Characterization of H7N9 influenza A viruses isolated from humans

Abstract

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Figure 1. Pathological findings in infected macaques

Shown are pathological findings in the trachea (a–d, m–p), bronchus (e–h, q–t), and lungs (i–l, u–x) of macaques infected with Anhui/1 (a–l) or Dk/GM466 (m–x) at 3 dpi (a, b, e, f, i, j, m, n, q, r, u, v) or 6 dpi (c, d, g, h, k, l, o, p, s, t, w, x) with HE staining (a, c, e, g, i, k, m, o, q, s, u, w) or immunohistochemistry for influenza viral antigen (b, d, f, h, j, l, n, p, r, t, v, x). HE staining of the lung of an uninfected macaque is shown (y). Original magnification: x 400 (a–h, m–t), x 200 (i–l, u–y).

Figure 2. Respiratory droplet transmission in ferrets

Ferrets were infected with 5 ×10⁵ PFU of Anhui/1, Dk/GM466, or CA04 (inoculated ferrets). One day later, three naïve ferrets (contact ferrets) were each placed in a cage adjacent to an infected ferret. Nasal washes were collected from infected ferrets on day 1 after inoculation and from contact ferrets on day 1 after co-housing, and then every other day (up to 9 days) for virus titration.

Figure 3. Glycan microarray analysis and HA structural analysis

(a) Localization of amino acid changes in a virus from a ferret infected via respiratory droplets. Shown is the three-dimensional structure of A/Netherlands/219/2003 (H7N7) HA (PDB ID: 4DJ6) in complex with human receptor analogues. (b) Close-up view of the globular head. Mutations that increase affinity to human-type receptors are shown in cyan. Mutations that emerged in Anhui/1 HA during replication and/or transmission in ferrets are shown in red (T71I), green (A135T), and blue (R131K). The human receptor analogue [derived from its complex with H9 HA (PDB ID: 1JSI); shown in orange] is docked into the structure. Images were created with MacPymol [http://www.pymol.org/]. (c–g) Receptor specificities of recombinant viruses possessing A(H7N9) HAs (Anhui/1, Shanghai/1, Hangzhou/1) were compared with representative avian (Vietnam/1203) and human (Kawasaki/173) isolates in a glycan microarray containing α2–3 and α2–6 sialosides. Error bars represent standard deviations calculated from 6 replicate spots of each glycan. A complete list of glycans is found in Table S9.

Figure 4. Virus sensitivity to antivirals in mice

Mice were intranasally inoculated with 10³ or 10⁴ PFU (50 μl) of Anhui/1 (a, d), CA04 (b, e), or recombinant Anhui/1 virus possessing Shanghai/1-NA-292K (c, f). At 2 h after infection, mice were treated with oseltamivir phosphate, zanamivir, laninamivir octanoate, favipiravir, PBS, or distilled water. Body weights were monitored daily (a–c). Three mice per group were euthanized at 3 and 6 dpi, and the virus titres in lungs were determined by plaque assays in MDCK cells (d–f). Statistically significant differences between virus titres of control mice and those of mice treated with antiviral drugs were determined by using Welch’s t-test or Student’s t-test on the result of the F-test. The resulting p-values were corrected by using Holm’s method (asterisk, p<0.05). Mean titres, or individual titres when virus was not recovered from all three animals (double-asterisk), are shown.