV and ZIKV particles — detected in breast milk [17].

In this study, we used *in vivo* (mouse) and *in vitro* (human cells) models to investigate YFV transmission to neonates via oral exposure and breastfeeding. We demonstrate that both vaccine and wild-type strains infect the mammary gland, cross the mammary epithelium, and are excreted in murine milk as infectious free particles and cell-associated virus. *In vivo*, YFV primarily targets mammary stromal and immune cells, however human mammary epithelial cells are also permissive to infection, suggesting two epithelial-crossing mechanisms in mammary glands: infected immune cell transmigration (“Trojan horse” strategy), and epithelial infection with viral release. We also show that neonatal mice are susceptible to YFV via oral route, with neuroinvasion following intragastric exposure. Using a human intestinal epithelium model, we demonstrate that both strains infect and cross this barrier without disrupting its integrity. Finally, we confirm YFV transmission to suckling pups via breastfeeding — albeit infrequently — validating this route of transmission for YFV.

# METHODS

## Animal model

Experiments used A129 mouse model (129S2/SvPas-Ifnar1<sup>tm1Agt</sup>), deficient in interferon-α/β receptor (IFNAR1<sup>-/-</sup>). Mice were housed and bred in the Institut Pasteur (Paris, France), in animal facilities accredited by the French Ministry of Agriculture for breeding and performing experiments on live rodents (authorization A75150101).

## Virus strains

Three YFV strains were used: wild-type strains Asibi (GenBank: AY640589.1) and Dakar/HD1279 (GenBank: MN106242.1), and vaccine strain 17D-204 (GenBank: MN708488.1).

## Mouse infection
Non-lactating females were subcutaneously inoculated with 1-3 × 10⁵ plaque-forming units (PFU) of YFV (Asibi, Dakar/HD1279 or 17D-204 strains). Blood and mammary glands were collected at various days post-inoculation (dpi). Lactating females received subcutaneously 1-3 × 10⁵ PFU of YFV (Asibi or 17D-204). Maternal blood, breast milk and mammary glands were collected 4-6 dpi [18]. For breastfeeding-mediated transmission experiments, pups blood and organs were collected 7-9 dpi. For oral infection of neonatal mice, A129 pups received 2 × 10⁴ to 1 × 10⁵ PFU of YFV (Asibi or 17D-204) by intragastric route. Blood and brains were collected at 6 dpi. Protocols and animal monitoring are detailed in supplementary materials.

## Ethics statement

Animal experiments complied with French and European regulations on care and protection of laboratory animals (EC Directive 2010/63, French Law 2013-118, 6 February 2013). All experiments were approved by the Ethics Committee n°89 and registered by the French "Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation" (MESRI) under the reference "APAFIS#31829-2021052812549296" (date of approval: 31 May 2021). Use of genetically modified mice (A129) was approved by the institutional instances and the MESRI under the reference n°9319 (date of approval: 6 December 2021).

Full protocols are available in the supplementary materials.

## RESULTS

### YFV disseminates to the mammary glands of non-lactating and lactating A129 mice.

To investigate the early steps of YFV transmission via breastfeeding, A129 mice — a well-established model of YFV infection [19] — were used to assess viral dissemination to mammary glands and subsequent excretion into breast milk. Infection kinetics were first evaluated in non-lactating females for ethical and practical reasons, with key findings later validated in lactating mice. Mice were subcutaneously inoculated with wild-type Asibi or Dakar/HD1279 strains, or vaccine 17D-204 strain (1-3 × 10⁵ PFU), modelling a local infection as occurs following mosquito bite. Clinical signs, weight, and viremia were monitored, and mammary glands collected at various days post-infection (dpi) (Figure 1A). No mortality was observed. Only wild-type strains induced mild symptoms and limited weight loss (~5% for Dakar), while infection with 17D-204 strain remained asymptomatic (Supplementary Figure S1A-B). Viremia was confirmed in all groups by the presence of viral RNA (vRNA) in the plasma, except in three 17D-204 cases, where infection was confirmed in the spleen (Figure S1C). In non-lactating mice (6-22 weeks-old), infectious virus was detected by plaque assay in mammary glands for all strains, peaking between 4-6 dpi with notable inter-individual variation (Figure 1B), including within an age-matched group (8-12 weeks; Figure S1E). Titers declined by 7 dpi, suggesting limited persistence in this tissue. When pooling all time points, mammary infection
was significantly more frequent with wild-type Asibi (73.8%) and Dakar (84.2%) than with 17D-204 (46.7%) (Figure S1D, a), suggesting limited dissemination of the vaccine strain. Using the identified peak infection times (5-6 dpi), presence of vRNA and infectious particles in mammary glands of lactating females was confirmed (Figure 1C-D), with marked inter-individual variability. RT-qPCR detection was significantly more frequent in Asibi-infected mice (100%) than in 17D-204-infected ones (69.2%) (Figure S1D, b), further supporting reduced dissemination of the vaccine strain. Notably, infectious virus was detected less frequently in lactating than in non-lactating glands, likely due to technical challenges in viral quantification, as milk and tissue remodelling may reduce detection sensitivity. These results show that both wild-type and vaccine YFV strains can reach the mammary glands in mice under both lactating and non-lactating conditions.

