The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route

Abstract

Influenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine.

Importance: Neuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks.

FIG 1

NA activities of Dk78/Eng62N2 and variant viruses after low-pH treatment. Dk78/Eng62N2, Dk-rec1, and Dk-rec6 were exposed to citric acid buffer from pH 6 to 3 for 1 h. After the solution containing the viruses was neutralized by sodium hydroxide to pH 7, an NA assay was performed according to the method established by Aymard-Henry et al. (16). The results are shown as the percentage of NA activity relative to the NA activity of each virus after treatment at pH 6. The values are the means of three independent experiments. The error bars indicate standard deviations. An asterisk indicates a statistically significant increase compared to the activity of Dk78/Eng62N2 (P < 0.05).

FIG 2

NA activities of mutant NA proteins expressed in 293 cells after treatment at pH 3. Plasmids expressing each NA protein were transfected into 293 cells and at 48 h posttransfection, the cells were exposed to citric buffer at pH 6 or pH 3 for 10 min. After the cells were washed, the NA assay was performed according to the method established by Aymard-Henry et al. (16). The NA activity after the treatment at pH 3 is shown relative to that after treatment at pH 6. The values are the means of three independent experiments. The error bars indicate standard deviations. An asterisk indicates a statistically significant increase compared to the activity of Dk78/Eng62N2 NA (P < 0.05).

FIG 3

NA activities of NA proteins expressed in 293 cells. pCAGGS plasmids encoding NA genes were transfected into 293 cells. At 48 h posttransfection, cells expressing NA proteins were collected and the NA activity was measured by use of the method established by Aymard-Henry et al. (16). The NA activity is shown relative to that of Dk78/Eng62N2 NA. The values are the means of three independent experiments. The error bars indicate standard deviations.

FIG 4

Locations of amino acid differences between NA protein from Dk78/Eng62N2 and Dk-rec6. The positions of amino acid residues 165 (blue), 356 (green), and 431 (red), as well as the enzymatic active site indicated by the position of the bound sialic acid (cyan), are shown on the NA structure of A/Tokyo/3/67 (H2N2) (19).