Chinese sacbrood virus mediates m6A modification to target and suppress the expression of hemolymph maintenance gene AF9, exacerbating bee infections
Authors: Bai Hua , Ma Yueyu , Qiu Huitong , Qi Yang , Huang Yingshuo , Guo Yaxi , Sun Li , Li Ming , Fei Dongliang , Ma Mingxiao , Liu Yuming 
Published: February 2025

ABSTRACT

The Chinese sacbrood virus (CSBV) severely threatens the beekeeping industry, wherein 3- to 5-day-old larvae in the critical differentiation stage are highly susceptible to low levels of CSBV exposure. Once infected, larvae cannot undergo normal pupation, but the pathogenic mechanism remains unclear. Previous studies have shown that m6A modification plays an important regulatory role in larval development during the critical differentiation stage. However, it is unknown whether CSBV infection affects the pupation of honeybee larvae by altering m6A modification. Here, a novel immunoregulatory factor, AF9, was identified in honeybee larvae through combined methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis following CSBV infection. Enzyme-linked immunosorbent assay (ELISA) quantification of m6A methylation in total RNA and MeRIP-qPCR further revealed that CSBV infection of honeybee larvae inhibits the expression of
AF9
via m6A modification, thereby hindering the host innate immune response and promoting CSBV replication. MeRIP-qPCR was then used to demonstrate that AcMETTL3 targets and modifies
AF9
mRNA, thereby inhibiting
AF9
expression. Homology and functional analysis of human-derived
AF9
(MLLT3) suggested that AF9 exerted a similar effect as MLLT3 on honeybee hemolymph functioning. dsRNA was then fed to silence genes, followed by RNA extraction and expression analysis from hemolymph. Downregulation of
AF9
expression led to decreased numbers of live cells in the hemolymph of honeybee larvae and a reduction in phenoloxidase activity, thereby inhibiting the host immune response. Finally, an
Apis mellifera
pupation infection model was constructed to further explore the antiviral activities associated with
AmAF9. AmAF9
exerted a similarly significant antiviral effect against deformed wing virus (DWV) and acute bee paralysis virus (ABPV) infections in
Apis mellifera
pupae. These results indicate that CSBV infection promotes overall m6A modification in the host and inhibits the expression of
AF9
through AcMETTL3 targeting, leading to host immunosuppression and exacerbating honeybee infection. Similarly,
AF9
is stably expressed in
Apis mellifera
and exhibits the same antiviral effect, making it a broad-spectrum target in honeybee viruses.

IMPORTANCE

The Chinese sacbrood virus (CSBV) poses a serious threat to the health of
Apis cerana
colonies, yet its specific pathogenic mechanism remains unclear. This study shows that infection with CSBV can enhance overall m6A modification levels in
Apis cerana
larvae and suppress the expression of
AF9
by promoting targeting of AcMETTL3, thereby inhibiting the innate immune response and exacerbating CSBV infection. Further analyses indicated that
AF9
functions similarly as the mammalian homologous gene
MLLT3
by maintaining normal functions of hemolymph. Moreover,
AF9
can also significantly inhibit infections by common
Apis mellifera
viruses. In summary, a new mechanism is detailed here by which CSBV escapes the host’s innate immune response by enhancing m6A modification to target and suppress the immune response gene
AF9
. This study also provides new insights into the mechanisms by which bee viruses inhibit host immune responses and suggests that
AF9
may serve as a potential new broad-spectrum antiviral target in bees.