Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Effects of a hemagglutinin D222G substitution on the pathogenicity of 2009 influenza A (H1N1) virus in mice

The surface glycoprotein hemagglutinin (HA) of influenza virus initiates the infection process by binding to sialic acid receptors on upper respiratory cells in the host. In contrast to avian influenza viruses, which bind to sialic acids connected by an α2-3 linkage to the penultimate galactose, human influenza viruses prefer sialic acids with an α2-6 linkage. Recently, there have been multiple cases of severe human infections associated with an HA D222G mutant influenza virus. In this study, we have investigated the pathogenic effects of the HA D222G substitution in a 2009 pandemic H1N1 virus in mice. Compared with the A/Korea/01/2009 (K/09) virus, the HA D222G mutant showed reduced growth in cells and reduced binding avidity to human and turkey red blood cells. In a BALB/c mouse infection model, infection with the HA D222G mutant virus resulted in less body weight loss when compared to the parental K/09 virus. Altogether, our data suggest that the HA D222G substitution in the K/09 virus might be deleterious to viral fitness.     

Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health

Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.     

Characterization of an influenza A virus in Mexican swine that is related to the A/H1N1/2009 pandemic clade

In the spring of 2009, swine-origin influenza H1N1pdm09 viruses caused the first influenza pandemic of this century. We characterized the influenza viruses that circulated early during the outbreak in Mexico, including one newly sequenced swine H1N1pdm09 virus and three newly sequenced human H1N1pdm09 viruses that circulated in the outbreak of respiratory disease in La Gloria, Veracruz. Phylogenetic analysis revealed that the swine isolate (A/swine/Mexico/4/2009) collected in April 2009 is positioned in a branch that is basal to the rest of the H1N1pdm09 clade in two (NP and PA) of the eight single-gene trees. In addition, the concatenated HA-NA and the complete whole-genome trees also showed a basal position for A/swine/Mexico/4/2009. Furthermore, this swine virus was found to share molecular traits with non-H1N1pdm09 H1N1 viral lineages. These results suggest that this isolate could potentially be the first one detected from a sister lineage closely related to the H1N1pdm09 viruses.     

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.     

Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi, India

Genetic analysis of pandemic 2009 influenza A (H1N1; H1N1pdm09) virus was undertaken to understand virus evolution during 2009 and 2010 in India. Surveillance of influenza viruses from July 2009 to December 2010 revealed major peaks of circulating H1N1pdm09 viruses in August–September and December–January 2009 and then in August–September 2010. To understand the diversity of the H1N1pdm09 virus, selected specimens (n = 23) from 2009 or 2010 were characterized by nucleotide sequence determination of the HA1 subunit of the HA gene. Phylogenetic analysis revealed that 22 clustered with clade 7 viruses characterized by S203T mutations, whereas one virus from 2010 fell within clade 6. None of the viruses from either 2009 or 2010 formed a monophyletic group, suggesting a continuum of independent introduction of circulating viral strains. Amino acid analysis revealed minor amino acid changes in the antigenic or receptor‐binding domains. Importantly, we observed mutations that were also present in 1918 pandemic virus, which includes S183P in 4 and S185T mutation in 3 of 13 viruses analyzed from 2010, while none of the 2009 viruses carried these mutations. Whether antibody‐mediated pressure is imposing such changes remains to be determined. Continued genetic surveillance is warranted to monitor pathogenicity as the virus evolves to acquire new features. J. Med. Virol. 84:386–393, 2012. © 2011 Wiley Periodicals, Inc.     

Immunization with a low dose of hemagglutinin-encoding plasmid protects against 2009 H1N1 pandemic influenza virus in mice

A vaccine against the novel pandemic influenza virus (2009 H1N1) is available, but several problems in preparation of vaccines against the new emerging influenza viruses need to be overcome. DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. To evaluate the ability of a DNA vaccine encoding the hemagglutinin (HA) of 2009 H1N1 to generate humoral responses and protective immunity, BALB/c mice were immunized with various doses of 2009 H1N1 HA-encoding plasmid and anti-HA total IgG, hemagglutination inhibition antibodies and neutralizing antibodies were assayed. The total IgG titers against HA correlated positively with the doses of DNA vaccine, but immunization with either a low dose (10 μg) or a higher dose (25-200 μg) of HA plasmid resulted in similar titers of hemagglutination inhibition and neutralizing antibodies, following a single booster. Further, 10 μg plasmid conferred effective protection against lethal virus challenge. These results suggested that the DNA vaccine encoding the HA of 2009 H1N1 virus is highly effective for inducing neutralizing antibodies and protective immunity. DNA vaccines are a promising new strategy for the rapid development of efficient vaccines to control new emerging pandemic influenza viruses.     

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009. © 2009 Wiley‐Liss, Inc.     

Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice

A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and a virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6 months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.     

