Coinfection with Fowl Adenovirus Serotypes 1 and 4 (FAdV-1 and -4) Enhances FAdV-4 Replication through FAdV-1-mediated Upregulation of HSPA2 Expression

Fowl adenoviruses (FAdVs) are widely distributed in poultry populations around the world, and many diseases are associated with FAdV infection in chickens. This study documented the first characterization of coinfection with fowl adenovirus serotypes 1 and 4 (FAdV-1 and -4) associated with hydropericardium hepatitis syndrome (HHS) in Chinese layer flocks, revealing a novel viral cooperation mechanism. Two novel strains (CH/SX/201805-1 and -4) were identified and isolated, with whole-genome sequencing showing CH/SX/201805-1 clustering with FAdV-1 (99.7% identity to FAdV-A-61/11z), whereas CH/SX/201805-4 displayed characteristic ORF19/27/29 deletions mirroring emergent Chinese FAdV-4 variants. Experimental coinfection in SPF chickens resulted in 87.5% mortality, which was 16.7% greater than that resulting from infection alone, with exacerbated pathology. In vitro coinfection experiments demonstrated concurrent viral replication within same LMH cells, a previously unreported phenomenon, where FAdV-1 increased FAdV-4 replication efficiency 21-fold (P<0.001). Transcriptomic profiling revealed heat shock protein A2 (HSPA2) as the most differentially expressed gene, which was upregulated 2.8-fold during coinfection compared with infection with FAdV-4 alone. Functional validation through HSPA2 knockdown reduced FAdV-4 replication, establishing that FAdV-1 potentiates FAdV-4 through HSPA2-mediated host modulation. These findings provide the first evidence of HSPA2-dependent interserotype synergy in avian adenoviruses and can be used to develop a cellular model for FAdV coinfection studies. These insights redefine the understanding of FAdV pathogenesis and create new avenues for targeted intervention strategies against emerging poultry adenoviral coinfections. Overall design: LMH cells were seeded in 6-well plates and divided into four groups: the FAdV-1 alone group, the FAdV-4 alone group, the coinfection group and the control group. The cells were inoculated with CH/SX/201805-1 (MOI = 1), CH/SX/201805-4 (MOI = 1), or a mixture of both (MOI = 0.5 each). At 12 hpi, total RNA was extracted via TRIzol (TaKaRa, Tokyo, Japan) and sequenced via a NovaSeq 6000 sequencer (Illumina Inc., San Diego, CA) by Beijing Biotechnology Company Ltd. (Beijing, China). The raw reads were processed via fastp (https://github.com/OpenGene/fastp), and the sequences were mapped to the Gallus gallus genome (NCBI GCF_000002315.6) via HISAT2.2.4. DEGs were identified via edge R with a log2 (fold change) = 1 and P < 0.05.