Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

Abstract

H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs.IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health.

Detection and characterization of an H4N6 avian-lineage influenza A virus in pigs in the Midwestern United States

“…Virus was isolated from nasal swabs in 4 out of 5 H4-challenged pigs and duration of shedding varied from 4–7 days post infection. A recent study characterized 32 contemporary H4 viruses that were isolated from live poultry markets in China, and consistent with previous findings, all viruses could replicate in mice without adaptation (Liang et al, 2016). Additionally, they determined from a subset of 10 H4 viruses that 6 efficiently transmitted in guinea pigs, an animal model used to assess the potential risk of IAV to infect and circulate in mammals.…”

“…MO/15 encodes L226 in the HA1, previously shown to be a major determinant of infectivity of primary swine and human respiratory epithelial cells in ONT/99_1 H4N6 (Bateman et al, 2008). MO/15, like ONT/99_1, also contained an asparagine at position 193 in the HA1, which was recently shown to be critical for a Eurasian lineage avian H4N2 strain to bind to α-2,6-linked sialic acids and is a possible molecular marker for predicting α-2,6-linked sialic acids binding for avian H4 viruses (Liang et al, 2016). Both MO/15 and ONT/99_1 contained PB1 gene segments that encode full-length 90 amino acid PB1-F2 proteins, although they vary at position 66.…”

“…Avian H4 influenza viruses are detected frequently in wild waterfowl (Liang et al, 2016; Shelton et al, 2011). Although infrequently detected in mammals in nature, several H4 strains tested in the guinea pig model were able to replicate and transmit to direct contacts and by respiratory droplet without further adaptation (Liang et al, 2016).…”

“…Although infrequently detected in mammals in nature, several H4 strains tested in the guinea pig model were able to replicate and transmit to direct contacts and by respiratory droplet without further adaptation (Liang et al, 2016). Including this report, there are now four documented cases of H4 viruses detected in pigs (Hu et al, 2012; Karasin et al, 2000; Su et al, 2012).…”

“…We then manually subsampled to include different spatial and temporal representative sequences. Eurasian lineage reference strains were selected from a publication examining H4 avian influenza viruses isolated from live poultry markets in China (Liang et al, 2016). Nucleotide sequences were aligned using MAFFT v7.222 (Katoh and Standley, 2013) and then manually corrected in MEGA7 (Kumar, Stecher, and Tamura 2015).…”