A Novel Reassortant Avian H7N6 Influenza Virus Is Transmissible in Guinea Pigs via Respiratory Droplets

Abstract

Since 2013, H7N9 and H5N6 avian influenza viruses (AIVs) have caused sporadic human infections and deaths and continued to circulate in the poultry industry. Since 2014, H7N6 viruses which might be reassortants of H7N9 and H5N6 viruses, have been isolated in China. However, the biological properties of H7N6 viruses are unknown. Here, we characterize the receptor binding preference, pathogenicity and transmissibility of a H7N6 virus A/chicken/Hubei/00095/2017(H7N6) (abbreviated HB95), and a closely related H7N9 virus, A/chicken/Hubei/00093/2017(H7N9) (abbreviated HB93), which were isolated from poultry in Hubei Province, China, in 2017. Phylogenetic analyses demonstrated that the hemagglutinin (HA) gene of HB95 is closely related to those of HB93 and human-origin H7N9 viruses, and that the neuraminidase (NA) gene of HB95 shared the highest nucleotide similarity with those of H5N6 viruses. HB95 and HB93 had binding affinity for human-like α2, 6-linked sialic acid receptors and were virulent in mice without prior adaptation. In addition, in guinea pig model, HB93 was transmissible by direct contact, but HB95 was transmissible via respiratory droplets. These results revealed the potential threat to public health posed by H7N6 influenza viruses and emphasized the need for continued surveillance of the circulation of this subtype in poultry.

Adaptive amino acid substitutions enable transmission of an H9N2 avian influenza virus in guinea pigs

“…Human receptor-binding specificity is an important factor for cross-species transmission of AIVs 41,42 . We thus measured the receptor binding specificity of the two viruses as previously described 36 . Briefly, the receptor binding affinity was determined by evaluating the ability of WT and MA to agglutinate four types of cRBCs.…”

“…The ethics statement was described in our previous work 36 . Briefly, all animal studies were conducted in strict accordance with the Guidelines of Animal Welfare of World Organization for Animal Health and the protocols approved by the Hubei Provincial Animal Care and Use Committee (approval number SYXK 2016-0004).…”

“…The viral gene sequences were acquired as described in our previous work 36 . In brief, viral RNA was extracted from allantoic fluid using TRIzol reagent (Invitrogen) and reverse transcribed into cDNAs using the primer Uni12 (5′-AGC RAA AGC AGG-3′) primers, an RT reagent kit and viral genes were amplified using a PCR kit (Takara, Japan) according to the manufacturer’s protocol.…”

“…The receptor-binding specificities of the WT and MA viruses were determined by HA assays with 1% chicken red blood cells (cRBCs) as described in our previous work 36 . For the HA assay, sialic acid residues were enzymatically removed from cRBCs by incubating the cells with 50 mU of Vibrio cholerae neuraminidase (VCNA, Roche, San Francisco, CA) at 37 °C for 1 h, followed by resialylation using either α2-,6-N-sialyltransferase or α2-,3-N-sialyltransferase (Sigma-Aldrich, St. Louis, MO) at 37 °C for 4 h. The sample was then washed two times with phosphate-buffered saline (PBS), centrifuged at 1500 rpm for 5 min each time, adjusted to a final working concentration (1%) with PBS, and stored at 4 °C.…”

“…Guinea pig experiments were performed as described in our previous work 36 . Hartley strain female guinea pigs weighing 300 to 350 g (Merial Vital Laboratory Animal Technology Company, Beijing, China), confirmed to be seronegative for influenza viruses prior to the experiment, were used in these studies.…”