Novel preening-mediated transmission route of avian influenza viruses in aquatic birds

Wild aquatic birds are reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic ones. High viral loads in feces of infected birds allow fecal-oral transmission. However, this route does not fully account for the efficiency of avian influenza virus (AIV) spread since dilution of infectious feces in water progressively decreases the chances of virus/host interaction. We investigated whether preen oil gland secretion, by which all aquatic birds make their feathers waterproof, could support a natural concentration mechanism of AIVs from water to birds’ bodies, thus favouring virus spread and persistence in the aquatic environment. First, we detected consistently both viral genome and infectious AIVs on swabs taken by rubbing preened feathers of 345 wild mallards and examined by reverse transcription (RT)-PCR and virus isolation (VI) assays. Second, in two laboratory experiments using a quantitative real-time (qR) RT-PCR, we demonstrated that feather samples (n=5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces, as shown by statistical analysis. Third, we experimentally coated 7 mallards with a preen oil-AIV mix, and housed them with a control, uncoated, duck. Through self- and/or allopreening behaviour, all birds ingested the virus, as shown by virus detection in both oro-pharyngeal and cloacal samples. Virus isolation from cloacal swabs and virus-specific antibody response confirmed the occurrence of mallards’ infection. Infectious AIVs were isolated from ducks’ body surface until 32 days after the experimental coating.Our field and experimental results indicate that AIVs can be naturally concentrated and carried on the feather surface of infected ducks (i.e., those VI-positive from both cloacal and feathers swabs) and uninfected ones (i.e., those VI-positive from feathers only). In such a context, the natural preening behaviour, by which waterbirds spread preen oil all over their plumage (self-preening) or other birds’ plumage (allo-preening), could facilitate the ingesion of AIV particles stuck on birds’ feathers, thus promoting a preening-mediated infection route. Our findings also suggest that during the time period between the virus adhesion to the bird’s body and the infection (possibly due to self- and/or allopreening), the virus could move in nature with the host by an undescribed circulation mechanism. We demonstrate here a novel viral transmission route that adds to, and possibly contributes to explain the knowledge of longdistance movements and long-term infectivity of lowly and highly pathogenic AIVs in nature.