Residue 41 of the Eurasian avian-like swine influenza a virus matrix protein modulates virion filament length and efficiency of contact transmission

Abstract

Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity.

Importance: We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model.

FIG 1

Schematic diagram of recombinant viruses. Genotypes of each recombinant virus used in the study are depicted. Blue and red bars represent genes from A/PR/8/34 (H1N1) (PR8) and A/Swine/Spain/53207/04 (H1N1) (SPN04) viruses, respectively. The yellow bar indicates the presence of a point mutation encoding a proline-to-alanine change at position 41 of the matrix protein.

FIG 2

Characterization of recombinant viruses in HTBE and MDCK cells. (A) MDCK cells were infected at a low MOI (0.002) with recombinant PR8, rSPN04, or WT SPN04 viruses. Released virus was collected from the supernatant and enumerated by plaque assay at the indicated time points. (B) HTBE cells were infected at a low MOI (0.001) with recombinant NL602, SPN04-P, or SPN04-A viruses and incubated at 37°C in 5% CO₂ for 96 h. Released virus was collected from the apical surface of differentiated cells and enumerated by plaque assay. (C) MDCK cells were infected at a low MOI (0.002) with recombinant PR8, SPN04-P, or SPN04-A viruses. Released virus was collected and enumerated as described for panel A. (D) MDCK cells were infected at a low MOI (0.002) with recombinant PR8, PR8 SPN04 M41P, PR8 SPN04 M41A, PR8 SPN04 M41P+NA, PR8 SPN04 M41A+NA, PR8 SPN04 M41P+NA+HA, or PR8 SPN04 M41A+NA+HA viruses. Each virus possessing a proline at position 41 had marginally delayed kinetics of growth relative to the viruses possessing an alanine at that position. Moreover, PR8 SPN04 M41P and PR8 SPN04 M41P+NA viruses grew to slightly lower peak titers than the isogenic virus possessing alanine at position 41. Released virus was collected from the supernatant and enumerated as described for panel A. The plaque size of each recombinant virus was measured at 48 h postinfection following immunostaining of infected MDCK cells. (E to H) Values for the diameters of at least 125 plaques were plotted for each pair of recombinant viruses as indicated. Statistically significant differences between plaque sizes are denoted by an asterisk (P < 0.05).

FIG 3

A proline at residue 41 of matrix protein increases the filament lengths of recombinant viruses. Virion morphologies are shown in representative SEM images (A, C, E, G, I, K, M, and O) or TEM images (B, D, F, H, J, L, N, and P). Adherent MDCK cells were infected at an MOI of 5.0, or mock infected, and incubated for 18 h before fixing and staining. Reassortant viruses possessing a proline at position 41 of the matrix protein (as indicated) exhibited formation of long filamentous virions (A, B, E, F, I, and J). Those viruses possessing alanine at position 41 displayed virions consisting of considerably shorter filaments (C, D, G, H, K, and L). SPN04-P virus displayed pleiomorphic virions, including highly filamentous examples (M and N), whereas SPN04-A produced virions with a markedly reduced length, of approximately 1 μm or less (O and P). Magnification, ×10,000 (A, C, E, G, I, K, M, and O) or ×40,000 (B, D, F, H, J, L, N, and P). Bars, 1.00 μm (A, C, E, G, I, K, M, and O), 0.2 μm (F), or 0.5 μm (B, D, H, J, L, N, and P).

FIG 4

A proline at residue 41 of matrix protein increases neuraminidase activity of select recombinant viruses in vitro. The neuraminidase activity of each virus (as indicated) was measured through the cleavage of the fluorogenic substrate MUNANA. Virus was incubated in the presence of MUNANA at 37°C for 45 min, and the relative fluorescence was measured at 1-min intervals. Plots displaying (tightly overlapping) triplicate samples of each virus are shown. Each graph is representative of at least three replicate experiments.

FIG 5

A proline at residue 41 of matrix protein attenuates growth and transmission of recombinant viruses in guinea pigs. (A) Three out of four guinea pigs inoculated with 1,000 PFU of SPN04-P virus were productively infected, and one transmission to a cage mate occurred on day 6 postinfection. (B) No guinea pigs were productively infected following inoculation with 100 PFU of SPN04-P virus. (C) All guinea pigs inoculated with 1,000 PFU of SPN04-A virus were infected, and two transmission events occurred on day 6 postinfection. (D) Two out of 4 guinea pigs inoculated with 100 PFU of SPN04-A became infected, and one transmission to a cage mate occurred on day 8 postinfection. (E to H) Eight guinea pigs infected with either PR8 SPN04 M41P (E) or PR8 SPN04 M41P+NA (G) virus did not transmit the virus to cage mates. In contrast, guinea pigs infected with PR8 SPN04 M41A (F) or PR8 SPN04 M41A+NA (H) virus displayed an earlier peak of replication and were permissive for 4/8 transmission events. (I) Guinea pigs infected with PR8 SPN04 M41P+NA+HA showed 1/8 transmission events. (J) Animals infected with PR8 SPN04 M41A+NA+HA virus were subject to 5/8 transmission events, although replication kinetics were similar between the two strains (compare graphs in panels I and J). Virus titers were determined by plaque assay from nasal washes collected every 2 days after infection from inoculated (1,000 PFU; dashed lines) and contact-exposed (solid lines) guinea pigs. Virus titers were determined by plaque assay of nasal washes collected every 2 days after infection from inoculated (100 or 1,000 PFU; dashed lines) and contact-exposed (solid lines) guinea pigs. swSp04-A contains the consensus M segment of the Eurasian avian-like swine virus lineage.