**YFV infectious virus is released into murine breast milk, as free viral particles and cell-associated virus.**

To investigate viral excretion into milk, lactating A129 mice were inoculated with YFV Asibi or 17D-204 strains via subcutaneous route (Figure 2A), and successful infection was confirmed in plasma (Figure S2A) or spleen (Figure S1C, c). Viral RNA was detected in breast milk collected at 5-6 dpi for both strains in 80-90% of animals (Figure S2D, a), ranging from $2 \times 10^3$ to $7.1 \times 10^5$ (Asibi) and $2.5 \times 10^4$ to $4.7 \times 10^6$ vRNA copies/mL (17D-204) (Figure S2C, a), reflecting a high rate of viral shedding into milk, without correlation between plasma and milk viral loads (Figure S2B). Infectious virus was also detected, with higher titers at 5 dpi — generally greater for 17D-204 (up to $1.5 \times 10^5$ PFU/mL) than Asibi (up to $3 \times 10^4$ PFU/mL) — though differences were not statistically significant (Figure 2B). Titers declined by 6 dpi, with infectious virus undetectable in the Asibi group and lower detection in 17D-204 samples. To characterize the form of virus in milk, murine milk samples were fractionated by centrifugation into cream, whey and cellular components (Figure 2A). vRNA was consistently detected in whey and purified milk cells (Figure S2C, b-c), and infectious virus was found in whey (Figure 2C) and in milk cells after co-culture with permissive cells (Figure 2D), indicating the presence of both free viral particles and cell-associated infectious virus in breast milk. These findings demonstrate that both wild-type and vaccine YFV strains are excreted into breast milk as free and cell-associated infectious virus. No strain-specific differences were observed in viral load, infectious titer, or the proportion of positive milk samples (Figure S2D), which contrasts with the dissemination patterns seen in mammary tissue. This viral excretion into breast milk requires crossing the mammary epithelium, via mechanisms yet to be elucidated.

**YFV infects mainly immune and stromal cells in the mammary glands of non-lactating mice in vivo.**

The mammary gland comprises epithelial structures within a fibro-adipose matrix, featuring various cell types. Stromal cells (including adipocytes and fibroblasts) provide structural support, and endothelial and immune cells manage vascularization and immune surveillance
[20]. The pseudostratified epithelium includes luminal cells (milk secretion) and contractile myoepithelial cells (milk ejection). YFV presence in breast milk suggests mammary epithelium viral crossing, potentially through infected immune cell transmigration or epithelial cell infection followed by viral release into the milk or shedding of infected cells. To study YFV tropism, mice were subcutaneously inoculated with YFV (Asibi and Dakar strains at 1 × 10⁵ PFU). Infection was confirmed in plasma (Figure S3A), and mammary glands were analyzed at 6 dpi using enzymatic dissociation and flow cytometry or immunohistochemistry (Figure 3A). Mammary cells were categorized into endothelial (CD31⁺), immune (CD45⁺), epithelial (CD24⁺), and stromal cells (CD31⁻CD45⁻CD24⁻CD49f⁻). Luminal (CD24highCD49flow) and myoepithelial cells (CD24lowCD49fhigh) were further identified within the epithelial subset (Figure S3B). Flow cytometry revealed YFV infection in a small proportion of mammary cells, predominantly in stromal and immune cells (~4 and 1.5% of infected cells, respectively), with minimal detection in endothelial and epithelial cells (Figures 3B, S3C). Immunohistochemistry of mammary glands confirmed presence of YFV-positive cells mainly in the stromal compartment, likely infiltrating immune cells or resident stromal cells (Figure 3C). Furthermore, histological analysis of mammary glands from Asibi-infected mice revealed the presence of cellular infiltrates in periductal stromal regions, compatible with a leukocytic infiltration and local inflammatory response (Figure S4). These findings identify mammary stromal and immune cells as possible primary YFV targets in vivo, suggesting that infected immune cell transmigration may facilitate viral crossing of the mammary epithelium. Nevertheless, direct epithelial cell infection cannot be excluded, as it might occur below detection thresholds or be influenced by technical limitations, as being among the most difficult cells to dissociate. Moreover, epithelial cells constitute the mammary barrier and are predominant in human and murine milk (up to 98 and 88% of milk cells, respectively [18, 21]), and, although rare, their infection could represent an alternative route for viral passage through the mammary epithelium. To investigate this possibility, we performed in vitro experiments showing that human primary mammary epithelial cells (HMEpiC, Figure S5B) and two human mammary epithelial cell lines — luminal MCF7 and myoepithelial MDA-MB-231 (Figure S5C) — support productive YFV replication for both wild-type and vaccine strains. These results suggest the possibility that the mammary epithelium may contribute to viral excretion into breast milk, either through free virus or infected cell shedding.

