Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in mallard ducks

The highly pathogenic (HP) influenza viruses H5 and H7 are usually nonpathogenic in mallard ducks. However, the currently circulating HP H5N1 viruses acquired a different phenotype and are able to cause mortality in mallards. To establish the molecular basis of this phenotype, we cloned the human A/Vietnam/1203/04 (H5N1) influenza virus isolate that is highly pathogenic in ferrets, mice, and mallards and found it to be a heterogeneous mixture. Large-plaque isolates were highly pathogenic to ducks, mice, and ferrets, whereas small-plaque isolates were nonpathogenic in these species. Sequence analysis of the entire genome revealed that the small-plaque and the large-plaque isolates differed in the coding of five amino acids. There were two differences in the hemagglutinin (HA) gene (K52T and A544V), one in the PA gene (T515A), and two in the PB1 gene (K207R and Y436H). We inserted the amino acid changes into the wild-type reverse genetic virus construct to assess their effects on pathogenicity in vivo. The HA gene mutations and the PB1 gene K207R mutation did not alter the HP phenotype of the large-plaque virus, whereas constructs with the PA (T515A) and PB1 (Y436H) gene mutations were nonpathogenic in orally inoculated ducks. The PB1 (Y436H) construct was not efficiently transmitted in ducks, whereas the PA (T515A) construct replicated as well as the wild-type virus did and was transmitted efficiently. These results show that the PA and PB1 genes of HP H5N1 influenza viruses are associated with lethality in ducks. The mechanisms of lethality and the perpetuation of this lethal phenotype in ducks in nature remain to be determined.     

Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus.     

Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.     

PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.     

Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.     

Naturally Occurring Mutations in the PA Gene Are Key Contributors to Increased Virulence of Pandemic H1N1/09 Influenza Virus in Mice

We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus''s 50% mouse lethal dose (LD₅₀), by almost 1,000-fold.     

Pandemic 2009 H1N1 Influenza A Virus Carrying a Q136K Mutation in the Neuraminidase Gene Is Resistant to Zanamivir but Exhibits Reduced Fitness in the Guinea Pig Transmission Model

Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.     

Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.     

Genetically engineered live attenuated influenza A virus vaccine candidates.

We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.     

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.     

Oseltamivir-Resistant Variants of the 2009 Pandemic H1N1 Influenza A Virus Are Not Attenuated in the Guinea Pig and Ferret Transmission Models

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.Oseltamivir resistance was rare until 2008, when resistant seasonal H1N1 viruses were found circulating in the general Scandinavian population (15). Soon after, studies from other countries in Europe also reported the isolation of oseltamivir-resistant viruses, and eventually, oseltamivir resistance was recognized as a global phenomenon (9, 27). Prior to 2008, resistant viruses were primarily isolated from patients with nonresponsive influenza virus infections or from infected patients who received a low-dose prophylaxis regiment prior to symptom onset. At the time, these resistant isolates accounted for 1% of the circulating H1N1 virus. Drug resistance mutations were identified during oseltamivir development, including a histidine-to-tyrosine mutation at position 275 (H275Y) in N1 neuraminidase (NA). This mutation in particular was shown to attenuate virus growth and pathology in ferrets (17). Additionally, oseltamivir-resistant viruses with a nearby mutation in N2 neuraminidase transmitted less efficiently than oseltamivir-sensitive viruses in the guinea pig transmission model (4). Surprisingly, the seasonal 2008 H1N1 viral isolates that spread around the world had the same tyrosine mutation, which was previously associated with iatrogenic infections and attenuation. Furthermore, epidemiological studies concluded that this resistant virus developed independently of drug selection, suggesting that compensatory adaptations allowed an attenuating mutation to become permissible (3, 18). The ability of resistant 2008 isolates to perform on par with nonresistant 2008 isolates in growth curves, in mean plaque size, and in a transmission model was recently confirmed (2). Currently, 99% of seasonal H1N1 viruses are oseltamivir resistant; however, the prevalence of these viruses is very low due to replacement by a novel reassortant H1N1 virus (6, 8). This novel reassortant was originally identified in Mexico by doctors concerned about a jump in the number of influenza cases during the month of March in 2009 (7). Later referred to as swine-origin influenza virus, novel H1N1 virus, or 2009 pandemic H1N1 virus, this virus would continue to efficiently transmit around the world, even during the summer months of the northern hemisphere. Its robust transmission was later confirmed in aerosol transmission models, in which 86% of ferrets and 100% of guinea pigs exposed to infected animals contracted pandemic influenza (22, 28, 31). Oseltamivir was used broadly during the outbreak, treating those with complications and prophylactically treating close contacts of confirmed cases. The use of oseltamivir in this manner provided ample opportunity for oseltamivir-resistant viruses to develop. More than 225 cases of oseltamivir-resistant infections have been confirmed from the beginning of the pandemic, including four incidents of suspected aerosol transmission (21, 32, 33). Fortunately, these clinical isolates never progressed into stable transmission in the general public. This study seeks to evaluate if introducing the H275Y mutation into the pandemic 2009 H1N1 virus attenuates virus replication in vitro or in vivo using the guinea pig model and the ferret model to test aerosol transmission efficiency. Furthermore, this study evaluates if a reassortant between the circulating novel H1N1 virus and seasonal neuraminidase (NA) forms a well-adapted, resistant virus capable of efficient transmission.Currently, oseltamivir is the drug of choice for treating novel H1N1 complications and outpatient prophylaxis. Therefore, it is of great importance to study the in vitro replication and transmission phenotypes of oseltamivir-resistant novel H1N1 viruses to understand why broad oseltamivir resistance has not occurred or whether we should expect it to occur in the future.     

T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

A Single Amino Acid in the Stalk Region of the H1N1pdm Influenza Virus HA Protein Affects Viral Fusion,Stability and Infectivity

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.     

Mass Spectrometry-Based Comparative Sequence Analysis for the Genetic Monitoring of Influenza A(H1N1)pdm09 Virus

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.     

Reassortment and mutations associated with emergence and spread of oseltamivir-resistant seasonal influenza A/H1N1 viruses in 2005-2009

A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007-2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005-2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007-2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008-2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes.