Co-acquisition of two sugar beet poleroviruses by M.
persicae increased the transmission efficiency of both
viruses

Hélène Schlaefli, Aurélie Marmonier, Souheyla Khechmar, Catherine Reinbold, Claire Villeroy, Quentin Chesnais, Martin Drucker, Véronique Brault

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To cite this version:

Hélène Schlaefli, Aurélie Marmonier, Souheyla Khechmar, Catherine Reinbold, Claire Villeroy, et al. Co-acquisition of two sugar beet poleroviruses by M. persicae increased the transmission efficiency of both viruses. Virology, 2025, 610, pp.110594. 10.1016/j.virol.2025.110594. hal-05117459

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# Co-acquisition of two sugar beet poleroviruses by *M. persicae* increased the transmission efficiency of both viruses

Hélène Schlaefli, Aurélie Marmonier, Souheyla Khechmar, Catherine Reinbold, Claire Villeroy, Quentin Chesnais, Martin Drucker, Véronique Brault*

INRAE, Université de Strasbourg, SVQV, Colmar, France

## ARTICLE INFO

**Keywords:**

Poleroviruses

Sugar beet

Coinfection

Aphid transmission

## ABSTRACT

Beet mild yellowing virus (BMYV) and beet chlorosis virus (BChV) are two closely related poleroviruses infecting sugar beet plants, which induce leaf yellowing and cause high yield losses. Poleroviruses are phloem-limited and strictly transmitted by aphids in a circulative and persistent manner. In nature, sugar beet plants can be coinfect ed by these two viruses, but the outcome of BMYV and BChV coexistence in a plant on virus accumulation and aphid transmission has never been addressed before. In this study, we showed that the accumulation of each virus was not affected by the presence of the other, despite coinfecting about 40 % of infected phloem cells. Both viruses crossed the gut epithelium at the same site in *Myzus persicae* without any evidence of competition for receptor binding. On the contrary, when aphids simultaneously acquired both viruses from an artificial medium containing equal amounts of each virus, the transmission efficiency increased for both viruses. In contrast, only BMYV exhibited enhanced transmission when acquired from a coinfected plant. This suggests a cooperative effect at the inoculation step facilitating virus delivery in sugar beet cells by aphids. Our results shed light on fine-tuned virus-virus and virus-vector interactions, with potential implications for epidemiology and disease management.

## 1. Introduction

Since the ban on neonicotinoid insecticides in 2018, sugar beet yel-
lowing has re-emerged as a major plant disease across all sugar beet
growing countries in Europe (https://www.legifrance.gouv.fr/jorf/id/
JORFTEXT000033140744/), with a major impact on sugar beet yield.
Sugar beet yellowing is caused by a complex of viruses comprising beet
mild yellowing virus (BMYV) and beet chlorosis virus (BChV) both
belonging to the *Polerovirus* genus in the *Solemoviridae* family, beet
yellows virus (BYV, *Closterovirus* genus, *Closteroviridae* family), and beet
mosaic virus (BtMV, genus *Potyvirus*, *Potyviridae* family). Sugar beet
plants can also be infected with beet western yellows virus-USA (BWYV-
USA, *Polerovirus*), a virus not yet reported in Europe (Beuve et al., 2008;
Hossain et al., 2021; Stevens et al., 2005). All these viruses induce leaf
yellowing which decreases the photosynthetic capacity of the plant
leading to lower root weight, sugar content and processing quality.
However, extent of damage depends on the year, the sugar beet variety,
the moment of infection and other undetermined factors (Stevens et al.,
2004). BMYV, BChV, BYV, BWYV and BtMV are all transmitted by
aphids, mainly *Myzus persicae*, using different transmission modes, i.e.,

persistent and circulative for BMYV, BChV and BWYV, semi-persistent
and non-circulative for BYV, and non-persistent and non-circulative
for BtMV (Brault et al., 2007; Gray and Gildow, 2003, 2003; Kozlowska-Makulska et al., 2009; Schliephake et al., 2000; Stevens et al., 2005).
In nature, these viruses can occur individually in sugar beet plants
exhibiting yellowing symptoms, but mixed infections have been reported with a frequency that varies depending on the year (Hossain et al., 2021). Interactions between sugar beet viruses may have agro-nomical consequences. For instance, coinfection with BYV and BtMV reduced sugar beet biomass in greenhouse conditions and resulted in an increased accumulation of both viruses (Wintermantel, 2005). Additive effects of BtMV when combined with BWYV or BYV on yield losses have also been reported in the field, and a higher yield loss was also observed when BYV co-occurred with BMYV (Russell, 1963). In contrast, a moderate impact on yield loss was monitored in plots co-inoculated with BYV and BChV (Hossain et al., 2021).

BMYV and BChV are closely related poleroviruses which possess
positive-sense single stranded RNA genomes of about 5.6 kb encapsi-
dated into icosahedral particles of about 30 nm composed of the major
coat protein (CP) and a minor component referred to as the readthrough

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: veronique.brault@inrae.fr (V. Brault).</footnote>
protein (RT*) (Mayo and Ziegler-Graff, 1996; Stevens et al., 2005). Alignment of the full-length genomic sequences of BMYV (Guilley et al., 1995), and BChV (Hauser et al., 2002) reveals high sequence homologies within the 3' part of their genomes. This region encompasses the major coat protein (CP encoded by ORF3), the movement protein (encoded by ORF4), and the readthrough domain (encoded by ORF5) sequences. In addition, this region contains a small ORF, ORF3a, upstream of ORF3, which is involved in long-distance virus movement and which is found in both viruses (Smirnova et al., 2015). In contrast, the 5' end of both genomes displays less than 30 % sequence homology (Hauser et al., 2002). High sequence similarity between BMYV and BChV genomes is potentially responsible for the occurrence of virus recombinants observed in coinfect ed plants (Kozlowska-Makulska et al., 2015), although a polymerase template switch during the *in vitro* cDNA synthesis for viral RNA analysis that could result in chimeric genomes, cannot be completely discarded (Cocquet et al., 2006). Poleroviruses are phloem-limited and localization of BChV genomes in phloem cells of sugar beet plants has been observed by *in situ* hybridization (Esau and Hoefert, 1971; Khechmar et al., 2024; Mayo and Ziegler-Graff, 1996; Mutterer et al., 1999). Sugar beet poleroviruses have wide host ranges, but BMYV and BChV display differences in host specificity (Beuve et al., 2008; Hauser et al., 2002; Stevens et al., 1994). BChV and BMYV are mainly transmitted by *Myzus persicae* but can also be propagated from plant to plant by *Macrosiphum euphorbiae* and, in rare occasions, by *Aphis fabae* (Kozlowska-Makulska et al., 2009; Schliephake et al., 2000).

The present study was conducted to gain more insights into the
interaction between two sugar beet poleroviruses (BMYV and BChV) by
addressing the effect of coinfection on virus accumulation, localization
in the plants, and transmission by *M. persicae*. We observed that the
presence of both viruses in sugar beet plants had no effect on virus
accumulation and did not modify the cellular localization of either virus,
which remained phloem-limited. However, co-acquisition of both vi-
ruses by *M. persicae* from an artificial medium resulted in a higher
transmission efficiency of each virus, suggesting a cooperative effect at
the virus inoculation stage. This stimulating effect on transmission was
only observed for BMYV when acquired from coinfect ed plants.

## 2. Methods

2.1. Virus, aphids, and plants

The beet chlorosis virus strain BChV-2a was collected in England
from sugar beet (Hauser et al., 2002). The beet mild yellowing virus
strain 2itb is a French isolate also collected from sugar beet (Guilley
et al., 1995). Both viruses were purified from infected spinaches (Spi-
nacia oleracea) collected 4–5 weeks after inoculation using viruliferous
aphids, and following the procedure described in (van den Heuvel et al.,
1991). The *Myzus persicae* (Sulzer) (Hemiptera: Aphididae) biotype
WMp2 originated from the Netherlands (Reinink et al., 1989). Aphids
were reared on Chinese cabbage (Brassica rapa L. *pekinensis var. Granaat*)
in a growth chamber under 20 ± 1 °C, and 16 h photoperiod. The
commercial variety of sugar beet (Beta vulgaris cv Auckland; SesVan-
derHave, Belgium) was kindly provided by the French Technical Insti-
tute of Sugar Beet (Paris). Plants were grown in greenhouses until the
first two true leaves developed (2 weeks after sowing). Plants were then
grown in laboratories and growth chambers under 21 ± 1 °C and 14 h of
light and 10 h of darkness.

## 2.2. Virus transmission by aphids

To analyze virus transmission efficiency of each virus alone or mixed,
two techniques were used: (I) non-viruliferous *M. persicae* were fed on an
artificial medium (Harrewijn, 1983) containing purified BChV or BMYV
virions, or a mixture of both purified viruses. After a 24 h acquisition
period, one aphid was transferred onto each 3-week-old sugar beet
plants for a 72 h inoculation period. Aphids were then killed by an

insecticide (Pirimor). Plants were grown for three weeks before being
tested by DAS-ELISA first, and then by IC-RT-PCR or RT-PCR to distin-
guish the two poleroviruses; (II) alternately, non-viruliferous *M. persicae*
were fed on sugar beet plants infected with BChV or BMYV alone or with
both viruses. For each transmission experiment, three source plants for
the same condition (inoculated only with BMYV, BChV or co-inoculated
with BMYV and BChV 3–4 weeks before used) were selected after being
tested by DAS-ELISA. Non-viruliferous aphids were deposited either on
whole infected plants, or on detached leaves laid on a 1 % agarose in
H₂O in petri dishes. After a 24 h acquisition period, three aphids were
transferred onto each 3-week-old sugar beet plants for 72 h that were
thereafter processed as described above.

