ABSTRACT Respiratory influenza D virus (IDV), first identified in 2011, has since been detected globally in bovine populations. Considering the high thermal and acidic stability of IDV, coupled with the observations showing its replication in the intestines of mice and fecal shedding in goats, we investigated the enteric tropism, gastrointestinal (GI) replication, and fecal-oral transmission of IDV in guinea pigs. Oral inoculation of IDV in guinea pigs revealed measurable viral replication in the intestines of infected animals, with fecal shedding detected at 3, 5, and 7 days post-inoculation. These findings were further corroborated by RNAscope-based in situ hybridization, virus isolation, and deep sequencing. Sequence analysis identified three mutations present in the hemagglutinin-esterase fusion (HEF) protein of IDV isolated from the small intestines of infected animals. These HEF mutations (A203T, G210R, and T251A) may play a role in facilitating IDV adaptation to the enteric tract of guinea pigs for replication fitness. In contrast, a low level of IDV replication was observed in the intestines of sentinel animals of both direct-contact and fecal exposure groups. Furthermore, only one of the three animals seroconverted in the direct oral inoculation group, with no seroconversion detected in both oral contact and fecal-contact sentinel groups. Taken together, the results of our study demonstrate that IDV can replicate in the intestinal tract but has a limited ability in fecal-oral transmission in guinea pigs. These findings further support a theme that bovine influenza D virus infects and transmits among animals through the respiratory tract. IMPORTANCE This study investigates whether bovine influenza D virus (IDV) can replicate in the gastrointestinal tract and how it may contribute to disseminating IDV through an oral-fecal transmission route. By experimentally infecting guinea pigs, we observed that IDV can replicate in the intestinal tract of infected animals following direct oral exposure. Furthermore, we showed that IDV sheds into the feces of some guinea pigs, with the seroconversion detected. Virus isolation from the infected intestines, coupled with next-generation sequencing, revealed three mutations occurring in the hemagglutinin-esterase fusion protein of IDV, which may relate to viral adaptation to the GI tract for better replication. However, low levels of IDV replication were detected in the intestines of sentinel animals from both direct-contact and fecal exposure groups, with none of the seroconversions found in both transmission groups. These findings suggest that bovine IDV has limited ability for fecal-oral transmission in this animal model.
Naveed et al. (Tue,) studied this question.