HA Of Influenza Research Articles

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus.

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.

Phylogenetic analysis of HA and NA genes of influenza A viruses in immunosuppressed inpatients in Beijing during the 2018–2020 influenza seasons

BackgroundInfluenza A viruses have undergone rapid evolution with virulent; however, complete and comprehensive data on gene evolution and amino acid variation of HA and NA in immunosuppressed patients was few. In this study, we analysed molecular epidemiology and evolution of influenza A viruses in immunosuppressed population, and immunocompetent population were used as controls.MethodsFull sequences of HA and NA of A(H1N1)pdm09 and A(H3N2) were acquired through reverse transcription-polymerase chain reaction (RT-PCR). HA and NA genes were sequenced using the Sanger method and phylogenetically analysed using ClustalW 2.10 and MEGA software version 11.0.ResultsDuring the 2018–2020 influenza seasons, 54 immunosuppressed and 46 immunocompetent inpatients screened positive for influenza A viruses by using the quantitative real-time PCR (qRT-PCR) were enrolled. 27 immunosuppressed and 23 immunocompetent nasal swab or bronchoalveolar lavage fluid samples were randomly selected and sequenced using the Sanger method. A(H1N1)pdm09 were detected in 15 samples and the remaining 35 samples were A(H3N2) positive. By analyzing the HA and NA gene sequences of these virus strains, we found that all A(H1N1)pdm09 viruses shared high similarities to each other and the HA and NA genes of these viruses exclusively belonged to subclade 6B.1A.1. Some NA genes of A(H3N2) viruses were not in the same clade as those of A/Singapore/INFIMH-16-0019/2016 and A/Kansas/14/2017, which may have led to A(H3N2) being the dominant strain in the 2019–2020 influenza season. Both A(H1N1)pdm09 and A(H3N2) viruses showed similar evolutionary lineages patterns of HA and NA between immunosuppressed and immunocompetent patients. Compared with the vaccine strains, there were no statistically significant of HA and NA genes and amino acid sequences of influenza A viruses in immunosuppressed and immunocompetent patients. However, the oseltamivir resistance substitution of NA-H275Y and R292K have been observed in immunosuppressed patients.ConclusionsA(H1N1)pdm09 and A(H3N2) viruses showed similar evolutionary lineages patterns of HA and NA between immunosuppressed and immunocompetent patients. Both immunocompetent and immunosuppressed patients have some key substitutions, which should be of note monitored, especially those with potential to affect the viral antigen.

Phylogenetic analysis and clinical characteristics of the co-occurring mutations in HA and NA genes of influenza A(H1N1)pdm09 viruses during 2015\u20132017 in Beijing, China

BackgroundInfluenza A(H1N1)pdm09 viruses have undergone rapid evolution, and in recent years the complementary and antagonistic effects of HA and NA have gathered more attentions; however, the effects of co-occurring mutations in HA and NA on the patients’ clinical characteristics are still poorly understood. In this study, we analyzed molecular epidemiology and evolution of A(H1N1) pdm09, explored co-occurring mutations of HA and NA, and investigated effect of co-occurring mutations on patients’ clinical features.MethodsA(H1N1)pdm09 was confirmed by reverse transcription-polymerase chain reaction. HA and NA genes were sequenced and phylogenetically analyzed. Clinical characteristics of the co-occurring mutations were analyzed statistically.ResultsBy analyzing the HA and NA gene sequences of 33 A(H1N1)pdm09 viruses during the 2015–2017 influenza season, we found that all the viruses shared high similarities to each other and the HA genes of these viruses exclusively belonged to subclade 6B.1A. Several unreported substitutions in HA and NA proteins were observed, furthermore, co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). Sore throat was significantly associated with co-occurring mutations in HA and NA of A(H1N1)pdm09 (χ2, P < 0.05).ConclusionsCo-occurring mutations in HA and NA were detected in A(H1N1)pdm09 isolated during 2015–2017 in Beijing. Symptomatically, sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09. Therefore, studying the effect and mechanism of co-occurring mutations in HA and NA on patients’ clinical features is of note needed.