2.3. Virus detection

2.3.1. DAS-ELISA

Both viruses were detected in sugar beet plants by double-antibody
sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with a
turnip yellows virus (TuYV)-specific polyclonal antiserum at 1/200e
dilution (Loewe Biochemica, Sauerlach, Germany) 3 weeks post-
inoculation (Clark and Adams, 1977). Because of high sequence simi-
larities between the structural proteins of TuYV (previously referred to
as BWYV) and those of BChV and BMYV viruses (Hauser et al., 2002),
the TuYV antiserum is able to detect, by DAS-ELISA, BChV, BMYV with
equal sensitivity (Fig. S1) as well as several other closely-related pole-
roviruses. For each plant, 200 mg of leaf sample were collected from 2 to
3 upper leaves, ground in one mL of buffer and deposited in a single well.
Absorbance values were measured at 405 nm with a spectrophotometer
Infinite F50 equipped with the software Magellan reader (Tecan,
Männedorf, Switzerland). Samples were considered infected when the
OD405 nm was twice the mean value of non-infected control plants (i.e., 3
technical replicates) plus three times the standard deviation.

2.4. RT-PCR and IC-RT-PCR

To discriminate between the two poleroviruses, two procedures were
developed. For RT-PCR, total RNA was extracted from 100 mg collected
from 2 to 3 upper leaves pooled for each plant tested using NucleoSpin
RNA Plant Kit (Macherey-Nagel, Hoerdt, France). The RT-PCR was
performed using the virus-specific primers (Table S1) by the one-step
procedure (One step RT-PCR kit, Qiagen, Hilden, Germany) following
instructions of the supplier. The primers final concentrations were
adjusted depending on the virus (0.6 µL for each primer at 25 µM for
BChV and 1.2 µL for each primer at 25 µM for BMYV). To detect BMYV
and BChV by immunocapture IC-RT-PCR, a first step similar to DAS-
ELISA was performed. Briefly, the ground leaf sample was deposited
in coated ELISA plate (dilution Loewe antiserum 1/200°). After three
washes, 10 µL of RNase free water were added to each well, and the plate
was sealed before being incubated at 80 °C for 20 min, followed by 5 min
on ice. RT-PCR one step (One step RT-PCR kit, Qiagen, Hilden, Ger-
many) was then performed on 4.5 µL using the aforementioned primers
concentrations.

2.5. Virus quantification by quantitative RT-PCR (RT-qPCR)

Quantitative RT-qPCR was performed with SYBR Green. Fragments
of 3–4 young non-inoculated leaves (100 mg) were collected for each
plant at specific times, ranging from 3 to 7 weeks post-inoculation. Total
RNA was extracted using NucleoSpin RNA Plant Kit (Macherey-Nagel,
Hoerdt, France). After quantification (Nanodrop, 2000; ThermoFisher
Scientific, Illkirch, France), 200 ng of total RNA were converted into
cDNA using a specific reverse primer (Table S1) and the M-MLV reverse
transcriptase kit (Promega, Charbonnières-les-Bains, France). The for-
ward and reverse primers (Table S1) were used to amplify the cDNA
using a CFX cycler (Biorad, Schiltigheim, France) programmed as fol-
lows: 3 min 95 °C, 40 amplification cycles for 10 s at 95 °C, 30 s at 60 °C.
Melt curve analysis was performed from 60 °C to 95 °C with 5 s of 0.5 °C increments. Dilution series of 10⁸ to 10³ viral cDNA copies obtained from RNA extracted from purified virions were used for calibration. The comparison between the Ct values of the calibration standard and the samples provided an absolute quantification of BMYV or BChV genomes.

## 2.6. Virus observation using transmission electron microscopy (TEM)

For ultrastructural TEM observations, *M. persicae* reared on pepper seedlings (*Capsicum annuum*) were allowed to acquire purified virus (90 ng/µL for BChV and 100 ng/µL for BMYV) included in an artificial medium for 48 h–72 h (Bruyère et al., 1997), before being processed as described in (Alliaume et al., 2018; Reinbold et al., 2001).