**Oral inoculation of A129 mouse pups with YFV results in systemic infection and can lead to neuroinvasion.**

After excretion in milk, YFV must cross the infant's digestive barrier (tonsils, intestinal epithelium, etc.) to allow transmission via breastfeeding. To assess potential oral transmission of YFV, susceptibility of 4- to 7-days-old mouse pups after intragastric inoculation with wild-type (Asibi) or vaccine (17D-204) strains (2 × 10⁴ to 1 × 10⁵ PFU) was evaluated. At 6 dpi, blood and brain samples were collected to evaluate systemic infection and neuroinvasion (Figure 4A). Plasma vRNA was detected for both strains, with significantly higher infection rates for Asibi
(29.3%) than for 17D-204 at either viral dose (8.5% and 9.7%, respectively) (Figure 4B). Infectious virus was found in pups with high plasma viremia (>5 × 10⁵ vRNA copies/mL), titers being higher for Asibi (up to 2.8 × 10⁶ PFU/mL) than for 17D-204 (up to 3.7 × 10³ PFU/mL) (Figure 4C). Neuroinvasion occurred for both strains. In Asibi-infected pups, vRNA and infectious virus were detected in the brain of 64.7% and 47% of plasma-positive animals, respectively (Figure 4D-E). For 17D-204, brain infection was observed in 1/5 and 1/3 plasma-positive pups at 2 × 10⁴ and 1 × 10⁵ PFU, respectively, though the limited sample size precludes conclusions on neuroinvasion efficiency. These findings demonstrate that oral YFV inoculation can lead to systemic infection and neuroinvasion in some pups, supporting the plausibility of breastfeeding-associated transmission and neurological complications observed in human cases involving YFV vaccine strains [9–12].

**YFV can infect and cross an *in vitro* model of human intestinal epithelium without disrupting its integrity.**

Oral infection suggests that YFV can cross a digestive barrier, such as the tonsillar or intestinal mucosa. To investigate whether the intestinal epithelium could allow YFV entry following oral exposure, a previously established *in vitro* model of human intestinal epithelium based on Caco-2/TC7 cells cultured on Transwell inserts was used. After 14–29 days, tight polarized monolayers (transepithelial electrical resistance (TEER) >250 Ω.cm²) were apically inoculated at MOI 1 with YFV (Asibi, Dakar, 17D-204 strains). Barrier integrity was monitored by TEER measurement and tight junction protein ZO-1 immunostaining. Intracellular RT-qPCR and plaque assays of apical and basolateral media were used to assess viral replication, production, and epithelial crossing (Figure 5A). ZO-1 immunoreactivity at 48 hpi revealed intact tight junctions in YFV-infected monolayers (Figure 5B, a). Similarly, TEER values remained stable in YFV-infected cells, contrary to EDTA-treated controls (Figure 5B, b), indicating preserved barrier integrity. Intracellular vRNA increased significantly over time for all strains, peaking at 72 hpi (~3 × 10⁷ copies/µg) and confirming YFV replication in differentiated enterocytes (Figure 5C). Infectious titers showed significant increase apically and basolaterally for all strains, though the increase for Asibi on the basolateral side was not statistically significant (Figure 5D). Basolateral titers reached high levels by 72 hpi (Asibi: 5.1 × 10³, Dakar: 1.5 × 10⁶, 17D-204: 5.9 × 10³ PFU/mL), supporting productive infection in the basolateral compartment, though passive transcytosis of viral particles cannot be excluded. These results support the intestinal epithelium as a potential portal of entry for both wild-type and vaccine YFV strains via productive infection of enterocytes.

