Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens


NA is important for aerosol transmission of H9N2 AIV in chickens

To determine whether NA also influences SD01 transmission in chickens, we first tested
two recombinant viruses: recombinant SD01 (rSD01) virus recovered using reverse genetics;
and r01/NASS virus, in which the NA of virus SD01 was replaced by that of SS94 (Figure 1A).

Figure 1. Gene schematic diagrams and 3D-structure of H9N2 neuraminidase used in this study.
(A)
Gene schematic diagrams of SD01, SS94 and recombinant virus. The blue and red bars
indicate the genes originating from SD01 and SS94, respectively. The positions of
four amino acid regions in the NA gene are shown at the top of the diagram and differences
in the mutants are shown as amino acid abbreviation. Amino acids in NA of SD01 and
SS94 are marked up by yellow and black letters respectively. + indicates that the
virus could be transmitted among chickens via aerosols, ? indicates could not. (B) The 3D-structure of H9N2 neuraminidase generated using PyMOL software shows the locations
of mutations (PDB access number: 1ivd). The left is the structure before the mutation,
and the right is the structure after the mutation. * indicates the positions that
will be studied. Amino-acids 368–370 are close to the HB site, while 313–381 are on
the opposite side of the globular head.

As shown in Table 2, virus was detected in the oropharyngeal and cloacal swab samples of all rSD01 and
r01/NASS inoculated chickens at 4 dpi. rSD01 and r01/NASS virus were also detected
in direct contact chickens at 2–14 dpi or 4–10 dpi. rSD01 virus was detected in the
oropharyngeal and cloacal swab samples of six aerosol contact chickens at 4 dpi, and
ten chickens at 8 dpi. No virus was detected in aerosol contact chickens for r01/NASS.
Seroconversion was observed for all inoculated chickens, with average antibody titers
increasing until 21 dpi (Figures 2A and B). Seroconversion was also observed for rSD01 infected direct contact chickens
and for aerosol contact chickens (Figure 2A). In contrast, seroconversion was not observed for aerosol contact chickens exposed
to r01/NASS (Figure 2B). Virus aerosols were detected in the air of rSD01 isolators from 4–10 dpi, but
not for r01/NASS isolators (Figure 3). These results indicate that recombinant virus rSD01 was detected in the air and
transmitted by aerosols between chickens, but recombinant virus r01/NASS, in which
the NA of virus SD01 was replaced by that of virus SS94, was not detected in the air
and was not aerially transmitted. These results suggest that the NA gene is an important
determinant in virus transmission by aerosols.

Table 2. Number of chickens infected by recombinant virus in independent experiments*

Figure 2. Seroconversion of chickens in the transmission experiments. In every independent experiment, sera of ten chickens in every group were collected
at 7 day intervals and seroconversion was confirmed by hemagglutination inhibition
(HI) assay. Each color bar represents the antibody titers of every chicken. Repeat
seroconversion experiments of r01/NASS and r01/NA381 were performed. The results also
indicate that no seroconversion was observed in aerosol contact chickens. 1–10 represents
ten inoculated chickens, 11–20 represents ten direct-contact chickens and 21–30 represents
ten aerosol-contact chickens. The blue, red and green colors express antibody titers
detected on 7, 14 and 21 dpi respectively.

Figure 3. Virus titers of airborne H9N2 AIV in an isolator. Air samples were collected simultaneously from the space of two isolators using an
AGI-30 liquid sampler operated continuously for an optimized time of 30 min at an
airflow rate of 12.5 L/min. Virus titer in the air was expressed as values of EID50/L air. Each color bar represented the every recombinant virus concentration in the
air collected every two days from the beginning of 2 dpi in an independent experiment.
No airborne virus was detected in the experiment for r01/NASS and r01/NA381 viruses.

Mutations D368E, S370L, E313K and G381D in NA of SD01 virus abolished airborne transmission
in chickens

The NA protein of influenza A virus is not only required for virion release and spread
but also impacts on virion infectivity and membrane fusion 24]. Although studies indicated that amino acid deletion in the stalk region contributes
to the high virulence and pathogenicity of H5N1 isolates, in our study, there was
no difference between airborne transmissible viruses SD01 and F98, and non-transmissible
SS94 which all owned 3-amino acid (aa: 61–63, N2 number) deletion in their NA stalk
region. So we investigated whether amino acid changes in the head of the NA protein
(aa: 78–469) affected the replication and transmission of mutant viruses in chickens.
Multiple sequence alignment of the NA proteins of H9N2 AIV isolated in China and submitted
to NCBI from 1994 to 2013 revealed that SS94 and A/Chicken/Guangdong/1997 had acidic
amino acid K at position 313, whilst almost all other strains had acidic E313 or D313
35]. SS94 and three Shandong H9N2 AIV had an acidic D381 residue, while all other viruses
had neutral G381 or N381 residues (see Additional file 1). The 3D-structure of neuraminidase was generated using PyMOL software (DeLano Scientific
LLC, San Carlos, CA, USA). The G381D mutation was in a random coil region, and the
conversion from a non-polar to polar amino acid may result in changes to protein hydrophilicity.
When a G381D mutation occurred simultaneously with an E313K mutation in the ?-fold
region, the protein structure was changed significantly, with the distance between
the two reduced amino acids (Figure 1B).