## 2.7. Probe synthesis and in situ hybridization by SABER-FISH

For virus localization, sugar beet plants were infected using viruliferous aphids fed on an artificial medium containing each of the purified virus or a mixture of both in conditions described above. Three weeks later, SABER-FISH technique, which is a multiplexable nucleic acid-based detection method was applied to detect viral genomes. This technique includes a signal amplification step enabling rapid and highly sensitive target revelation. The protocol, originally used for frozen retinal tissue as described by Kishi (Kishi et al., 2019), was adapted to virus detection in sugar beet plants. For detection of BMYV and BChV,

16 (BMYV) and 20 (BChV) specific primers were designed with Oligo-Miner (Beliveau et al., 2018). A TTT and a concatemer sequence were added at the 3'-end of each primer (concatemer 27 for BChV and 28 for BMYV, see Table S2). Probes were obtained by primer extension reaction and a SABER-FISH protocol adapted to paraformaldehyde-fixed, parafin-embedded leaf sections was carried out as described (Khechmar et al., 2024). Processed sections were mounted in Fluoroshield medium and observed with a Zeiss Axio Imager M2 microscope equipped with a Hamamatsu Orca-Flash 4.0LT black-and white camera. Images were acquired with a 5x or 20x objective and with Zeiss 43 HE or AHF Cy5 ET bandpass filter sets for epifluorescence acquisition in the dsRED or Far Red channels, respectively, or with brightfield or DIC settings for transmission acquisition. Images were processed with ZEN 2.5 or Image J 1.54 software. Final figures were assembled with Microsoft PowerPoint.

# 3. Results

## 3.1. Kinetics of BChV and BMYV accumulation in mono- or coinfected plants

To address whether virus accumulation was impacted in sugar beet plants coinfected with BMYV and BChV, we performed RT-qPCR on mono- or coinfected plants. Three weeks post-inoculation and up to 7 weeks, leaf samples from the youngest leaves were collected every week,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Accumulation of BChV and BMYV in mono- or coinfected sugar beet plants. Leaf samples of infected plants were collected 3–7 weeks post-inoculation and virus accumulation was measured by RT-qPCR. The number of BMYV or BChV genome copies are normalized to ng of total plant RNA. Sugar beet plants were inoculated with one or the two viruses (BMYV or BChV) using aphids fed on an artificial medium containing one or both purified viruses for 24 h. A) BMYV accumulation in mono- or coinfected sugar beet leaves was not statistically different (Nonlinear Mixed-Effect Models, mono-vs. coinfection *P* = 0.145, time *P* = 0.161, infection:time *P* = 0.756); B) BChV accumulation in mono- or coinfected sugar beet leaves was not statistically different (Nonlinear Mixed-Effect Models, mono-vs. coinfection *P* = 0.059, time *P* = 0.730, infection:time *P* = 0.626). Box plots show median (line), 25–75 % percentiles (box), 10–90 % percentiles (whisker), and outliers (dots). Eight BChV-infected plants, five BMYV-infected and eight BChV and BMYV coinfected plants were analyzed. The coinfected sugar beet leaves are the same in A and B.</figcaption></figure>
mixed and processed for total RNA extraction and RT-qPCR. When BMYV or BChV accumulation was compared at each time point between mono- or coinfected plants, no significant differences were observed all along the experiment between mono- and coinfected plants (Fig. 1A and B). However, accumulation of BChV in mono- or coinfected plants was about twice that of BMYV in mono- or coinfected plants (Fig. 1A and B).

## 3.2. Localization of BChV and BMYV in mono and coinfected plants

We then assessed whether coinfection affected tissue localization of BChV or BMYV. For this, SABER-FISH was performed on mono- and coinfected plants at three weeks after virus inoculation. In cross-sections of BMYV mono-infected leaves, fluorescence corresponding to BMYV genomes was observed exclusively in phloem cells, as expected (Fig. 2A). The label was found in the phloem of primary (midribs) and secondary veins (Fig. 2A). Similarly, in BChV mono-infected plants, fluorescent signals corresponding to BChV genomes were observed in the phloem of primary and secondary veins (Fig. 2B). Coinfection did not change the tissue distribution of BMYV and BChV (Fig. 2C). Both viruses were confined to phloem cells, and no escape in non-phloem cells was observed (Fig. 2C). Both viruses were found together in some phloem cells, as exemplified by the yellow color in the last panel in Fig. 2C. To further evaluate the percentage of coinfected cells and mono-infected cells in coinfected plants, ten to eleven sections from four coinfected plants were processed in two independent experiments (Table 1). Despite the high variability between the individual plants analyzed, we observed a much lower percentage of cells mono-infected with BMYV (6% of all infected cells containing only BMYV) compared to BChV (55% of infected cells containing only BChV) (Table 1). On average, about 40% of the infected cells were coinfected, with a high variety of coinfection ranging from 0% (plant #3) to 72% (plant #1). (Table 1). Nonetheless, this suggests that both viruses can coexist in the same cells and that there is no exclusion between the two viruses at the cellular level in sugar beet plants.