**Limited but detectable transmission of YFV to suckling pups through breastfeeding.**

After validating the sequential steps of milk-borne YFV transmission, breastfeeding transmission experiments were performed in our mouse model to confirm this route of infection. Lactating A129 mice nursing 1- to 13-day-old pups were inoculated post-partum with YFV (Asibi and 17D-204 strains) at 3 × 10⁵ PFU. At 7 and/or 9 dpi, samples from suckling pups (blood, spleen,
liver) were collected to assess infection (Figure 6A). Dams' infection was confirmed by viremia (Figure S6A). Among all pups, three were tested positive for YFV RNA in plasma by RT-qPCR — 1/16 fed by an Asibi-infected dam (P1, 4-5 days old) and 2/30 by 17D-204-infected dams (P2, 7-10 days old; P3, 4 days old) (Figure 6B), demonstrating that YFV breastfeeding transmission can occur, albeit infrequently. Infected pups were further analysed on plasma, spleen, and liver by RT-qPCR and plaque assay (Figure 6C), with confirmation of RT-qPCR amplicons by gel electrophoresis (Figure 6D). P1 showed high viral loads and infectious titers in all tissues at 9 dpi, with detection of the YFV-specific 193 bp amplicon, confirming systemic infection via breastfeeding. P2 and P3 had lower plasma RNA levels at 7 dpi; and at 9 dpi, only P2 showed low vRNA in the spleen and a positive viral amplicon. These results demonstrate that both wild-type and vaccine YFV strains can be transmitted through breastfeeding in mice, although such transmission appears to be a rare event.

DISCUSSION

Despite the availability of an effective live-attenuated vaccine, YFV remains a major public health concern. While mosquito transmission is primary, cases of vaccine-strain YFV transmission via breastfeeding, leading to meningoencephalitis in infants [9–12], have prompted updated recommendations [13]. Detection of wild-type YFV RNA in human breast milk during outbreaks further raised concerns about potential non-vectorial transmission [14]. However, confirming this risk in endemic settings is difficult due to constant mosquito exposure. To address this, we conducted the first proof-of-concept study using A129 mice to investigate YFV transmission through breastfeeding and characterize underlying mechanisms.

We showed that both wild-type and vaccine YFV strains reach mammary glands in mice and are shed in breast milk at similar titers, though the vaccine strain disseminated to fewer glands. At 5-6 dpi, infectious virus was detected in 38.5% (Asibi) and 23.1% (17D-204) of lactating mice – less frequently than in non-lactating mice (82.8% and 42.9%, respectively), possibly due to milk presence and physiological changes affecting detection. Infectious virus of both YFV strains was detected in breast milk, whey and cell fraction, indicating the presence of free viral particles and cell-associated infectious virus. Despite reduced mammary dissemination, the vaccine strain reaches breast milk at levels similar to the wild-type strain, suggesting different mammary crossing mechanisms. *In vivo*, YFV primarily targeted mammary stromal and immune cells, however mammary epithelial cells are also permissive *in vitro*. These findings suggest two potential routes into milk: direct epithelial infection with viral release or shedding, and infected immune cells transmigration ("Trojan horse" strategy) – though other mechanisms cannot be excluded. Elucidating these mechanisms remains challenging and poorly defined, even for established breastfeeding-transmitted viruses like HIV, HTLV-1, and CMV [17]. Organoid models may help clarify epithelial susceptibility but may not fully recapitulate dynamics required to study viral crossing mechanisms [22].
Crucially, both strains infected pups after oral exposure, with higher rates for Asibi (29.3%) than 17D-204 (8.5–9.7%), highlighting oral YFV transmission. Neuroinvasion occurred with both strains, especially Asibi (64.7% of infected pups). Together with reported neurological symptoms in cases of breastfeeding transmission of vaccine strains in humans [9–12], our findings raise concerns about the neurovirulence of wild-type YFV following oral exposure. Using a human intestinal epithelium model, we found that differentiated enterocytes support productive viral replication, and apical-to-basal viral crossing *in vitro*. While transcytosis cannot be ruled out, paracellular passage seems unlikely given preserved barrier integrity. Finally, as proof of concept, we confirmed rare but detectable YFV transmission through breastfeeding to suckling pups *in vivo*.

Overall, our findings highlight phenotypic differences between wild-type and vaccine YFV strains in dissemination and transmission efficiency. Both reach the mammary glands and are shed in milk at similar titers, despite less frequent gland infection with the vaccine strain. Oral infection experiments suggest higher neonatal susceptibility to the wild-type strain, indicating strain-specific differences in epithelial barriers crossing. These differences may be linked to the 20 amino acid differences — mainly in the envelope protein — between Asibi and 17D [4], which may influence entry pathways and barrier crossing mechanism.

