Highly pathogenic avian influenza H7N9 viruses with reduced susceptibility to neuraminidase inhibitors showed comparable replication capacity to their sensitive counterparts

Since the fifth epidemic wave, a highly pathogenic avian influenza (HPAI) H7N9 virus has emerged and caused human infections in China. HPAI H7N9 viruses isolated from human cases collected from Dec. 1, 2016, to Apr. 27, 2019, were obtained from the Chinese National Influenza Surveillance Network, which covers 32 provinces in mainland China (including the autonomous regions/municipalities directly under the Central Government), including 408 network laboratories and 554 sentinel hospitals. Virus isolation was conducted in a biosafety level 3 facility by inoculating 0.2-ml original samples allantoically into 9~11-day-old specific-pathogen-free (SPF) embryonated chicken eggs. After sequencing, HPAI H7N9 sequences were analyzed to identify amino acid substitutions related to potentially reduced susceptibility to NAIs. The criteria for screening potential drug resistance sites of HPAI H7N9 virus were as follows: 1) NAI resistance sites in other subtypes of influenza virus that have been reported previously and 2) 19 highly conserved residues in the NA active site of all influenza A, which may be related to resistance.

Madin-Darby canine kidney (MDCK) cells and human embryonic kidney (293 T) cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), HEPES (10 mM, Invitrogen), penicillin (100 units/ml), and streptomycin (100 μg/ml, Invitrogen).

A virus containing amino acid substitutions in sites related to potentially reduced susceptibility to NAIs in HPAI H7N9 viruses, including R292 K, E119V, A246T and H274Y, was constructed in this study. An HPAI H7N9 virus, A/Guangdong/17SF006/2017 isolated from humans that had resulted in fatal outcomes, was used as the backbone virus, and its NA contained 292 K. All eight gene segments of influenza virus A/Guangdong/17SF006/2017 (H7N9) were artificially synthesized (Sangon Biotech) and cloned into the pHW2000 vector. The wild-type A/Guangdong/17SF006/2017 (H7N9) virus encoded 292 K, 119E, 246A and 274H in the NA protein. First, a single-point mutation was introduced to convert 292 K in NA to 292R. Then, in the 292R background, single-point mutations 119 V, 246 T or 274Y in the NA gene were carried out. 292R, 119 V, 246 T or 274Y substitutions were generated with the following primers:

Mutations were introduced into the NA plasmid using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The presence of the introduced mutations and the absence of additional unwanted mutations were verified by sequencing of the entire cDNA.

The recombinant viruses were generated by reverse genetics as previously reported. In general, the eight gene segments were cloned into the pHW2000 vector and cotransfected into 293 T/MDCK cocultured monolayers at 37 °C for 24 h [14]. Then, Opti-MEM supplemented with trypsin (1 μg/ml) (Sigma-Aldrich) was added. Culture supernatant was collected at 72 h and inoculated into 9~11-day-old SPF embryonated chicken eggs. A hemagglutination test was used for the detection of productive virus infection. Virus stocks were sequenced for verification, and virus titers were determined on MDCK cells.

The NA inhibitors oseltamivir carboxylate (oseltamivir; Hoffman-La Roche) and zanamivir (GlaxoSmithKline) were prepared in sterile distilled water and stored in aliquots at − 30 °C until use.

Inhibition of NA enzyme activity by the 2 NA inhibitors was assessed in the fluorescence-based NA inhibition assay using the NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems, ThermoFisher) as previously described [15]. IC₅₀ values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad Prism 5 software. The IC₅₀ values reported are the means (±SD) of IC₅₀ values measured for at least 2 independent tests with 2 duplicates for each sample. Interpretation of IC₅₀ values (obtained by comparing the test virus with the drug-sensitive reference virus) was performed using the WHO AVWG criteria for influenza A viruses: a < 10-fold increase in IC₅₀ represents normal inhibition, a 10~100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.

MDCK cells were infected at an MOI of 0.001 with the recombinant viruses for multicycle replication under the conditions of 5% CO₂ and 37 °C. After 1 h of incubation, cells were washed twice with PBS, and infection medium containing TPCK-trypsin was added. At time points 0, 18, 24, 48, 72 and 96 h post-infection, supernatants were collected, and 3 biological repeats were set up for each sample. Viral titers were determined on MDCK cells.

The existing NA protein crystal structure of A/Anhui/1/2013 H7N9 virus (PDB ID: 4MWJ) in Protein Data Bank (PDB) was used, based on which a crystal structure model of the NA monomer was obtained. DeepView v4.1.0 software was used to display and label drug resistance-related mutations.

All experiments involving HPAI H7N9 viruses were performed by qualified personnel in a BSL-3 laboratory.