<table><caption>**Table 1**
Number of cells infected by one or both viruses in sections of coinfected sugar beet leaves.</caption><thead><tr><th></th><th>Plant #1</th><th>Plant #2</th><th>Plant #3<sup>c</sup></th><th>Plant #4</th><th>%<sup>d</sup></th></tr></thead><tbody><tr><td>BMYV</td><td>0<sup>a</sup> (0 %)<sup>b</sup></td><td>1 (4 %)</td><td>0 (0 %)</td><td>12 (23 %)</td><td><strong>6 %</strong></td></tr><tr><td>BChV</td><td>24 (28 %)</td><td>17 (71 %)</td><td>43 (100 %)</td><td>29 (56 %)</td><td><strong>55 %</strong></td></tr><tr><td>BMYV + BChV</td><td>61 (72 %)</td><td>6 (25 %)</td><td>0 (0 %)</td><td>11 (21 %)</td><td><strong>38 %</strong></td></tr></tbody></table>

<sup>a</sup> Number of cells observed on 10 to 11 sections of each plant showing a fluorescent signal for the indicated virus.

<sup>b</sup> %: percentage of labeled cells observed in mentioned plant containing each virus alone or both.

<sup>c</sup> Coinfection of plant #3 with BMYV and BChV was confirmed by IC-RT-PCR.

<sup>d</sup> %: percentage of labeled cells/total number of fluorescent cells. Two experiments were performed (plants #1 to 3 are from one experiment and plant #4 from another experiment).

## 3.3. Effect of coinfection with BChV and BMYV on virus transmission by *M. persicae*

Since BMYV and BChV can infect the same plant, and are both transmitted by *M. persicae*, it is conceivable that coinfection may affect virus transmission by aphids. In particular, these viruses share high amino acid identities between their structural proteins, with 91.5% and 94.0% identity for CP and RT* respectively, two proteins involved in polerovirus aphid gut receptors recognition (Mulot et al., 2018). Therefore, BMYV and BChV could compete for the same intestinal receptors which could reduce internalization of each virus into the hemolymph, and thereafter the transmission rate of each virus. To address whether virus competition at the gut level could be envisaged, we identified the intestinal uptake site of BChV and BMYV in *M. persicae* by transmission electron microscopy (TEM). Aphids were fed on purified suspensions of BMYV or BChV before being processed for TEM observations. The viruliferous status of the aphids was verified by transferring part of the potentially viruliferous aphids on non-infected plants (1–3 aphids per plant) for virus inoculation (Table S3). As shown in Table 2 and Fig. 3(A–D), BChV and BMYV virus particles were observed as free

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** SABER-FISH detection of BChV and BMYV genomes in sugar beet plants mono-infected with BChV or BMYV or coinfected with both viruses. Representative images of cross sections of sugar beet leaves infected with BMYV (A), BChV (B), BMYV and BChV (C) or non-infected (D). The first column shows BMYV label (red), the second BChV label (green), the third bright field acquisition and the fourth column shows merges. Scale bar (200 µm) is indicated on each image. In the last right panel, a close-up corresponding to the white square is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<caption>**Table 2**</caption>

Identification of *M. persicae* intestinal part in which virus particles of BChV or BMYV were observed by TEM after *in vitro* acquisition of viruses.

<table><thead><tr><th rowspan="2"></th><th>stomach</th><th colspan="3">posterior midgut</th><th colspan="2">hindgut</th></tr><tr><th>lumen</th><th>lumen</th><th>invaginations</th><th>cytoplasm</th><th>lumen</th><th>cytoplasm</th></tr></thead><tbody><tr><td>BChV</td><td>3/7<sup>a</sup></td><td>13/14</td><td>9/14</td><td>5/14</td><td>3/6</td><td>0/6</td></tr><tr><td>BMYV</td><td>1/8</td><td>12/15</td><td>8/15</td><td>5/15</td><td>2/7</td><td>0/7</td></tr><tr><td>controls<sup>b</sup></td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/3</td><td>0/3</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Transmission electron microscopy (TEM) observations of BChV and BMYV virus particles in the posterior midgut of *M. persicae*. BChV particles were observed in the lumen (A) or in the cytoplasm of the posterior midgut cells (B). BMYV particles were observed in the lumen (C) or in the cytoplasm of the posterior midgut cells (D). Arrows indicate the presence of virus particles. (E) microvilli and (F) cytoplasm of posterior midgut cells of non-viruliferous aphids. The scale bars represent 200 nm.</figcaption></figure>

particles in the lumen of the digestive tube of *M. persicae*, but also in intestinal invaginations of the apical plasmalemma of posterior midgut cells, and in single or tubular vesicles in the cytoplasm of these cells, but never in the cytoplasm of hindgut cells. No virus-like particles were observed in the posterior midgut or hindgut cells of non-viruliferous aphids (Fig. 3E and F). So, we showed that BMYV and BChV are internalized at the same intestinal sites in *M. persicae*.