Mice are not natural hosts and are naturally resistant to YFV infection except when extremely young or infected intracranially [23–26]. Therefore, A129 model, lacking the type I interferon receptor, is widely used, despite its inherent limitations for studying YFV pathogenesis and immune responses, as absence of type I IFN signaling can enhance viral replication and dissemination. Some aspects of infection such as neuroinvasion or tissue tropism may be more pronounced than in immunocompetent hosts, and direct extrapolation to human infection should be made with caution. Nevertheless, A129 model remains biologically relevant [19], as YFV itself partially inhibits interferon signaling in humans — but not in mice — through NS5-mediated STAT2 degradation. Importantly, it provides a sensitive and discriminative system to compare viral strains *in vivo* and to assess antiviral or vaccine efficacy under controlled conditions [27–32]. This discriminative capacity is further supported by the present findings on YFV and by our previous work on ZIKV transmission in the same model. ZIKV, another *Orthoflavivirus*, was transmitted via breastfeeding in A129 mice with markedly higher efficiency (39–90%) [18,33] than YFV (~6%), reaching substantially higher milk titers (up to 10⁹ vs. 1.5 × 10⁵ PFU/mL) [18] and showing greater oral infectivity (64% vs. 8.5% and 29.3% for wild-type and vaccine YFV, respectively) [33]. These findings suggest a markedly greater lactogenic transmission potential for ZIKV and emphasize the biological relevance of the A129 model for studying milk-borne transmission efficacy of *Orthoflaviviruses*.

Breastfeeding remains essential for infant health and is still recommended in ZIKV-endemic areas [34]. However, confirmed vaccine-strain YFV transmission via breastfeeding in humans [9–12], our findings reinforce the need to include this route among potential transmission risks
for both YFV and ZIKV and to reassess current public health recommendations, especially in the absence of clear guidelines during maternal infection.

## SUPPLEMENTARY DATA

Supplementary materials are available online at ______. These materials, provided by the authors to support and enhance the reader's understanding, have not been copyedited and remain the sole responsibility of the authors. For any questions or comments, please contact the corresponding author.

## AUTHOR CONTRIBUTIONS

- Conceptualization, P.-E.C. and A.V.;

- Methodology, J.P., S.D., A.C., H.L. and A.V.;

- Formal analysis, J.P.;

- Investigation, J.P., S.D., P.J., R.K., A.C. and A.V.;

- Validation, H.L., A.V. and P.-E.C;

- Resources, A.V. and A.C.;

- Writing – original draft preparation, J.P.;

- Writing – review & editing, J.P., S.D., A.C., H.L., A.G., P.-E.C. and A.V.;

- Supervision, A.V. and P.-E.C.;

- Visualization, J.P., P.-E.C. and A.V.;

- Project administration, P.-E.C. and A.V.;

- Funding acquisition, P.-E.C. and A.V.;

All authors have read and agreed to the published version of the manuscript

**ACKNOWLEDGMENTS:** We thank the staff of the Institut Pasteur ABSL-3 animal facility and the animal caretakers for their assistance with mouse experiments and occasional help with sampling; Nolwenn Jouvenet (Institut Pasteur, Paris, France) for providing viral strains, protocols, and plasmids; Valérie Choumet (Institut Pasteur) for generously providing antibodies; and Pascal Campagne (Bioinformatics and Biostatistics HUB, Institut Pasteur) for his support in improving the statistical analyses; and Etienne Simon-Lorrière (Institut Pasteur) for kindly performing the sequencing of the viral strains used in this study. We also thank Biorender, which was used for the creation of some figures (figures made using Biorender under a paid license).
**FINANCIAL SUPPORT:** This work was supported by the French Ministry of Education and Research, with J.P. and S.D. receiving PhD fellowships, and by the Institut Pasteur, where R.K. held a fixed-term doctoral contract as doctoral student.

**POTENTIAL CONFLICTS OF INTEREST:** All authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

**MEETINGS WHERE PART OF THE DATA HAS PREVIOUSLY BEEN PRESENTED**

« 26ème Journées Francophones de Virologie » in Brussels (Belgium), 10-12 April 2024.

- « Adapting to change: Emerging Infectious Diseases in a Shifting Climate » in Paris (France), 16-18 October 2024.

- « 27ème Journées Francophones de Virologie » in Lyon (France), 23-25 April 2025.

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**Figure 1:** YFV disseminates to the mammary glands of non-lactating and lactating A129 mice.

