The short stalk length of highly pathogenic avian influenza H5N1 virus neuraminidase limits transmission of pandemic H1N1 virus in ferrets

Abstract

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.

Fig 1

Sequence alignment of human, avian, and engineered NAs. Sequences of N1 NA are shown by full-length amino acid sequence (A), nucleotide sequence of region covering stalk deletion (B), and amino acid sequence of stalk region (C). The first sequence, A/England/195/2009 (E195 NA), is the natural full-length sequence of a prototypic pandemic NA, followed by the natural sequence from A/turkey/Turkey/1/05 NA (SStyNA), from which nucleotides 145 to 204 are deleted. The third sequence (ck/JilinNA) is from the N1 NA of A/chicken/Jilin/hl/04 virus and was the sequence, without a stalk deletion, found in GenBank to have the highest nucleotide identity with SStyNA. The final sequence (LStyNA) was engineered by inserting nucleotides 145 to 204 from ck/JilinNA into SStyNA. Amino acids are colored as bars in panel A and block outlined in panel C where they differ among the NA sequences in this set (blue, positively charged; red, negatively charged; green, polar; yellow, nonpolar). Nucleotides in panel B are block outlined in gray where E195 NA differs from ck/JilinNA. The green bar at the bottom shows the length of the stalk domain.

Fig 2

Multicycle virus replication of SStyNA is attenuated in cell culture. Multicycle virus replication was assessed in MDCK cells (A) and HNE cells (B) both at 32°C following infection at an MOI of 0.01. The mean viral titer of each virus (n = 3) is indicated with a symbol, and error bars represent 95% confidence intervals. Note that the 95% confidence intervals for LStyNA at 48 and 72 hpi in MDCK cells and at 72 hpi in HNE cells do not show because they were very small. Asterisks indicate the P values for the differences between SStyNA and LStyNA: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 3

Short-stalk NA limits influenza virus transmission in ferrets. Donor ferrets were inoculated with 10⁶ PFU of either SStyNA (Red) or LStyNA (Blue) on day zero and housed in separate cages. The day after inoculation, one DC ferret was cohoused with each donor and one RD ferret was housed in an adjacent cage separated by two perforated panels. Viral titer from nasal wash was determined daily by plaque assay. dpi, days postinfection. “G” refers to one donor, one DC, and one RD-exposed ferret in shared or adjoining cages, respectively.

Fig 4

Viruses with short-stalk NA are eluted slowly from red blood cells. Concentrated viruses were diluted to equal HAU and then mixed with RBCs at 4°C to promote hemagglutination. After HA was established, the plate was moved to 32°C to promote NA enzyme activity. The loss of HA was assessed every 20 min and determined by the appearance of an RBC pellet. Experiments were performed three times with different batches of virus, and graphs are representative of the pattern seen with chicken (A) and human (B) RBCs.

Fig 5

Virions with short-stalk NA aggregate more than virions with long-stalk NA. HNE cells were infected with either SStyNA or LStyNA virus (MOI of 20) and incubated at 32°C. Twenty-four hours later, released virions were collected in a small volume, negatively stained with uranyl acetate, and then visualized using an electron microscope. Virions were considered aggregated if two or more were clearly touching. (A) Representative EM images show that SStyNA virions tended to aggregate (left), and LStyNA virions did not (right). Supernatant from uninfected wells lacked virions (not shown). (B) The average percentage of virions aggregated determined by counting over 150 virions per well from three separate wells for each virus. Symbols represent individual wells; the middle line is the mean, and error bars denote 95% confidence intervals. ***, P < 0.001.

Fig 6

Mucus inhibits infectivity of virions with short-stalk NA more than long-stalk NA. Infectivity remaining was determined as the percentage of virions that infected MDCK cells (MOI, 0.00001) in the presence of mucus, compared to the absence of mucus, after 2 h of incubation at 32°C followed by plaque assay. Error bars denote 95% confidence intervals. **, P < 0.01; ****, P < 0.0001; n = 6.

Fig 7

NA and HA maximum likelihood phylogenetic trees. Maximum likelihood phylogenetic trees were constructed from 1,150 paired H5N1 full-length sequences. The HA tree (left) shows that HPAI H5N1 viruses with long-stalk NAs are mostly isolates that belong to early clades (0, 3, 4, 5, 6, 7, 8, and 9) and one isolate from clade 2.3.2. The NA tree (right) shows that stalk truncation has occurred five times in this data set (annotated in gray and black, with the year of the earliest virus isolate and number of amino acids that were deleted), while stalk elongation was not observed. Taxon names have been removed for clarity (see Fig. S1A and B in the supplemental material for full virus names). Branch colors are as follows: red, avian HPAI LSNA; pink, mammalian HPAI LSNA; gold, avian LPAI LSNA; black, avian and mammal HPAI SSNA; gray, avian LPAI SSNA. Years indicate the date of virus isolation. The inset shows that the LSNA of dk/Zhejiang/2245/2011 was most closely related to NAs within the European LPAI clade. Inset nodes are labeled with bootstrap values. Virus names of interest and HA clades are noted. Dk/Zhejiang/2245/2011 is indicated with a red dot. del, deletion.