Pleiotropic Effects of Influenza H1, H3, and B Baloxavir-Resistant Substitutions on Replication, Sensitivity to Baloxavir, and Interferon Expression.

Baloxavir is an anti-influenza endonuclease inhibitor that targets the polymerase acidic (PA) protein of influenza A and B viruses. Our knowledge regarding the pleiotropic effects of baloxavir resistance-associated substitutions is limited. We generated recombinant A/California/04/09 (H1N1)-, A/Hong Kong/218849/2006 (H3N2)-, and B/Victoria/504/2000-like viruses that contained PA substitutions identified in baloxavir clinical trials and surveillance that could potentially be associated with baloxavir resistance. We characterized their susceptibility to baloxavir, impact on polymerase activity, viral growth, and ability to induce interferon (IFN) and IFN-stimulated genes expression in vitro. Four PA substitutions, H1N1 I38L/T, E199D, and B G199R, significantly reduced the sensitivity of the recombinant viruses to baloxavir (14.1-fold). We confirmed our findings by using the luciferase-based ribonucleoprotein minigenome assay and by using virus yield reduction assay in Calu-3 and normal human bronchial epithelial (NHBE) cells. We observed that I38L and E199D resulted in decreased viral replication of the H1N1 wild-type virus (1.4-fold) but the H1N1 I38T and B G199R substitutions did not significantly alter replication capacity in Calu-3 cells. In addition, H1N1 variants with PA I38L/T and E199D induced significantly higher levels of IFNB1 gene expression compared to the wild-type virus (4.2-fold). In contrast, the B variant, G199R, triggered the lowest levels of IFN genes in Calu-3 cells (1.6-fold). Because baloxavir is a novel anti-influenza therapeutic agent, identifying and characterizing substitutions associated with reduced sensitivity to baloxavir, as well as the impact of these substitutions on viral fitness, is paramount to the strategic implementation of this novel countermeasure.

Laninamivir-Interferon Lambda 1 Combination Treatment Promotes Resistance by Influenza A Virus More Rapidly than Laninamivir Alone.

Each year, 5% to 20% of the population of the United States becomes infected with influenza A virus. Combination therapy with two or more antiviral agents has been considered a potential treatment option for influenza virus infection. However, the clinical results derived from combination treatment with two or more antiviral drugs have been variable. We examined the effectiveness of cotreatment with two distinct classes of anti-influenza drugs, i.e., neuraminidase (NA) inhibitor, laninamivir, and interferon lambda 1 (IFN-λ1), against the emergence of drug-resistant virus variants in vitro We serially passaged pandemic A/California/04/09 [A(H1N1)pdm09] influenza virus in a human lung epithelial cell line (Calu-3) in the presence or absence of increasing concentrations of laninamivir or laninamivir plus IFN-λ1. Surprisingly, laninamivir used in combination with IFN-λ1 promoted the emergence of the E119G NA mutation five passages earlier than laninamivir alone (passage 2 versus passage 7, respectively). Acquisition of this mutation resulted in significantly reduced sensitivity to the NA inhibitors laninamivir (∼284-fold) and zanamivir (∼1,024-fold) and decreased NA enzyme catalytic activity (∼5-fold) compared to the parental virus. Moreover, the E119G NA mutation emerged together with concomitant hemagglutinin (HA) mutations (T197A and D222G), which were selected more rapidly by combination treatment with laninamivir plus IFN-λ1 (passages 2 and 3, respectively) than by laninamivir alone (passage 10). Our results show that treatment with laninamivir alone or in combination with IFN-λ1 can lead to the emergence of drug-resistant influenza virus variants. The addition of IFN-λ1 in combination with laninamivir may promote acquisition of drug resistance more rapidly than treatment with laninamivir alone.

Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses.

Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses. IMPORTANCE: Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the United States since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U.S. swine population in the mid-1990s, but they are different from both these ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.

Adaptation of influenza B virus by serial passage in human airway epithelial cells.

