Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned

Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model▿ †

The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host

Neuraminidase inhibitor susceptibility and neuraminidase enzyme kinetics of human influenza A and B viruses circulating in Thailand in 2010-2015.

Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934 (A/PR/8, H1N1) or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses' replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined

Inner filter effect (IFE) from light absorption at the excitation and emission wavelengths of the 4-MU product.

<p>(A) Absorbance spectra of MUNANA and 4-MU at 0.01 mg/mL and 0.1 mg/mL concentrations in enzyme buffer. Optical density was measured using Synergy 2 multi-mode microplate reader in a UV-transparent 96-well plate. (B) Absorbance spectra of MUNANA and 4-MU at 0.1 mg/mL concentration. (C) 4-MU fluorescence measured in the presence of different concentrations of MUNANA (15–2000 µM) shows similar impact of MUNANA-associated spectroscopic interference across 4-MU concentrations of 10–80 µM.</p

Flowchart for the determination of NA enzyme kinetic parameters using whole influenza virus preparations.

<p>Flowchart for the determination of NA enzyme kinetic parameters using whole influenza virus preparations.</p

Calculation of the spectroscopic correction factor.

<p><b>Abbreviations:</b> MUNANA, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid; 4-MU, 4-methylumbelliferone; RFU, relative fluorescence units.</p>a<p>Fluorescence of MUNANA alone in enzyme buffer at 1.95–2000 µM concentrations (shown in column 1).</p>b<p>Fluorescence of 4-MU alone in enzyme buffer at 19 µM concentration. Mean (± SD) 4-MU fluorescence in enzyme buffer was 10222±90 RFU.</p>c<p>Fluorescence of 4-MU in enzyme buffer at 19 µM concentration in the presence of 1.95–2000 µM MUNANA concentrations (shown in column 1).</p>d<p>Values (in RFU) of MUNANA fluorescence (column 2) subtracted from the values (in RFU) obtained in the mixture of 4-MU and MUNANA (column 4).</p>e<p>Correction factor calculated as the ratio of 4-MU fluorescence (in RFU) in the mixture with MUNANA substrate (column 5) and 4-MU fluorescence (in RFU) alone (mean value from column 3).</p

Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate

<div><p>The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining <i>K_m</i> and <i>V_max</i> kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.</p></div

Influenza A/CA/04/09 (H1N1pdm09) virus dilution selection for determination of NA enzyme kinetics parameters.

<p>Two-fold dilutions of A/CA/04/09 (H1N1pdm09) virus were prepared in enzyme buffer. The graph shows virus dilutions (1∶16 to 1∶1024) that generated linearly increasing amounts of 4-MU over the reaction time (<i>R</i>²>0.99). Fluorescence intensity was recorded every 60 s for 60 min at 37°C with the MUNANA substrate at a final concentration of 100 µM. Blank signal value determined from reactions without virus was subtracted from RFU generated in the NA enzymatic reactions. The virus dilution of 1∶64 was selected in this case for further studies on the basis of linearity of the curve (dotted line), signal-to-noise ratio ≥10, and conversion of ≤15% of the total amount of MUNANA after 60 min.</p

Standard curve of 4-methylumbelliferone (4-MU) fluorescence.

<p>Fluorescence intensity was measured using a Synergy 2 multimode microplate reader at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Relative fluorescence units (RFU) obtained at low 4-MU concentrations (0–2.0 µM) are shown in the insert. Each data point represents the mean ± standard deviation (SD) of 10 independent measurements.</p