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Exploring the reassortment ability of the 2009 pandemic H1N1 (A/H1N1pdm09) influenza virus with other circulating human or avian influenza viruses is the main concern related to the generation of more virulent or new variants having implications for public health. After different coinfection experiments in human A549 cells, by using the A/H1N1pdm09 virus plus one of human seasonal influenza viruses of H1N1 and H3N2 subtype or one of H11, H10, H9, H7 and H1 avian influenza viruses, several reassortant viruses were obtained. Among these, the HA of H1N1 was the main segment of human seasonal influenza virus reassorted in the A/H1N1pdm09 virus backbone. Conversely, HA and each of the three polymerase segments, alone or in combination, of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone. Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or H11N6 avian viruses or carried different combination of avian origin polymerase segments, exerted a higher replication effectiveness than that of the parental viruses. These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic avian influenza viruses should not be misjudged in the prediction of the next pandemic.

Recent studies showed that low-pathogenic (LP) avian influenza viruses (AIVs) stuck on wild ducks' feathers during migration can be infectious. In addition, experimental reproduction of AIV infection by allopreening route, showed that viruses stuck on ducks’ feathers could be infectious several weeks after the end of enteric viral shedding. In this work we investigated whether infectious AIVs covering feathers may evade the duck’s immune system. Cloacal swabs, feather swabs and serum samples were collected from 202 wild mallards trapped in Central Italy during the wintering and spring migration (December 2006-March 2007). Both feather and cloacal swabs were screened by reverse transcription (RT)-PCR, specific for virus matrix gene, and then tested by virus isolation (VI) assays. Sera were tested for the presence of antibodies against influenza A virus nucleoprotein by NP-ELISA. Seroprevalence for influenza A virus was 89% (179/202); 51.4% (92/179) and 19.5% (35/179) of these seropositive mallards, tested RT-PCR positive for AIVs on feather and in cloaca respectively. LPAIVs were isolated from 12 feather swabs and from 11 cloacal swabs. NP-ELISA positive sera from 10 ducks found to be H10N7 VI-positive on feathers and VI-negative from cloaca were also investigated by hemagglutination inhibition assay (HI), using as antigen an LPAIV H10N7 strain isolated from feather swabs in this study. HI results showed that one of 10 mallards was immune against homologous H10N7, showing an HI titre of 1:32-64. Our results show evidences suggesting that birds carrying viruses on their feathers might have a post-infection immunity and play an active role in spreading AIVs infection in nature.

Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the presence and the possible health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall 147 cloacal swabs were sampled in the Sardinia Region from June 2009 to September 2011. Samples were obtained from 12 different taxonomic orders including 16 family and 40 species of birds. Based on the endangered host status or on the ecology of the host/virus interaction, samples were divided into three groups of species: endemic and/or endangered (n. 17 species), potential reservoir (n. 21 species) and potential spill-over (n. 2 species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus M-gene amplification. Forty-one serum samples were tested for antibodies against influenza A virus nucleoprotein by NP-ELISA and for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, while two weak serological positive results were detected by NP-ELISA in a Mallard duck (Anas platyrhynchos) and in a Yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study period, suggests a naїve status in these avian populations. A possible occurrence of AIV exposure of susceptible endemic species, might represent a cause of transition from compensatory to additive mortality which can contribute to increase a possible extinction process.

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.