Because a large amount of data was generated to compare the antigenic cross-reactivity of 10 groups of sera with 27 viruses by MN, by HI with cRBCs, and by HI with hRBCs (), a visualization method for the titers was needed. Influenza antigenic cartography is analogous to geographic cartography; it projects influenza antigens onto a two-dimensional map on the basis of their titers. The distances between the antigens can then be measured just as geographic distances are measured on a geographic map. Influenza antigenic cartography can thus be used to identify antigenic variants and is useful for influenza vaccine strain selection. Maps representing our HI cRBCs, MN, and HI hRBCs data are shown in , , and , respectively.
Arithmetic mean normalized HI and MN titers
In the MN assay (), all clade 1 viruses grouped together with the exception of 04-VNM and 03-HKG. In clade 2.2, 05-MNG was the only outlier. Interestingly, clade 2.3.2 and 2.3.4 strains remained within 2 log2 of one another, although 08-HKG (clade 2.3.4) was somewhat distant from its counterparts. Ancestral viruses A, B, C, and D were distant from all isolates except the clade 1 03-HKG, (1 to 2 log2 away). When all 27 viruses were considered, ancestral A strain was positioned centrally and therefore was putatively the best candidate diagnostic reagent for detection of anti-H5N1 antibodies by MN. However, when clear outliers (04-VNM, 03-HKG, and the ancestral viruses), were excluded, a 2.2 strain (e.g., 05-SAU, 06-NGA, 07-EGY, or 08-EGY) appeared to be a better candidate diagnostic reagent.
Cartography of HI with cRBCs () showed a pattern very similar to that of MN cartography. The same outlier strains were detected in clades 1 and 2.2. In clade 2.3.4, in contrast, 08-HKG appeared near its counterparts while 06-HKG was 1 log2 away. Ancestral strains A, B, and D were not as tightly clustered in the cartograph of HI with cRBCs () than in the cartography of MN (). Ancestral strain A and 06-HKG were located in the center of the overall map, but 2.2 strains again appeared to be better candidate diagnostic reagents for most strains when the outliers (the clade 0 97-HKG, clade 1 03-HKG and 04-VNM, and clade 4 06-CHN viruses) were excluded.
Cartography of HI with hRBCs was much less dispersed; all points on the map fit within eight squares (<3 to 4 log2 apart, ). Differences between strains were therefore less distinct. 07-KHM and 04-VNM (clade 1) were very closely related. Clade 2.2 strains were within 2 log2 of each other, and 05-MNG was no longer a clade outlier. In clade 2.3.4, the Lao strains still clustered closely, while strains from Hong Kong were distant from the Lao viruses and even more distant from each other. A central strain was harder to identify in , but clade 2.2 strains still remain in the middle of the map. The smaller antigenic distances may suggest broader cross-reactivity in HI assays with hRBCs.
Interclade cross-reactivity and subtype specificity.
Clades 0, 1, 2, and 4 were not spatially distinct in any of the three antigenic cartographs. For example, the clade 2 viruses were not closer to one another than to clade 1 or 4 strains. Interestingly, 04-VNM (clade 1), 02-CHN (clade 4), 05-MNG (clade 2.2), and 06-CHN (clade 2.1) clustered together in the MN assay (<1 log2 apart, ). The four ancestral strains and 03-HKG (clade 1) were consistently separate from the other tested viruses in the HI cRBCs and MN assays ( and ).
In HI assays with cRBCs and with hRBCs, the ancestral viruses (A to D), 04-VNM, and 02-CHN did not cross-react with any non-H5 antisera (HI titers < 10) except H13 in the HI-hRBC assay (HI titers 10 to 40; homologous HI titers, 200 to 720 [data not shown]).