Time dependent proinflammatory responses shape virus interference during coinfections of influenza A virus and influenza D virus

Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. Results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STEC) with IAV 24- or 48-hours prior to IDV inoculation, and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 hours. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 hours between the two viral infections, regardless of their order. The interference pattern at 24- and 48-hours correlated with proinflammatory responses induced by the first infection, which was about 24-hours slower for IDV than IAV. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response and is therefore dependent on the time between infection, and the order of infection. ImportanceBoth IAV and IDV are enzootic in pigs, and feral pigs have a higher risk for both IAV and IDV exposures than IDV exposure alone. This study suggests that in coinfection with IAV and IDV either virus can interfere with the replication of the other virus by stimulating proinflammatory responses; however, the proinflammatory response was 24 hours slower for IDV than IAV. In vitro there was no interference during simultaneous coinfection, regardless of infection order. Coinfection of IDV and IAV in pigs did not show enhanced pathogenesis, compared with those infected only with IAV. This study can facilitate our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.