(A) Non-lactating (6- to 22-week-old) and lactating (10- to 22-week-old) A129 mice were either MOCK-inoculated or inoculated with YFV via subcutaneous (SC) injection. Non-lactating females were inoculated with PBS (MOCK, n=16) or with 1 to 3 × 10⁵ PFU of the Asibi (n=42), Dakar/HD1279 (n=19) or 17D-204 strains (n=30) of YFV (7 independent experiments). Lactating females were inoculated 1 to 13 days post-partum with PBS (MOCK, n=7) or with 1 to 3 ×10⁵ PFU of the Asibi (n=16) or 17D-204 strains (n=13) (5 independent experiments). At various days post-infection (dpi), blood samples and thoracic mammary glands were collected to assess mice infection and mammary gland dissemination, respectively (Illustration created with Biorender). (B) YFV dissemination to thoracic mammary glands of non-lactating mice was assessed by plaque assay on Vero cells to detect infectious viral particles. (C-D) Viral dissemination to thoracic mammary glands of lactating mice was assessed by RT-qPCR using NS3-specific primers to determine the presence of viral RNA genome (C) and by plaque assay on Vero cells (D). *The number of infected mammary glands relative to the total number of tested mammary glands is shown in italics above each time point.* Results are expressed as the mean ± standard deviation. The dashed lines indicate the limit of detection or specificity (determined from MOCK control conditions), below which values were considered non-specific or not detected. The highest of these two thresholds was chosen as the final limit (ND). Statistical tests: Kruskal-Wallis test with Dunn's multiple comparisons was used all in panels, comparing means values between all strains at all time-points. \* = p < 0.05. Only statistically significant differences (p < 0.05) are indicated.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2:** YFV infectious virus is released into murine breast milk, both as free viral particles and cell-associated virus.</figcaption></figure>

(A) Lactating A129 mice (10-22 weeks old) were either MOCK-inoculated (n=7) or inoculated with YFV at 1 to 12 days post-partum via subcutaneous (SC) injection of 1 to 3 ×10⁵ PFU of the Asibi (n=17) or 17D-204 (n=15) strains of YFV (6 independent experiments). Four to 6 days after inoculation (dpi), blood and breast milk samples were collected. For some milk samples, a portion was centrifuged to separate cream, whey, and cells. Whey was further clarified by three
centrifugations to enhance purity, while the cell pellet underwent three wash-spin cycles in PBS to remove free viral particles (Illustration created with Biorender). (B-C) Detection of YFV in whole breast milk (B) and whey (C) was assessed by plaque assay on Vero cells to determine the presence of infectious viral particles. (D) Detection of infectious cell-associated virus in the milk cell pellet was performed by infectious center assay. *The number of infected samples relative to the total number of tested samples per condition is shown in italics above each time point.* Results are expressed as the mean ± standard deviation. The dashed lines indicate the limit of detection, below which values were considered as not detected (ND). Statistical tests: Kruskal-Wallis test with Dunn's multiple comparisons was used in all panels, comparing means values between all strains at all time-points. \* = p < 0.05. Only statistically significant differences (p < 0.05) are indicated.

<figure><img src="image_6.png" /><figcaption>**Figure 3:** YFV infects mainly immune and stromal cells in the mammary glands of non-lactating mice *in vivo*.</figcaption></figure>

(A) Non-lactating A129 mice (11-19 weeks old) were either MOCK-inoculated or inoculated with YFV via subcutaneous (SC) injection of $1 \times 10^5$ PFU of the Asibi or Dakar strains of YFV. At 5-6 days after inoculation (D5-D6), blood and thoracic and/or abdominal mammary samples were collected to assess mice infection and viral tropism in the mammary tissue. Mammary glands were analyzed by flow cytometry following enzymatic dissociation and immunostaining, (2 independent experiments; MOCK n=7, Asibi n=7, Dakar n=5) or by immunohistochemistry after fixation, paraffin embedding, sectioning and immunostaining (2 independent experiments; MOCK n=1, Asibi n=4) (Illustration created with Biorender). (B) For flow cytometry, thoracic
and abdominal mammary glands from 3-4 mice per group were pooled and enzymatically digested. Cell suspensions were stained with fluorochrome-conjugated antibodies against CD31 (PE), CD45 (APC), CD24 (BV421), and CD49f (PECy7), then fixed, permeabilized, and stained for YFV using mouse polyclonal antibodies (ascitic fluid) and an AF488-conjugated secondary antibody. Samples were analyzed by flow cytometry. Panel (a) shows representative plots; panel (b) quantifies YFV+ cells (2 independent experiments, one dot = one experiment). The dashed line indicates the limit of specificity (determined from MOCK control conditions), below which values were considered as non-specific (ND). (C) For immunohistochemistry, mammary glands from Asibi-infected mice were fixed in 10% formalin, paraffin-embedded, sectioned (5 µm), and stained for YFV using the same primary antibody and an AF546-conjugated secondary. Fluorescence microscopy was used for analysis. Red = YFV; blue = nuclei (DAPI). L: lumen of lactiferous ducts, lined by mammary epithelium and surrounded by stroma. Both images are from the same animal and are representative of four independent animals.
A