To further address the effect of sugar beet plants coinfection with BChV and BMYV on virus transmission efficiency by *M. persicae*,

transmission experiments were performed using coinfected plants as virus source, and mono-infected plants as controls. These plants were used 3–4 weeks after prior inoculation with the corresponding viruses. BMYV transmission efficiency was statistically higher when aphids acquired BMYV from coinfected plants (Fig. 4A and Table S4). In contrast, no change in BChV transmission efficiency was observed when aphids acquired the virus from mono- or coinfected plants (Fig. 4A and Table S4). Interestingly, both viruses were transmitted together at a higher rate than expected (Fig. 4C and Table S4). The higher

<footnote>a Number of *M. persicae* in which virus particles were observed/total number of aphids observed at the location.</footnote>

<footnote>b Controls: non-viruliferous aphids.</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Aphid transmission efficiency of BMYV and BChV when acquired from mono- or coinfected sugar beet plants. *M. persicae* acquired virus from mono or coinfected leaves for 24 h. Three aphids were then transferred per test plant for a 72 h inoculation period. A DAS-ELISA assay was performed 3 weeks later to detect infection, and IC-RT-PCR to discriminate between BChV and BMYV. (A) Percentage of BMYV-infected plants when aphids acquire BMYV from mono- (orange), or coinfected (hatched) plants. (B) Percentage of BChV-infected plants when aphids acquire BChV from mono- (blue), or coinfected (hatched) plants. The difference was statistically significant for BMYV transmission, but not for BChV transmission (Pearson's Chi-squared test; BMYV: df = 1, p-value = 0.000398; n = 228, 11 independent experiments; BChV: df = 1, p-value = 0.399; n = 228, 11 independent experiments; Table S4). (C) Percentage of observed and expected coinfected test plants calculated for each individual experiment from the transmission efficiency of each virus from mono-infected plants ([% BMYV * % BChV]/100 = expected % of coinfected plants). The difference between expected and observed was statistically significant (Chi-squared test for given probabilities; df = 1, p-value = 2.436e-08; n = 228, 11 independent experiments; Table S4). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

transmission efficiency of BMYV from coinfected plants did not correlate with a higher BMYV accumulation in coinfected plants as shown in Fig. 1A.

So, although these viruses are internalized at a similar site in the gut of *M. persicae*, and could have competed for cellular uptake in intestinal cells, a reverse phenomenon is observed with a stimulatory effect on BMYV transmission when acquired together with BChV.

In order to investigate the factors contributing to the increased BMYV transmission from coinfected plants, and to discriminate between an indirect plant effect on the virus transmission efficiency and a direct effect of virus co-acquisition by aphids, transmission experiments were conducted using an artificial medium containing similar quantities of each virus as the source. As controls, aphids were fed on an artificial

medium containing each virus alone. BChV transmission efficiency, when acquired alone, was higher compared to BMYV (~20–30 % of plants infected with BChV and ~4 % of plants infected with BMYV; Fig. 5 and Table S5). Interestingly, when the two viruses were acquired simultaneously by aphids from the artificial medium, a statistically significant increase was observed for the transmission efficiency of BChV (~50–55 % of plants infected with BChV or simultaneously with BChV and BMYV) and of BMYV (~16 % of plants infected simultaneously with BChV and BMYV) (Fig. 5 and Table S5). BMYV was never transmitted alone when acquired by the aphids from the medium containing a mixture of both viruses. This experiment also revealed that a single aphid fed on a mixture of purified BMYV and BChV particles can transmit both viruses.