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice

In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus and potential application to serodiagnosis

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were generated after fusing spleen cells from a mouse immunized with rHA1 protein derived from influenza strain A/California/06/09 H1N1 with a mouse myeloma cell line. Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4, and 9E12 were reactive in Western blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 inhibited haemagglutination of turkey red blood cells with recombinant NIBRG‐121 virus derived from A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with NIBRG‐121 was localized to the membrane/cytoplasm for four of the reactive Mabs. The differing reactivity of the Mabs in Western blots, immunostaining, EIA, and haemagglutination inhibition assay suggest that at least four of the five Mabs recognize different epitopes on HA1 of the pandemic influenza A/H1N1 2009 virus. Ferret antisera to pandemic influenza A/H1N1 2009 (A/England/195/09 and A/California/07/09 strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine (CELTURA®, Novartis Vaccines, Germany), inhibit binding of 1F5‐HRP to biotinylated rHA1 derived from A/California/06/09 in a competitive EIA, suggesting that the epitope recognized by this Mab also evokes an antibody response in infected ferrets and vaccinated humans. J. Med. Virol. 83:559–567, 2011. © 2011 Wiley‐Liss, Inc.     

唐山市甲型H1N1流感病毒血凝素基因序列分析

目的 分析2010—2016年唐山市甲型H1N1流感病毒血凝素(hemagglutinin,HA)基因序列进化特征.方法 选取唐山市3家哨点医院流感样病例分离到的24株甲型H1N1病毒,通过RT-PCR和测序方法获得HA基因的全长序列,运用分子生物学软件和统计学软件对序列进行拼接、比对和分析.结果 同源进化分析显示,24株甲型H1N1流感病毒HA基因与疫苗株A/California/7/2009的核苷酸和氨基酸的同源性分别为97.0%~99.0%和97.0%~98.5%.进化分析显示,2010—2016年唐山地区流行的甲型H1N1流感病毒属于1、7、6三个基因分支,其中6分支毒株分为6C、6B、6B.1和6B.2亚支.氨基酸位点分析显示,不同毒株与疫苗株比较存在8~16处氨基酸位点改变,其中7个变异涉及3个抗原表位:H138Q/Y和S203T突变位于Ca区,N125S、K153E、S162N、K163T/Q突变位于Sa区,S185T突变位于Sb区同时也位于受体结合部位;2015—2016流行季6B.1分支毒株抗原位点S162N突变增加了新的潜在糖基化位点.结论 与疫苗株比较,随着时间推移唐山地区甲型H1N1流感病毒发生了抗原漂变,未来仍应关注6B分支流行株的变化.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.     

Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets

A 2009 H1N1 influenza virus pandemic, which had its origin in swine, caused severe illness and mortality in humans. Inflammatory responses may be responsible for pathogenesis caused by infection with influenza viruses. To better understand the pathogenic mechanism, clinical signs and inflammatory responses in ferrets infected with the pandemic H1N1 were compared with those caused by seasonal H1N1 influenza virus. Ferrets infected with the 2009 pandemic H1N1 virus displayed higher body temperatures, greater reduction in body weight, and higher viral titers in the tracheae and lungs. Levels of inflammatory cytokines, including interleukin-6, interferon-alpha, and tumor necrosis factor-alpha, were higher in the lungs of ferrets infected with the 2009 pandemic H1N1. The data support the idea that increased pathogenesis caused by the 2009 pandemic H1N1 influenza virus may have been partially mediated by a higher induction of pro-inflammatory cytokines in the lungs of affected humans or animals.     

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009)

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.     

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.     

Identification of oseltamivir resistance among pandemic and seasonal influenza A (H1N1) viruses by an His275Tyr genotyping assay using the cycling probe method

Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.     

Adamantane- and oseltamivir-resistant seasonal A (H1N1) and pandemic A (H1N1) 2009 influenza viruses in Guangdong, China, during 2008 and 2009

Adamantane and oseltamivir resistance among influenza viruses is a major concern to public health officials. To determine the prevalence of antiviral-resistant influenza viruses in Guangdong, China, 244 seasonal A (H1N1) and 222 pandemic A (H1N1) 2009 viruses were screened for oseltamivir resistance by a fluorescence-based neuraminidase (NA) inhibition assay along with NA gene sequencing. Also, 147 seasonal A (H1N1) viruses were sequenced to detect adamantane resistance markers in M2. Adamantane-resistant seasonal A (H1N1) viruses clustering to clade 2C were dominant in 2008, followed by oseltamivir-resistant seasonal A (H1N1) viruses, clustering to clade 2B during January and May 2009. In June 2009, a lineage of double-resistant seasonal A (H1N1) viruses emerged, until it was replaced by the pandemic A (H1N1) 2009 viruses. The lineage most likely resulted from reassortment under the pressure of the overuse of adamantanes. As all viruses were resistant to at least one of the two types of antiviral agents, the need for close monitoring of the prevalence of antiviral resistance is stressed.