Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have higher neuraminidase activities than the spherical wild-type.

Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo

rPR8wt, rPR8 M1 N87S, and rPR8 M1 R101G viruses have the same red blood cell binding avidity.

<p>Chicken red blood cells were treated with a series of dilutions of <i>C. perfringens</i> neuraminidase. Treated red blood cells were then added to a standardized amount of each virus (8 HAU) and allowed to develop at 4°C. The assay was run in triplicate (standard deviation for each virus = 0). The highest concentration of neuraminidase that still allowed agglutination by each virus is plotted.</p

NA and M1 protein levels between rPR8wt, rPR8 M1 N87S, and rPR8 M1 R101G are similar when normalized to NP protein levels.

<p>Virus samples were concentrated via ultracentrifugation through a 30% sucrose cushion and resuspended in PBS. Samples were then deglycosylated and denatured, after which NP protein levels were standardized via Coomassie. For the Western blot, protein was detected using primary antibodies specific for NA, M1, and NP and fluorophore-conjugated secondary antibodies. All bands were quantified using Image Lab software (Bio-Rad). Error bars represent standard deviation.</p

There is little difference in plaque reduction among rPR8wt, rPR8 M1 N87S, and rPR8 M1 R101G viruses.

<p>Each virus was diluted to approximately 250 PFU. Trypsin-heat-periodate treated serum was diluted 1∶80, 1∶160, 1∶320, and 1∶640 in PBS. Virus was added to serum dilutions and incubated for 30 minutes at 37°C. Virus titer for each serum/virus sample was then quantified by plaque assay on MDCK cells. The number of plaques obtained following incubation with immune serum is plotted as a percentage of the plaques obtained following incubation with naïve serum. The mean of three replicates is plotted, and error bars indicate standard deviation. *p<0.05 compared to rPR8wt virus. The limit of detection for the plaque assays was 5 PFU/ml.</p

128 HAU of rPR8wt, rPR8 M1 N87S and rPR8 M1 R101G viruses comprise comparable infectious titers and genome copies.

a<p>rPR8wt to rPR8 M1 N87S comparison (p = 0.38), rPR8wt to rPR8 M1 R101G comparison (p = 0.40). A two-tailed Student <i>t</i>-test was used to assess significance.</p>b<p>rPR8wt to rPR8 M1 N87S comparison (p = 0.23), rPR8wt to rPR8 M1 R101G comparison (p = 0.10). To assess significance, a two-tailed Student <i>t</i>-test was applied to values of 2^(−Cq).</p><p>128 HAU of rPR8wt, rPR8 M1 N87S and rPR8 M1 R101G viruses comprise comparable infectious titers and genome copies.</p

No difference in virion stability at an elevated temperature was observed between rPR8wt, rPR8 M1 N87S, and rPR8 M1 R101G viruses.

<p>Each virus was diluted to 1×10⁶ PFU and incubated in triplicate at 50°C for one of the following lengths of time: 0, 15, 30, 60, and 120 min. Results shown are the average of four separate assays performed in triplicate (thus n = 12). The titers of each mutant virus were compared to that of the wt virus at each time point using Student’s <i>t</i> test. All p values were >0.05 except that obtained for wt vs. N87S viruses at the 0 h time point (p = 0.0094). The dotted line indicates the limit of detection.</p

rPR8 M1 R101G and rPR8 M1 N87S viruses have higher neuraminidase activity than rPR8wt virus.

<p>A) Neuraminidase enzyme kinetics. Virus input was standardized to 5×10⁵ PFU and concentrations of MUNANA substrate ranging from 1.17 µM to 150 µM were used. Fluorescence generated at each time point (every minute over the course of 1 hour) was detected using a Biotek Synergy H1 plate reader. The resulting fluorescence curves were then fitted to the Michaelis-Menton equation. B) That equivalent amounts of each virus were used in the MUNANA assay was confirmed by RT-qPCR for the viral NP segment. The arithmetic mean (n = 3) and standard deviation of 2^(−Cq) values were calculated and then converted back to a C_q scale by taking the log₂. Two biological replicates of each virus are included in white and grey bars; each biological replicate comprised three technical replicates.</p

rPR8 M1 N87S and rPR8 M1 R101G viruses have a higher neuraminidase activity than rPR8 wt virus in a MUNANA-based assay.

<p>rPR8 M1 N87S and rPR8 M1 R101G viruses have a higher neuraminidase activity than rPR8 wt virus in a MUNANA-based assay.</p

There are no differences in hemagglutination inhibition among rPR8wt, rPR8 M1 N87S, rPR8 M1 R101G viruses.

a<p>The reciprocal of the highest dilution of serum that prevented hemagglutination is shown for three replicates (A, B and C).</p><p>There are no differences in hemagglutination inhibition among rPR8wt, rPR8 M1 N87S, rPR8 M1 R101G viruses.</p