A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-β4 (TGF-β4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.In the waterfowl reservoir, influenza A viruses are enzootic and infections are usually asymptomatic. The viruses replicate preferentially in the intestinal tract and are transmitted by the fecal-oral route. Phylogenetic analyses of amino acid changes show that influenza viruses in wild aquatic birds have low evolution rates, suggesting that they are in evolutionary stasis. Upon transmission to domestic poultry, rapid evolution occurs (63, 79). The replication of influenza viruses of duck origin in chickens is generally limited to the respiratory and gastrointestinal tracts and causes mild or no symptoms. However, sustained replication of viruses of the H5 or H7 subtype may lead to the emergence of highly pathogenic influenza viruses, which cause devastating epizootics (for a review, see reference 5). Mutations acquired upon replication in domestic poultry might also increase the potential for adaptation of avian influenza viruses to other species, including humans (20, 21, 50), which raises public health concerns.The viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are major determinants in the interspecies transmission and adaptation of influenza A viruses to a new host (for a review, see reference 46). The HA binds to the sialic acids (SA) linked to cellular membrane glycoproteins or glycolipids, whereas the sialidase activity of the NA facilitates the release and diffusion of progeny virions. SA are usually attached to a galactose moiety via an α2,3 or α2,6 glycosidic linkage. Avian viruses bind preferentially to SA-α2,3-galactose, whereas human viruses bind preferentially to SA-α2,6-galactose (13, 37, 56). This receptor binding specificity correlates with the relative predominance of SA-α2,3-galactose and SA-α2,6-galactose at the sites of viral multiplication in ducks and in humans, respectively (14, 28), and involves specific residues in the receptor binding site of the HA (37, 70). Viruses isolated from terrestrial poultry bind preferentially to SA-α2,3-galactose, but they differ from duck viruses by an enhanced binding to receptors in which the penultimate saccharide is sulfated and/or fucosylated (22, 23). Changes in the receptor binding site or at potential glycosylation sites of the HA that could modulate the binding of the HA to the receptors have been associated with the adaptation of duck viruses to poultry (3, 21, 36). A deletion of about 20 amino acids in the stalk region of the NA has also been frequently detected concomitant with the adaptation of duck viruses to poultry (3, 4, 25, 36, 62). In particular, it is a feature of the highly pathogenic H5N1 viruses that have become endemic in poultry in southern Asia since 2003 and have been causing sporadic human cases (26). The stalk is a flexible region which separates the globular, enzymatically active head of the tetrameric NA from the hydrophobic transmembrane domain (Fig. ​(Fig.1A).1A). The biological significance of the selection of variants with a shorter NA stalk is still unclear. Shortening of the stalk was found to decrease the ability of the NA to release the virus from cells (2, 6, 17, 25, 36, 39), and it was suggested that such a decreased activity of the NA could counterbalance a reduced binding of the HA to sialic acids expressed in poultry (2, 40, 71). The NA stalk length was found to have little effect on the efficiency of replication of a highly pathogenic H5N1 virus in poultry (39). However, it is possible that the presence of a multibasic site on the H5, a strong determinant of virulence, might be masking the effect, if any, of a shortened NA stalk.Open in a separate windowFIG. 1.Wild-type and deleted variants of the MZ-NA protein. (A) Schematic representation of the wild-type MZ-NA protein (470 amino acids aa]). The domain corresponding to the stalk (amino acids 36 to 90) is represented in gray. The region of the stalk which is deleted in the MZ-delNA variant (amino acids 54 to 72) is represented by a hatched box. TM, transmembrane domain. (B) Amino acid sequence alignment of the NAs from MZ (H1N1), MZ-delNA (H1N1), A/Goose/Guandong/1/96 (H5N1), and A/Hong Kong/156/97 (H5N1) viruses. The domain corresponding to the stalk is highlighted in gray. The residues that are deleted in the NAs of MZ-delNA and A/Hong Kong/156/97 are represented by dashes.In the present study, we used a low-pathogenic duck influenza A(H1N1) virus isolate to investigate possible mechanisms involved in the selection of influenza viruses with a deletion in the stalk of the NA upon transmission from the waterfowl reservoir to chickens. Reverse genetics was used to produce a wild-type (wt) virus and a variant with a 19-amino-acid deletion in the stalk of the NA. The rationale for using an H1N1 virus was that a slight increase in viral fitness of the deleted variant, if any, would be more readily detected if the wild-type virus showed a low replication potential in chickens. In vitro and in vivo approaches were developed to examine the effect of the deletion in the stalk of the NA on the biological properties of the enzyme, on viral infectivity in avian cultured cells, and on viral infectivity and pathogenicity in chickens.