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Aphid transmission efficiency of BMYV and BChV when acquired from an artificial medium containing purified BMYV or BChV or a mixture of both viruses. *M. persicae* acquired virus from an artificial medium containing only purified BMYV (50 ng/µL), only BChV (50 ng/µL) or a mixture of both viruses (50 ng/µL of each virus) for 24 h. One aphid was then transferred per test plant for a 72 h inoculation period. A DAS-ELISA assay was performed 3 weeks later to detect infection, and IC-RT-PCR to discriminate between BChV and BMYV. (A) Percentage of BMYV-infected plants when aphids acquire BMYV only (orange), or a mixture of both viruses (hatched). (B) Percentage of BChV-infected plants when aphids acquire BChV only (blue), or a mixture of both viruses (hatched). The difference was statistically significant for BMYV transmission (Pearson's Chi-squared test; BMYV: df = 1, p-value = 0.001768; n = 164, 2 independent experiments and for BChV transmission (BChV: df = 1, p-value = 0.02467; n = 74, 2 independent experiments; Table S5). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
## 4. Discussion

BMYV and BChV are two closely related poleroviruses infecting sugar beet plants and are strictly transmitted by aphids, mainly *M. persicae*. Since the European ban on neonicotinoids in 2018, previously used in seed coating to control aphid populations, sugar beet poleroviruses-induced disease has re-emerged leading to a serious crisis in the sugar beet industry. The present study was dedicated to investigate the interactions of BMYV and BChV in sugar beet plants, particularly focusing on the impact on virus transmission by aphids. Both are transmitted by *M. persicae* in a persistent and circulative mode implying an active virus transport from the gut lumen to the hemolymph. We identified the posterior midgut cells as the intestinal uptake site for both viruses in *M. persicae*. Although the identification of intestinal receptors of BMYV and BChV has not been achieved, we previously observed that the reduction of ephrin receptor expression in *M. persicae* resulted in a lower capacity of these aphids to transmit BMYV, and the closely related TuYV (Mulot et al., 2018). Therefore, ephrin receptors localized at the apical plasmalemma of posterior midgut cells could be hijacked by BMYV and BChV particles to get access to intestinal cells' cytoplasm. Whether the two closely related sugar beet poleroviruses use the same intestinal, or different, receptors for intestinal uptake cannot be concluded at this time and would need further investigations. Our transmission experiments, using a mixture of similar amount of BMYV and BChV in an artificial medium for aphid feeding, showed no evidence of receptor competition. If such competition existed, a reduced transmission efficiency would have been expected, but this was not observed in our conditions. Similarly, no interference was detected during virus internalization into accessory salivary glands, which would have led to decreased transmission of at least one virus. These findings suggest that the two poleroviruses use different receptors at the posterior midgut and/or the accessory salivary glands, or share common receptors that are sufficiently abundant to avoid any competitive exclusion.

Addressing interactions between the two poleroviruses in coinfected sugar beet plants at the whole plant level did not reveal any synergism nor major antagonism regarding the accumulation of each virus. At the cellular level, BMYV and BChV were observed together in phloem cells which implies no cellular exclusion. No phloem escape of these two phloem-limited viruses was observed in BMYV/BChV coinfected plants, as expected. Indeed, phloem escape of phloem-limited viruses has only been reported in coinfection with a non-phloem limited virus. For example, coinfection of the polerovirus, potato leafroll virus, with a potyvirus infecting all tissues, potato virus A, resulted in phloem release of the polerovirus in *Nicotiana benthamiana* (Savenkov and Valkonen, 2001). Similarly, BMYV became mechanically transmissible to *N. benthamiana* when the inoculum was a mixture of BMYV and pea enation mosaic virus 2 (*Umbravirus*), a feature linked to BMYV phloem restriction alleviation (Mayo et al., 2000). The existence of sugar beet coinfected cells, as we observed here, is a prerequisite for recombination of the coinfecting viruses. Indeed, Kozlowska-Makulska et al. (2015) reported recombination between BMYV and BChV in the coat protein sequence in natural sugar beet infections. Recombination between virus strains or virus species may result in the formation of new, often more virulent strains or even virus species (Aguilera and Pfeiffer, 2019; McDonald et al., 2016; Wang et al., 2019), but whether BMYV-BChV recombinants would display specific biological features was not addressed in the study of Kozlowska-Makulska et al. (2015).

We also addressed BMYV and BChV interactions with regard to their transmission by *M. persicae*. Our results demonstrated that both viruses could be simultaneously acquired and inoculated by a single aphid. Interestingly, when BMYV was acquired from plants coinfected with BChV, its overall transmission efficiency was higher compared to acquisition from plants infected with BMYV alone, though this effect was not consistent across all the independent experiments conducted. In contrast, no such increase in virus transmission efficiency was observed for BChV when the virus was acquired from coinfected plants. This

finding rules out the hypothesis that a modification of the aphid feeding behavior on coinfected plants would account for the higher BMYV acquisition from infected plants, as both viruses are acquired during the same phloem feeding phase. Additionally, since no increase in BMYV accumulation was detected in coinfected plants at the time of virus acquisition, the virus titer cannot explain the enhanced BMYV transmission efficiency. It is conceivable that transencapsidation in coinfected plants could occur and potentially affect virus transmission efficiency. This is particularly conceivable for BMYV which is transmitted at a rate of 31.6% from mono-infected plants and at 47.8% from coinfected plants, a rate close to that of BChV from mono-infected plants (47.4%) (Table S4). Encapsidation of BMYV genome in BChV capsids could be responsible for such results. However, following the same reasoning, we would expect a lower transmission efficiency of BChV from coinfected plants, which was not the case.