Up until April 2019, 33 cases were confirmed to be infected with HPAI H7N9 virus based on the sequence of hemagglutinin, with illness onset dates from December 2016 to April 2019. Among them, 15 were fatal cases. Four potential NAI resistance sites, 292 K, 119 V, 246 T or 274Y in the NA protein, have been identified in HPAI H7N9-infected cases (Table 1), and the characterization of other viral proteins is shown in Additional file 1: Table S1. In total, 9 human cases were infected with HPAI H7N9 viruses containing potential NA resistance mutations (9/33, 27%), including 5 fatal cases and 4 recovered cases (Table 1).

In China, NAI_S (oseltamivir, zanamivir or peramivir) are used in hospitalized cases for influenza treatment, of which oseltamivir is the most common [16]. Among the 9 human cases infected with HPAI H7N9 viruses containing the above potential NA resistance mutations, 7 cases were treated with NAIs. The use of NAIs by the other 2 patients is unknown.

In this study, A/Guangdong/17SF006/2017 (H7N9) was used as a backbone (called rg006-NA292K), which contained NA 292 K – a mutation related to reduced susceptibility to NAIs. A NAI-sensitive virus containing NA 292R, called rg006-NA292R, was constructed first by reverse genetics based on rg006-NA292K. The other 3 amino acid substitutions (119 V, 246 T, 274Y) were introduced into the cDNA encoding the NA of rg006-NA292R. The recombinant viruses (called rg006-NA119V, rg006-NA246T and rg006-NA274Y) were generated by reverse genetics. The nucleotide changes were as follows: for E119V, E/GAA to V/GTA; for A246T, A/GCC to T/ACA; for H274Y, H/CAT to Y/TAT (Table 1). All the constructed plasmids were sequenced to ensure fidelity to the strain and/or the presence of the desired mutations. All rescued viruses had sufficient NA activity (data not shown) for NI assay testing.

The NAI assay was used to investigate the effect of the introduced amino acid substitutions on drug susceptibility. The resulting NA inhibition profiles for oseltamivir and zanamivir are shown in Table 2. A/Texas/12/2007 (H3N2 119E) and A/Texas/12/2007 (H3N2 119 V) were used as control viruses in the drug sensitivity test. The E119V mutation in N2 has been reported to significantly reduce the sensitivity of the virus to oseltamivir but not to zanamivir. The data in this study were consistent with previous reports. Rg006-NA292R virus, which encodes 119E, 246A and 274H in the NA protein, is sensitive to oseltamivir (mean IC₅₀ (nM) ± SD, 1.24 ± 0.01) or zanamivir (mean IC₅₀ (nM) ± SD, 1.50 ± 0.10). However, the substitution E119V in the NA protein induced a mean 90.77-fold increase in the IC₅₀ of oseltamivir (mean IC₅₀ (nM) ± SD, 112.55 ± 17.25). In addition, the substitution H274Y in the NA protein induced a mean 23.40-fold increase in the IC₅₀ of oseltamivir (mean IC₅₀ (nM) ± SD, 29.01 ± 0.18). Furthermore, the substitution A246T in the NA protein induced a mean 10.97-fold increase in the IC₅₀ of zanamivir (mean IC₅₀ (nM) ± SD, 16.46 ± 0.59). The 292 K in the NA protein induced a mean 3686.29-fold increase in the IC₅₀ of oseltamivir (mean IC₅₀ (nM) ± SD, 4571 ± 54.7) and a mean 21.68-fold increase in the IC₅₀ of zanamivir (mean IC₅₀ (nM) ± SD, 32.52 ± 4.53). 292 K in the NA protein of HPAI H7N9 virus induced high and multiple-drug resistance. In conclusion, the reverse genetically constructed HPAI H7N9 viruses containing 292 K, 119 V, 274Y or 246 T in NA could reduce the sensitivity of viruses to oseltamivir or zanamivir.

To test the effect of the NAI resistance mutations on the growth characteristics of the virus, we determined infectious titers in MDCK cells. MDCK cells were infected at an MOI of 0.001 with the recombinant viruses to allow multicycle replication. At time points 0, 18, 24, 48, 72 and 96 h post-infection, viral titers were determined on MDCK cells. Virus kinetics experiments showed that the rg006-NA119V, rg006-NA246T and rg006-NA274Y viruses have similar growth kinetics to viruses containing 119E, 246A and 274H in MDCK cells (Fig. 1). No significant difference in infectious titers in MDCK cells was observed (except for rg006-NA274Y at 96 h, P<0.05). This implies that the mutations studied did not have much of an effect on the growth characteristics of the viruses in MDCK cells, even if some mutations strongly decreased the NA activity of the virus (data not shown). The yields of rg006-NA292K were significantly lower than those of rg006-NA292R at 24, 48, 72 and 96 h (P<0.05). However, rg006-NA292K still maintains a considerable level of replication in MDCK cells. Overall, all tested NAI-resistant viruses were capable of replicating in MDCK cells. Rg006-NA119V, rg006-NA246T and rg006-NA276Y showed comparable replication capacity to their sensitive counterparts.