Effects of bacterial microflora of the lower digestive tract of free-range waterfowl on influenza virus activation

Abstract

Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a "trypsin-like" manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established.

Fig. 1.

SDS-PAGE analysis of polypeptide fragment patterns obtained following incubation of HA0 with supernatant material from protease-secreting bacterial isolates. [³⁵S]HA0 was incubated with bacterial supernatant material for 60 min, subjected to SDS-PAGE analysis, and autoradiographed as described in Materials and Methods. Lanes: 1, PBS negative control; 2, trypsin, 10 μg/ml; 3, Streptococcus hyointestinalis (isolate 95-11); 4, Aerococcus viridans (isolate 135-8); 5, Lysinibacillus sphaericus (isolate 135-12); 6, Bacillus amyloliquefaciens (isolate 135-4); 7, Kocuria kristinae (isolate 107-14); 8, Bacillus pumilus (isolate 136-9); 9, Enterobacter cloacae (isolate 99-3); 10, Cellulosimicrobium sp. (isolate 111-15); 11, Aeromonas sobria (isolate 124-1); 12, Aeromonas hydrophila (isolate 119-3). The numbered arrows indicate the established molecular masses for HA0 and trypsin hydrolysis products HA1 and HA2 (80, 58, and 26 kDa, respectively) (24).

Fig. 2.

Infectivity of influenza A/Indonesia/5/2005(H5N1) virus following incubation with concentrated supernatants from duck cloacal bacterial isolates. Uncleaved influenza A/Indonesia/5/2005 (H5N1) virus was incubated with respective bacterial supernatants for 60 min followed by layering onto MDCK monolayers for double-layer plaque assay analysis as described in Materials and Methods. Lanes: 1, trypsin, 10 μg/ml; 2, Lysinibacillus sphaericus (isolate 135-12); 3, Streptococcus hyointestinalis (isolate 95-11); 4, Aerococcus viridans (isolate 135-8); 5, Bacillus amyloliquefaciens (isolate 135-4); 6, Kocuria kristinae (isolate 107-14); 7, Enterobacter cloacae (isolate 99-3); 8, Aeromonas hydrophila (isolate 119-3); 9, Bacillus pumilus (isolate 136-9); 10, Cellulosimicrobium sp. (isolate 111-15); 11, Aeromonas sobria (isolate 124-1). The dotted line represents the PBS control (3.4 × 10³ PFU/ml).

Fig. 3.

Infectivity of trypsin-activated influenza A/Indonesia/5/2005(H5N1) virus following incubation with supernatant material from two lipase-secreting cloacal bacteria isolates. Trypsin-treated influenza A/Indonesia/5/2005 (H5N1) virus was incubated with bacterial supernatants for 60 min, followed by layering onto MDCK monolayers for double-layer plaque assay analysis as described in Materials and Methods. Lanes: 1, trypsin-only control (10 μg/ml); 2, supernatant material from Aeromonas hydrophila (isolate 119-3); 3, phospholipase C-only control (100 μg/ml); 4, supernatant material from Aeromonas sobria (isolate 124-1).

Fig. 4.

SDS-PAGE analysis of polypeptide fragment patterns obtained following incubation of trypsin-activated [³⁵S]HA0 with supernatant material from two lipase-secreting cloacal bacterial isolates. Preparation of radiolabeled HA0, HA0 cleavage with trypsin, SDS-PAGE analysis, and autoradiography were carried out as previously described in Materials and Methods. Lanes: 1, [³⁵S]HA0, untreated; 2, trypsin-only control (10 μg/ml); 3, phospholipase C-only control (100 μg/ml); 4, supernatant material from Aeromonas sobria (isolate 124-1); 5, supernatant material from Aeromonas hydrophila (isolate 119-3). The superscripted numbered arrows indicate the established molecular masses for HA0 and trypsin hydrolysis products HA1 and HA2 (80, 58, and 26 kDa, respectively) (24).