<figure><img src="image_7.png" /></figure>

B a

Infection of mammary cells
(Flow cytometry)

<figure><img src="image_8.png" /></figure>

b

Infection of mammary cells
(Flow cytometry)

<figure><img src="image_9.png" /></figure>

C

YFV

<figure><img src="image_10.png" /></figure>

Merge
YFV + Nuclei

<figure><img src="image_11.png" /></figure>

YFV

<figure><img src="image_12.png" /></figure>

Merge
YFV + Nuclei

<figure><img src="image_13.png" /></figure>
**Figure 4:** Oral inoculation of A129 mouse pups with YFV results in systemic infection and can lead to neuroinvasion.

(A) A129 mouse pups (4- to 7-days-old) were inoculated via the intragastric (IG) route with yellow fever virus (YFV) or with PBS as a negative control (MOCK). Pups received either PBS (MOCK, 2 litters, n = 5), 2 ×10⁴ PFU of the Asibi strain (10 litters, n = 58), or 2 ×10⁴ PFU (11 litters, n = 59) or 1 ×10⁵ PFU (4 litters, n = 31) of the 17D-204 strain of YFV (6 independent experiments). Blood and brain samples were collected at 6 days post-infection to assess viremia (B-C) and brain infection (D-E) (Illustration created with Biorender). (B-C) Viral infection in mouse pups was assessed in plasma (obtained by blood centrifugation) using NS3-specific RT-qPCR to quantify viral RNA load (B), and by plaque assay on Vero cells to determine infectious viral titers (C). (D-E) YFV presence in brain tissue was evaluated post-homogenization by RT-qPCR for viral RNA detection (D), and by plaque assay on Vero cells to quantify infectious virus (E). Results are presented as mean values. The dashed lines indicate the limit of detection or specificity (determined from MOCK control conditions), below which values were considered non-specific or not detected. The highest of these two thresholds was chosen as the final limit (ND). Percentages in bold (B-E) represent the proportion of positive samples out of the total; percentages in italic (D-E) represent the proportion of brain-positive samples among those positive in plasma by RT-qPCR. Statistical test: Fisher's exact test was used in panels (B-E). For statistical analyses: * = p < 0.05; ** = p < 0.005. Only statistically significant differences (p < 0.05) are indicated.
<figure><img src="image_14.png" /><figcaption>B</figcaption></figure>

Plasma
(viral genome)

<figure><img src="image_15.png" /><figcaption>C</figcaption></figure>

Plasma
(infectious viral particles)

<figure><img src="image_16.png" /><figcaption>D</figcaption></figure>

Brain
(viral genome)

<figure><img src="image_17.png" /><figcaption>E</figcaption></figure>

Brain
(infectious viral particles)

<figure><img src="image_18.png" /></figure>
**Figure 5:** YFV can infect and cross an *in vitro* model of human intestinal epithelium without disrupting its integrity.

(A) Caco-2/TC7 cells ($3 \times 10^4$) were seeded into the upper chamber of Transwell inserts (12 mm diameter, 3 µm pores) and cultured for 14–29 days to form a tight, polarized intestinal epithelial monolayer separating apical (upper) and basolateral (lower) compartments. Monolayer integrity was assessed by transepithelial electrical resistance (TEER) measurements. Cells were apically infected with YFV strains (Asibi, Dakar/HD1279 or 17D-204) at MOI 1, or left uninfected as controls (MOCK). At various time points post-infection, TEER and ZO-1 immunostaining were used to monitor barrier integrity (B), viral replication was quantified by RT-qPCR on intracellular RNA (C), and viral production and crossing of the epithelium was assessed by plaque assay on apical and basolateral supernatants (D) (Illustration created with Biorender).
(B)(a) At 48h post-inoculation, monolayer morphology was visualized by immunofluorescence of the tight junction protein ZO-1 (red). EDTA (12.5 mM; purple) was used as a positive control to disrupt barrier function. (b) TEER values were monitored over time. The yellow zone ($0–250 \text{ Ohm.cm}^2$) indicates the permeability threshold. Data shown are representative of two independent experiments and consistent across all replicates. (C) Viral RNA levels were measured at 2h, 48h, and 72h post-infection. RNA was extracted from Caco-2/TC7 cells, and YFV RNA was quantified using NS3-specific RT-qPCR. Results are expressed as viral RNA copies/µg of total RNA, calculated using a standard curve generated from 10-fold serial dilutions of a quantified YFV NS3 plasmid. (D) At 2h, 48h, and 72h post-infection, apical and basolateral supernatants were collected and infectious virus quantified by plaque assay on Vero cells. Results in panels (A), (C), and (D) are expressed as mean ± standard deviation. The dashed lines indicate the limit of detection or specificity (determined from MOCK control conditions), below which values were considered non-specific or not detected. The highest of these two thresholds was chosen as the final limit (ND). Data in panels (C) and (D) are representative of minimum three independent experiments, with each dot corresponding to the mean value from one experiment. Statistical test: Ordinary two-way ANOVA followed by Tukey's multiple comparisons test, performed on log₁₀-transformed data, was used in panel (C); Ordinary one-way ANOVA followed by Tukey's multiple comparisons test, performed separately for strain and apical/basal supernatants and on log₁₀-transformed data (using all individual data points, not only experiment means), was used in panel (D). For statistical analyses: \* = $p < 0.05$; \*\* = $p < 0.005$; \*\*\* = $p < 0.0005$; \*\*\*\* = $p < 0.0001$.
<figure><img src="image_19.png" /><figcaption>B a</figcaption></figure>

