Negative regulation of reassortant canine influenza virus replication and key site identification in porcine and ferret bronchial epithelial cell lines

The segmented nature and high mutability of the influenza virus RNA genome facilitate rapid mutation and reassortment, allowing the virus to breach host barriers and migrate between different species, potentially leading to unpredictable influenza outbreaks. With dogs emerging as new natural hosts for influenza virus, vigilant surveillance and scientific prevention strategies are imperative. Here, based on our previous isolation of 21 strains, which are reassortments of the canine influenza virus (CIV) H3N2 (KR/07) with gene segments from the influenza A pandemic (H1N1) 2009 virus strain (CA/09), the replication kinetics of these reassortants in immortalized mammalian respiratory epithelial cell lines from swine and ferret named hTERT-PBECs and hTERT-FBECs, alongside induced changes in cytokine expression, were investigated. Reverse genetics was utilized to generate the reassortment H3N2 canine influenza rKR/07-PB2/NP, which contains the PB2 and NP segments from CA/09. The viral titer of rKR/07-PB2/NP was significantly lower than those of the parental viruses KR/07 and CA/09. In addition, rKR/07-PB2/NP notably decreased expression levels of interleukin-1β (IL-1β) and interleukin-10 (IL-10) in both immortal cells, particularly in hTERT-PBECs. Our findings not only contribute to the understanding and exploring cross-species transmission mechanisms of influenza virus, but also provide new ideas for prevention and treatment of CIV.

流感病毒RNA基因组的分节特性与高变异性，使其能够快速发生突变与重配，进而突破宿主屏障并在不同物种间迁移，最终可能引发难以预测的流感暴发疫情。随着犬类成为流感病毒新的自然宿主，开展严密的病毒监测与科学的防控策略已迫在眉睫。本研究基于此前分离得到的21株重配病毒——这些病毒由犬流感病毒（canine influenza virus, CIV）H3N2（KR/07株）与2009年甲型H1N1流感大流行病毒株（CA/09）的基因片段重配而来——对这些重配病毒在猪源和雪貂源永生化哺乳动物呼吸道上皮细胞系hTERT-PBECs与hTERT-FBECs中的复制动力学，以及其诱导的细胞因子表达变化进行了研究。本研究利用反向遗传学技术构建了重配H3N2犬流感病毒rKR/07-PB2/NP，该病毒包含来自CA/09的PB2与NP基因片段。rKR/07-PB2/NP的病毒滴度显著低于亲本病毒KR/07与CA/09。此外，rKR/07-PB2/NP可显著下调两种永生化细胞中白细胞介素-1β（interleukin-1β, IL-1β）与白细胞介素-10（interleukin-10, IL-10）的表达水平，且在hTERT-PBECs中的下调效果尤为显著。本研究结果不仅有助于加深对流感病毒跨物种传播机制的理解与探索，同时也为犬流感病毒的防控与治疗提供了新思路。