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. To understand their adaptation capability, we examined the genetic changes that occurred following 15 serial passages of two influenza B viruses, B/Brisbane/60/2008 and B/Victoria/504/2000, in human epithelial cells. Thirteen distinct amino acid mutations were found in the PB1, PA, hemagglutinin (HA), neuraminidase (NA), and M proteins after serial passage in the human lung epithelial cell line, Calu-3, and normal human bronchial epithelial (NHBE) cells. These changes were associated with significantly decreased viral replication levels. Our results demonstrate that adaptation of influenza B viruses for growth in human airway epithelial cells is partially conferred by selection of HA1, NA, and polymerase mutations that regulate receptor specificity, functional compatibility with the HA protein, and polymerase activity, respectively.

Effect of influenza H1N1 neuraminidase V116A and I117V mutations on NA activity and sensitivity to NA inhibitors.

Neuraminidase inhibitors (NAIs) play a key role in the management of influenza. Given the limited number of FDA-approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority. Two NA mutations, V116A and I117V, are found in ∼0.6% of human, avian, and swine N1 isolates. Using the A/California/04/09-like (CA/04, H1N1) background, we examined the impact of V116A and I117V NA mutations on NAI susceptibility, substrate specificity, and replicative capacity in normal human bronchial (NHBE) cells and a human respiratory epithelial cell line (Calu-3). We compared the impact of V116A and I117V on the functional properties of NA and compared these mutations with that of previously reported NAI-resistant mutations, E119A, H275Y, and N295S. All NA mutations were genetically stable. None of the viruses carrying NA mutations grew to significantly lower titers than CA/04 in Calu-3 cells. In contrast, V116A, I117V, E119A, and N295S substitutions resulted in significantly lower viral titers (1.2 logs) than the parental CA/04 virus in NHBE cells. V116A conferred reduced sensitivity to oseltamivir and zanamivir (13.7-fold). When MUNANA, 3'SL, and 6'SL substrates were applied, we observed that V116A reduced binding ability for all substrates (13.9-fold) and I117V led to the significantly decreased affinity for MUNANA and 6'SL (4.2-fold). Neither mutation altered the catalytic efficiency (kcat/KM) in catalyzing 3'SL, but the efficiency in catalyzing MUNANA and 6'SL was significantly decreased: only ∼34.7% compared to the wild-type NA. The efficiencies of NAs with E119A, H275Y, and N295S mutations to catalyze all substrates were ∼19.4% of the CA/04 NA. Our study demonstrates the direct effect of drug-resistant mutations located inside or adjacent to the NA active site on NA substrate specificity.

The use of plant lectins to regulate H1N1 influenza A virus receptor binding activity.

We applied an in vitro selection approach using two different plant lectins that bind to α2,3- or α2,6-linked sialic acids to determine which genetic changes of the A/California/04/09 (H1N1) virus alter hemagglutinin (HA) receptor binding toward α2,3- or α2,6-linked glycans. Consecutive passages of the A/California/04/09 virus with or without lectins in human lung epithelial Calu-3 cells led to development of three HA1 amino acid substitutions, N129D, G155E, and S183P, and one mutation in the neuraminidase (NA), G201E. The S183P mutation significantly increased binding to several α2,6 SA-linked glycans, including YDS, 6'SL(N), and 6-Su-6'SLN, compared to the wild-type virus (↑3.6-fold, P < 0.05). Two other HA1 mutations, N129D and G155E, were sufficient to significantly increase binding to α2,6-linked glycans, 6'SLN and 6-Su-6'SLN, compared to S183P (↑4.1-fold, P < 0.05). These HA1 mutations also increased binding affinity for 3'SLN glycan compared to the wild-type virus as measured by Biacore surface plasmon resonance method. In addition, the HA1 N129D and HA1 G155E substitutions were identified as antigenic mutations. Furthermore, the G201E mutation in NA reduced the NA enzyme activity (↓2.3-fold). These findings demonstrate that the A/California/04/09 (H1N1) virus can acquire enhanced receptor affinity for both α2,3- and α2,6-linked sialic receptors under lectin-induced selective pressure. Such changes in binding affinity are conferred by selection of beneficial HA1 mutations that affect receptor specificity, antigenicity, and/or functional compatibility with the NA protein.

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses.

Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line (Calu-3) in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓˃20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes (IFNB1, IFNL1 and IFNL2/3) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.

Mutational pattern of influenza B viruses adapted to high growth replication in embryonated eggs.

Improved replication of influenza viruses in embryonated chicken eggs (CE) permits increased vaccine production and availability. We investigated the growth properties of influenza B viruses in relation to specific mutations occurring after serial passage in CE. In serial passage experiments yielding high growth variants of B/Victoria/504/2000, mutations predicted to alter amino acid (AA) composition occurred only near the receptor-binding pocket of the hemagglutinins (HA) and in no other genes. Two B/Victoria/504/2000 high growth variants had the same AA substitutions in HA (R162M and D196Y), but the higher yield variant had a third substitution (G141E), which also altered antigenic characteristics. In a serial passage experiment yielding a high growth variant of B/Hong Kong/330/2001, mutations predicted to alter AA composition occurred only in PB2 and NP in domains predicted to relate to RNP formation and function. Our results indicate that adaptation of influenza B viruses to high-yield replication by serial passage in CE requires few mutations either in internal or external genes. Specific modifications of genes or a combination of genes could be used to optimize or create influenza B viruses for specific growth substrates.

Simple and rapid strategy for genetic characterization of influenza B virus reassortants.

Genetic reassortment of influenza viruses is widely used for creating viruses with specific phenotypes. Reassortment of two influenza viruses, each with eight RNA segments potentially yields as many as 256 gene segment combinations. Therefore, confirmation that progeny viruses possess genomes corresponding to the specified phenotypes can be laborious and time-consuming. To establish a convenient method for genotyping influenza virus reassortants, we adapted single-strand conformation polymorphism analysis (SSCP) using standard laboratory equipment. By varying the concentration of polyacrylamide between 4-6% and the concentration of glycerol between 5-8% in the gel, together with adding PCR primers to the DNA sample during the denaturing step, optimal conditions can be found for SSCP with little effort. The described method has high accuracy and reliability, and provides a tool for rapid, cost-effective genetic screening and assessment of the purity and genetic stability of the reassortant viruses. This method should be useful in basic research applications and in preparing reassortant viruses for vaccine use.

Heterogeneity of the MDCK cell line and its applicability for influenza virus research.

Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA). All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1) and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2-3 and α2-6). The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed). HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to support replication of influenza A and B viruses.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination.

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Changes of the receptor-binding properties of influenza B virus B/Victoria/504/2000 during adaptation in chicken eggs.

Selection of high-growth virus variants of strain B/Victoria/504/2000 by serial passage in eggs resulted in three amino acid substitutions, G141E, R162M, and D196Y, in the vicinity of the receptor-binding pocket of viral hemagglutinin. Virus variants containing the identified amino acid substitutions, individually or in various combinations, were constructed using reverse genetics and analyzed for their receptor-binding properties using glycan microarray platform. Three different patterns of virus binding were revealed. A low-growth virus variant, corresponding to the original egg-derived virus B/Victoria/504/2000 prior to acquisition of amino acid changes G141E, R162M, and D196Y, had a clear preference for the oligosaccharide chains terminated with alpha2-6-linked sialic acid with very weak binding of the glycans terminated with alpha2-3-linked sialic acid. Amino acid substitutions R162M and D196Y had similar effects, resulting in viruses that bound with high efficiency almost all terminally sialylated glycans represented on the array regardless of the type of glycosidic linkage. In contrast, substitution of G141E alone, or in combinations with the other two amino acid substitutions, significantly restricted virus glycan-binding capabilities. All virus variants possessing this substitution lost the ability to bind glycans with alpha2-6 glycosidic linkage as well as most of the glycans with alpha2-3 glycosidic linkage. Linear penta- and heptasaccharide chains represented at the non-reducing end by alpha2-3 sialylated Type-II motif (LacNAc) were the only structures bound with high affinity by the virus variants with G141E substitution. In all cases when the effects on virus binding of individual amino acid substitutions differed, the effect of R162M was subordinate to the effect of either G141E or D196Y.

Generation of the influenza B viruses with improved growth phenotype by substitution of specific amino acids of hemagglutinin.

Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.