H6 influenza viruses pose a potential threat to human health

Abstract

Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature.

Importance: Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in the United States in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China; however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China and found that 34% of the viruses are able to bind human type receptors and that some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.

FIG 1

Characterization of the receptor-binding properties of H6 influenza viruses. (A) Hemagglutination titers of H6 influenza viruses with 0.5% cRBCs treated as follows: cBRCs, untreated; desialylated (Desial) cRBCs, treated with Vibrio cholerae neuraminidase; α-2,6 cRBCs, treated with VCNA and resialylated with α-2,6-glycans. The dashed line indicates the lower limit of detection. (B) Glycan-binding specificity of H6 viruses. The binding of the viruses to two different glycans (α-2,3-glycans, blue; α-2,6-glycans, red) was tested. The data shown are the means of three repeats; the error bars indicate standard deviations. Significant differences were detected between the affinities for the two glycans.

FIG 2

Phylogenetic analyses of the H6 viruses isolated from 2008 to 2011 in China. The phylogenetic trees were generated with the PHYLIP program of the ClustalX software package (version 1.81). The nine trees were generated based on the following sequences: HA nucleotides (nt) 18 to 1718, N2 nt 20 to 1429, N6 nt 20 to 1432, PB2 nt 28 to 2307, PB1 nt 25 to 2298, PA nt 25 to 2175, NP nt 46 to 1542, M nt 26 to 1007, and NS nt 27 to 864. The phylogenetic trees of HA (A), N2 NA (B), and N6 NA (C) were rooted to A/Turkey/Canada/1963 (H6N2), A/Turkey/England/1969 (H3N2), and A/Duck/England/1/1956 (H11N6), respectively. The phylogenetic trees of PB2 (D), PB1 (E), PA (F), NP (G), M (H), and NS (I) were rooted to A/Equine/Prague/1/56 (H7N7). Sequences of viruses with names in black were downloaded from available databases; viruses with names in other colors were sequenced in this study. The colors of the virus names in the NA, PB2, PB1, PA, NP, M, and NS trees match with those used in the HA tree. Abbreviations: AN, avian; BWT, blue-winged teal; CK, chicken; DK, duck; EC, Eastern China; EW, Eurasian wigeon; FJ, Fujian; GD, Guangdong; GS, goose; GX, Guangxi; HK, Hongkong; HuB, Hubei; HuN, Hunan; MDK, Muscovy duck; ML, mallard; SW, swine; TL, teal; WDK, wild duck; ZJ, Zhejiang.

FIG 3

Transmission of H6 avian influenza viruses in guinea pigs. Groups of three guinea pigs were inoculated i.n. with 10⁶ EID₅₀ of test virus, and 24 h later, three contact guinea pigs were placed in each cage. Nasal washes were collected every 2 days from all animals beginning 2 days p.i. for detection of virus shedding. (A) CK/GD/S1312/10 (H6N2); (B) DK/GD/S4192/08 (H6N2); (C) DK/HuB/S1114/09 (H6N2); (D) GS/GD/S1384/10 (H6N2); (E) CK/GD/S1311/10 (H6N6); (F) CK/HuN/S4495/10 (H6N6); (G) DK/GD/S3073/10 (H6N6); (H) DK/GD/S4251/10 (H6N6); (I) DK/GD/S1155/11 (H6N6); and (J) CK/HuN/S3003/09 (H6N6). Each color bar represents the virus titer from an individual animal. The horizontal dashed lines in these panels indicate the lower limit of detection. Asterisks indicate that transmission efficiency was significantly higher than that of the DK/GD/S4192/08 (H6N2), GS/GD/S1384/10 (H6N2), DK/GD/S3073/10 (H6N6), DK/GD/S1155/11 (H6N6), and CK/HuN/S3003/09 (H6N6) viruses.

FIG 4

Multicycle replication of H6 avian influenza viruses in A549 cells. A549 monolayers were inoculated at an MOI of 0.01 with virus, and the culture supernatants were collected at the indicated time points and then titrated in eggs. a, P < 0.01 compared with titers in CK/HuN/S3003/09 (H6N6)-infected cells; b, P < 0.05 compared with titers in CK/HuN/S3003/09 (H6N6)-infected cells; c, P < 0.01 compared with titers in CK/GD/S1312/10 (H6N2)-infected cells; d, P < 0.05 compared with titers in CK/GD/S1311/10 (H6N6)-infected cells.