Moreover, transencapsidation cannot occur *in vitro* and cannot account for the higher transmission efficiency that we observed when co-acquisition was performed using an artificial medium containing both viruses in equal amounts. Indeed, in this case the stimulatory effect on transmission efficiency was consistently observed for both viruses. This result suggests more a cooperative interaction, either during virus passage through the aphid or at the initiation of infection after coinoculation in the same plant cells. We believe the second hypothesis to be more likely. This cooperative effect could be caused by an additive effect of each virus's capacity to counteract plant defenses such as RNA silencing (Fig. 6). Given that the "stimulatory" effect on the transmission efficiency was only observed for BMYV when it was acquired from plants, we suspect that BMYV benefits more of this cooperation because of its lower ability to counteract plant defense compared to BChV. A weaker ability of BMYV to suppress plant defense could result in a lower number of infected cells and thus a lower ability to trigger plant infection. This would ultimately result in a lower BMYV transmission efficiency when compared to BChV, which was observed in our experimental conditions. In coinfected cells, this deficiency could be partially compensated by BChV infection (Fig. 6).

This hypothesis of cooperation between the two viruses is reinforced by our results showing that both viruses were transmitted together more frequently than expected when acquired from coinfected plants, suggesting a beneficial effect of being inoculated simultaneously. One of the potential candidate responsible for the cooperative effect between BMYV and BChV is the P0 protein, the RNA silencing suppressor of poleroviruses (Pazhouhandeh et al., 2006). BMYV-P0 was shown to display silencing suppressor activity in *N. benthamiana* and BChV-P0 from different French isolates were also recently shown to possess such activity [Véronique Ziegler-Graff, IBMP-CNRS Strasbourg, personal communication] in contrast to previous results (Kozlowska-Makulska, 2010). However, other unrelated events, not relying on the plant defense suppression activity of P0, could also be involved in the cooperative effect of BMYV and BChV, possibly occurring at the aphid-inoculation step.

## 5. Conclusion

We showed that two sugar beet poleroviruses, BChV and BMYV, can co-infect the same plant without any effect on their respective accumulation. Both viruses also coexist within the same cells, discarding cellular exclusion, but suggesting potential molecular interactions at the cellular level. In their aphid vector, *M. persicae*, BChV and BMYV particles were observed in posterior midgut cells indicating a common internalization pathway in intestinal cells. We found no evidence of competition for receptor binding in aphids. In contrast, we observed a significant increase in transmission efficiency when aphids acquired both viruses simultaneously. We suspect that the transmission facilitation occurs at the inoculation step, in the primary inoculated cells. This study highlights how plant coinfection with closely related viruses influences aphid transmission, with potential epidemiological impacts.
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Hypothetical model for explaining the cooperative effect of BMYV and BChV co-inoculation on the success of their coinfection. We hypothesize an additive effect on plant defenses suppression by each of the viruses, BMYV or BChV, in co-inoculated cells. The dampening of plant defenses could be caused by concert action of P0 or other BMYV and BChV proteins. The purple arrows represent the strength of the plant defense suppression by either or both viruses together. Below is represented an hypothetical number of cells that would be infected when BMYV is inoculated alone (on the left), when BChV is inoculated alone (in the middle), or when both viruses are inoculated together (on the right). The crossed cells represent cells inoculated by aphids but where the infection did not develop because of the inability of the virus to counter plant defenses. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

## CRediT authorship contribution statement

**Hélène Schlaefli:** Writing – original draft, Methodology, Investigation. **Aurélie Marmonier:** Writing – original draft, Supervision, Methodology, Investigation. **Souheyla Khechmar:** Writing – original draft, Methodology, Investigation. **Catherine Reinbold:** Writing – original draft, Investigation. **Claire Villeroy:** Resources. **Quentin Chesnais:** Writing – review & editing, Formal analysis. **Martin Drucker:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. **Véronique Brault:** Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

## Funding information

This work was financed by the PNRI project ProViBe awarded to VB and the ANR PRCI project ManiVir (ANR-23-CE20-0049-01) awarded to MD. SK received additional funding from INRAE SPE department. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

Plants were produced by the experimental unit of INRAE Grand Est-Colmar (UEAV).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virol.2025.110594.

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