<figure><img src="image_20.png" /></figure>

Tight junction integrity
(ZO-1 immunostaining)

<figure><img src="image_21.png" /><figcaption>b</figcaption></figure>

Transepithelial electrical resistance (TEER)

<figure><img src="image_22.png" /><figcaption>C</figcaption></figure>

Viral genome (intracellular)

<figure><img src="image_23.png" /><figcaption>D</figcaption></figure>

Infectious viral particles (apical and basal supernatants)
<caption>**Figure 6:** Limited but detectable transmission of YFV to suckling pups through breastfeeding.</caption>

(A) Lactating A129 mice (12- to 17-week-old) were inoculated subcutaneously (SC) 1 to 13 days postpartum with either PBS (MOCK, n=4) or 3 × 10⁵ PFU of Asibi (n=3) or 17D-204 (n=5) strains of YFV (3 independent experiments). Blood, spleen, and liver samples were collected from suckling pups at 7 (D7) and/or 9 (D9) days post-inoculation to assess transmission and viral dissemination (Asibi n=16, 3 litters; 17D-204 n =20, 5 litters) (Illustration created with Biorender). (B) Viral RNA levels in pup plasma were quantified by NS3-specific RT-qPCR. The same pups were analyzed at both time points: the 6 Asibi-inoculated pups sampled at 7 dpi are part of the 16 analyzed at 9 dpi, and the 20 pups inoculated with 17D-204 at 9 dpi are included in the 30 analyzed at 7 dpi. (C) Viral RNA and infectious titers in plasma, spleen, and liver were measured at 7 (D7) and 9 (D9) days post-infection in breastfed pups from dams infected with Asibi or 17D-204 (data shown on the same x-axis). Organs were homogenized prior to analysis by RT-qPCR and plaque assay. P1 (4–5 days old), P2 (7–10 days old), and P3 (4 days old) represent the three plasma-positive pups shown in panel (B). The dashed lines indicate the limit of detection or specificity (determined from MOCK control conditions), below which values were considered non-specific or not detected. The highest of these two thresholds was chosen as the final limit (ND). (D) In pups with plasma positive for YFV (C), the presence of viral RNA in spleen and liver was further evaluated by RT-qPCR following tissue homogenization. RT-qPCR-positive samples were then subjected to electrophoresis on a 2% TAE agarose gel to confirm amplification of the expected viral amplicons. A 100 bp Plus DNA ladder was used to estimate fragment size, with the expected amplicon corresponding to 193 base pairs (bp). Negative control included qPCR products from MOCK-infected pup samples (MOCK), while a plasmid containing YFV sequence (equivalent to 4 vRNA copies/µL) served as a positive control (YFV positive control). Full-size agarose gel electrophoresis images are shown in Supplementary Figure S4. MOCK: MOCK-inoculated pup 1; P1: Asibi-infected pup 1; P2: 17D-204-infected pup 1; P3: 17D-204-infected pup 2.
<figure><img src="image_24.png" /><figcaption>B</figcaption></figure>

<table><caption>C</caption><thead><tr><th rowspan="2">YFV Strain</th><th colspan="2">Plasma positive pups/Total pups</th></tr><tr><th>D7</th><th>D9</th></tr></thead><tbody><tr><td>Asibi</td><td>0/6<br>1 litter</td><td>1/16 (P1 pup)<br>3 litters</td></tr><tr><td>17D-204</td><td>2/30 (P2 and P3 pups)<br>5 litters</td><td>0/20<br>3 litters</td></tr></tbody></table>

<figure><img src="image_25.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_26.png" /></figure>
<figure><img src="image_27.png" /></figure>

Graphical Abstract