Avian influenza virus infection risk in humans with chronic diseases

Abstract

Saliva proteins may protect older people from influenza, however, it is often noted that hospitalizations and deaths after an influenza infection mainly occur in the elderly population living with chronic diseases, such as diabetes and cancer. Our objective was to investigate the expression level of the terminal α2-3- and α2-6-linked sialic acids in human saliva from type 2 diabetes mellitus (T2DM), liver disease and gastric cancer (GC) patients and assess the binding activity of these linked sialic acids against influenza A viruses (IAV). We observed that the expression level of the terminal α2-3-linked sialic acids of elderly individuals with T2DM and liver disease were down-regulated significantly, and the terminal α2-6 linked sialic acids were up-regulated slightly or had no significant alteration. However, in the saliva of patients with GC, neither sialic acid was significantly altered. These findings may reveal that elderly individuals with chronic diseases, such as diabetes and liver disease, might be more susceptible to the avian influenza virus due to the decreased expression of terminal α2-3-linked sialic acids in their saliva.

Figure 1. Relative expression levels of the terminal Siaα2-6Gal recognized by SNA and Siaα2-3Gal recognized by MAL-II in human saliva using lectin microarrays.

MH: male healthy groups, FH: female healthy groups, MD: male T2DM patients, FD: female T2DM patients, HB: hepatitis B, HC: hepatic cirrhosis, HCC: hepatocellular carcinoma, MG: male GC patients, FG: female GC patients. (A) Fluorescent images from a portion of SNA and MAL-II with three replicates. (B) Normalized fluorescent intensity of SNA and MAL-II from each clinical group compared with healthy groups based on the values of the ratio in pairs (*P < 0.05, **P < 0.01, and ***P ≤ 0.001).

Figure 2. Validation of the expression levels of the terminal α2-3/6-linked sialic acids in individual salivary samples.

(A) The layout of the salivary microarrays. A total of 210 individual salivary samples from male healthy group (MH, n = 30), female healthy group (FH, n = 30), male T2DM patients (MD, n = 30), female T2DM patients (FD, n = 30), as well as patients with HB (n = 30), HC (n = 30) and HCC (n = 30). Each sample was spotted in triplicate per block with two continuous blocks on one slide. (B) Scan images of Cy3-labeled SNA and MAL-II binding to the salivary microarrays. (C) Box plot analysis of the original data achieved from the salivary microarrays. Error bars represent 95% confidence intervals for means. Statistical significance of differences between groups was indicated by the p-value.

Figure 3. Validation of the terminal Siaα2-6Gal recognized by SNA and Siaα2-3Gal recognized by MAL-II in all salivary groups and a comparison of the binding pattern profiles of all salivary groups against three IAV strains A/Chicken/Guangxi/4/2009(H5N1), (A/Duck/Guangdong/17/2008(H5N1), A/Chicken/Fujian/S-1-521/2008(H9N2)) and the H1N1 influenza A vaccine.

The summed fluorescence intensities (SFIs) of the bands marked by red lines at approximately 25 kDa for all salivary groups from lane 1 to lane 4 were read by ImageQuant Tools. (A) Lane 1: male healthy group (MH); lane 2: female healthy group (FH); lane 3: male T2DM patients (MD); lane 4: female T2DM patients (FD). (B) The summed fluorescence intensities for SNA and MAL-II strains binding to samples from the T2DM salivary group. (C) The SFIs of the bands marked by red lines at approximately 25 kDa for three IAV strains and the H1N1 vaccine strains binding to the salivary protein samples from the T2DM group. (D) Lane 1: MH; lane 2: hepatitis B (HB); lane 3: hepatic cirrhosis (HC); lane 4: hepatocellular carcinoma (HCC). (E) The SFIs of the bands marked by red lines at approximately 25 kDa for SNA and MAL-II strains binding to the salivary protein samples from the group with liver disease. (F) The SFIs of the bands marked by red lines at approximately 25 kDa for three IAV strains and the H1N1 vaccine strains binding to the salivary protein samples from the group with liver disease. (G) Lane 1: MH; lane 2: FH; lane 3: male GC patients (MG); lane 4: female GC patients (FG). (H) The summed fluorescence intensities for SNA and MAL-II strains binding to the salivary protein samples from the group with GC. (I) The SFIs of the bands marked by red lines at approximately 25 kDa for three IAV strains and the H1N1 vaccine strains binding to the salivary protein samples from the group with GC.

Figure 4. Comparison of the binding pattern profiles of eight IAV strains and the H1N1 influenza A vaccine against the binding patters of salivary groups with T2DM.

MH: male healthy group, MD: male T2DM patients, FH: female healthy group, FD: female T2DM patients; The summed fluorescence intensities of the blotting analyses for IVA; lane 1 to lane 9 were read by ImageQuant Tools. (A) Lane 1: A/Chicken/Guangxi/4/2009(H5N1); Lane 2: A/Duck/Guangdong/17/2008(H5N1); Lane 3: A/Chicken/Fujian/S-1-521/2008(H9N2); Lane 4: H1N1 influenza A vaccine; Lane 5: A/Ostrich/Denmark/96-72420/1996(H5N2); Lane 6: A/Mallard/Jiangxi/16/2005(H5N2); Lane 7: A/Chicken/Hebei/1/2002(H7N2); Lane 8: A/Fowl/Rostock/45/1934(H7N1); Lane 9: A/Duck/Guangdong/S-7-134/2004 (H9N2). (B) The summed fluorescence intensities for the salivary samples from the T2DM group that bound to 8 IAV strains and the H1N1 vaccine.