To evaluate whether these amino acid mutations were related to H9N2 virus transmission
in chickens, we tested two mutants, r01/NAHB (D368E and S370L) which derives from
rSD01 by the two substitutions D368E?+?S370L in the hemadsorption site, and r01/NA381
(D368E, S370L, E313K and G381D) which derives from r01/NAHB by two further substitutions:
E313K?+?G381D (Figure 1A). r01/NAHB virus shedding was detected in inoculated and direct contact chickens
from 2 to 12 dpi, and in aerosol contact chickens, although fewer virus particles
were shed compared with rSD01. Virus shedding of r01/NA381 was also detected in inoculated
and direct contact chickens, but the number of infected chickens was fewer than those
infected by r01/NAHB, and no virus shedding was detected in aerosol contact chickens
(Table 2). Seroconversion was observed for all infected chickens (Figures 2C and D), with antibody titers increasing from 7 to 21 dpi. The antibody titers for
direct contact or aerosol contact chickens were appreciably lower than for inoculated
animals on the same day (Figures 2C and D).

Air samples were collected by an AGI-30 liquid sampler every other day from 2 dpi,
and virus titrated in eggs (Figure 3). Virus concentrations were expressed as EID50/L air. Viral aerosols were detected for rSD01 and r01/NAHB at concentrations of 265–13330
EID50/L air. Generally, airborne H9N2 AIV were first detected at 4 or 6 dpi, peaking at
8 dpi and declining at 10 dpi, with no viral aerosols detected at 12 dpi. Airborne
virus was not detected for r01/NASS and r01/NA381, which could be associated with
the reduced number of virus aerosols produced, and to assay sensitivity.

Recombinant virus r01/NAHB (which derives from rSD01 by the two substitutions D368E?+?S370L
in the HB site) was detected in the air and was aerially transmitted, which demonstrates
that the HB site has a modest impact on aerial transmission. Recombinant virus r01/NA381
(which derives from r01/NAHB by two further substitutions: E313K?+?G381D) was not
detected in the air and was not aerially transmitted, which demonstrates the importance
of the amino-acid pair 313–381 in aerial transmission. The NA of this virus is close
to that of SS94.

Replication of recombinant viruses in the respiratory tract of SPF chickens

Clarified homogenates of tracheas and lungs collected from three chickens at 5 dpi
were used for virus titration in SPF embryonated chicken eggs. There were no obvious
differences in viral titers between the lungs and tracheas of chickens infected with
the same virus (Table 3). However, virus titers for r01/NASS and r01/NA381-infected chickens were lower than
for those infected with rSD01 (Table 3). According to the study before, these two viruses also had similar characteristics
in transmissibility: the reduced virus shedding of infected chickens and no shedding
in aerosol contact chickens, and not being detected in the air. All strains were non-lethal
to chickens and did not induce serious clinical symptoms, other than slight inappetence
and inactivity, with clinical symptoms generally resolved within 10 days. Seroconversion
was observed for all viruses, and HI titers were between 64 and 128 (Table 3).

Table 3. Pathogenicity of H9N2 AIV to SPF chickensa

Substitutions or mutations in the NA gene affected virus NA activity

Replacement or mutations of the NA gene of SD01 were shown to decrease virus airborne
transmission efficiency. In the current study, we determined the kinetic parameters,
KM and Vmax, of viral NA using the fluorogenic substrate 4-MUNANA. Differences in KM values for NA of recombinant viruses were not considered to be statistically significant
(P??0.05), suggesting NA enzymatic activity was consistent across all viruses. Enzyme
activity was significantly higher (Vmax, P??0.05) for NA of rSD01 compared with NA of r01/NASS, r01/NAHB and r01/NA381 (Table 4). Virus elution times were used to evaluate the effect of NA substitutions or mutations
on virus release from CRBC. rSD01 virus was first to be eluted, followed by r01/NAHB,
with both r01/NASS and r01/NA381 viruses eluted much later (3.5 and 4 h, respectively).
These results indicate that NA gene substitution and mutations at 366–373HB, 313 and
381 played an important role in reducing virus NA activity. What is more, the results
also indicate that substitutions at residues 313–381 have more impact than those at
the HB site.

Table 4. Neuraminidase activities of H9N2 AIV

Mutations E368D, L370S, K313E and D381G in NA of r01/NASS confer virus airborne transmissibility
in chickens

r01/NASS-381 virus with mutations E368D, L370S, K313E and D381G in NA was generated
on the backbone of the r01/NASS virus which had lost airborne transmissibility in
chickens. We compared the transmissibility of r01/NASS-381 virus with r01/NASS virus
in chickens. As shown in Table 2, virus shedding was detected in both r01/NASS-381 and r01/NASS inoculated and direct
contact chickens, with a greater number of chickens infected by the former from 4
to 14 dpi. Two aerosol contact chickens were infected with r01/NASS-381 virus at 4
dpi; increasing to seven by 8 dpi. Altogether, fewer chickens were infected with r01/NASS-381
compared with rSD01 and r01/NAHB on the same day. Seroconversion occurred in aerosol
infected chickens, with average antibody titers of 4–32 recorded at 14 and 21 dpi
(Figure 2E). Air samples were collected from the isolators and viruses titrated in SPF embryonated
chicken eggs, as described previously. Airborne r01/NASS-381 virus was detected at
6 dpi, at a concentration of 265 EID50/L air, whilst no viral aerosol was detected at 12 dpi (Figure 3). Virus elution times of r01/NASS-381 virus released from CRBC was 2.5 h, which was
shorter than that of the r01/NASS virus. These results demonstrate that amino acid
mutations E368D, L370S, K313E and D381G in the NA protein of r01/NASS enhanced viral
NA activity and viral shedding from chickens, and restored airborne transmissibility
in chickens, and the HB site had a minor impact on these characteristics, while the
amino-acid pair 313–381 had a major effect.