Molecular evolution of influenza B virus during 2011\u20132017 in Chaoyang, Beijing, suggesting the free influenza vaccine policy

Two influenza B virus lineages, B/Victoria and B/Yamagata, are co-circulating in human population. While the two lineages are serologically distinct and TIV only contain one lineage. It is important to investigate the epidemiological and evolutionary dynamics of two influenza B virus lineages in Beijing after the free influenza vaccine policy from 2007. Here, we collected the nasopharyngeal swabs of 12657 outpatients of influenza-like illness and subtyped by real-time RT-PCR during 2011–2017. The HA and NA genes of influenza B were fully sequenced. The prevalence is the highest in the 6–17 years old group among people infected with influenza B. Yamagata-lineage virus evolved to two inter-clade from 2011–2014 to 2014–2017. The amino acids substitutions of HA1 region were R279K in strains of 2011–2014 and L173Q, M252V in strains of 2014–2017. Substitutions L58P, I146V were observed in HA1 region of Victoria-lineage virus in 2011–2012 and I117V, N129D were showed in 2015–2017. Phylogenetic analysis of NA showed Yamagata-Victoria inter-lineage reassortant occurred in 2013–2014. Influenza B mainly infect the school-aged children in Beijing and the free influenza vaccine inoculation does not seem to block school-age children from infection with influenza B. The antigen characteristics of circulating influenza B were different to the recommended vaccine strains. We concluded that the Victoria-lineage vaccine strain should been changed and the free influenza vaccine should be revalued.

Mutations in the H7 HA and PB1 genes of avian influenza a viruses increase viral pathogenicity and contact transmission in guinea pigs

ABSTRACT Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Differences in the ease with which mutant viruses escape from human monoclonal antibodies against the HA stem of influenza A virus

BackgroundBroadly protective human monoclonal antibodies that recognize the conserved epitopes in the HA of influenza A virus are being developed as therapeutic agents. Emergence of resistant viruses must always be considered when developing therapeutic agents against influenza. ObjectivesWe examined human hetero-reactive mAbs against the HA stem of influenza A virus for the ease with which escape mutant viruses emerged. Study designWe attempted to generate the mutant viruses escaped from the hetero-reactive anti-HA stem antibodies. We also evaluated their protective efficacy, binding affinity, and epitopes. ResultsWe obtained several human monoclonal antibodies (mAbs) that react with the HA of different HA subtypes of influenza A virus belonging to group 1. Upon attempting to generate escape mutant viruses, we found that the ease with which such viruses emerged differed among the mAbs; viruses barely escaped from two of the mAbs (clones S9-3-37 and F20C77), whereas escape from the third mAb (clone F5B7) occurred readily. Comparisons of the mAbs revealed that the HA stem epitopes, in vitro neutralization potency, binding affinity to H1-HA, and protective efficacy against lethal challenge with H1N1pdm09 virus were all comparable. ConclusionsThese results demonstrate the importance of determining the ease with which escape mutant viruses emerge when evaluating anti-HA stem antibodies as antiviral agents during preclinical testing.

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

Fast on-site diagnosis of influenza A virus by Palm PCR and portable capillary electrophoresis.

A method combining Palm polymerase chain reaction (PCR) and portable capillary electrophoresis (CE) was developed for rapid on-site analysis of influenza A (H1N1) virus. The portable CE system was suitable for rapid diagnosis which was able to detect a sample in ∼4 min after sample loading, while the 'Palm PCR' system allowed for high-speed nucleic acid amplification in ∼16 min. The analysis time from DNA sample to analysis of amplified target DNA molecule was only ∼20 min, which was significantly less than slab gel electrophoresis with other commercially available PCR machine. When the 100-bp DNA ladder was separated, the relative standard deviation values (n=5) for the migration times and peak areas of the 100 and 200-bp DNA molecules were 0.26 and 8.9%. The detection limits were 6.3 and 7.2 pg/μL, respectively. The combined method was also able to identify two influenza A-associated genes (the HA and NP genes of the novel H1N1 influenza). CE separation was achieved with a sieving matrix of 1% poly(vinylpyrrolidone) (Mr=1,300,000) in 1× TBE buffer (pH 8.45). The combined Palm PCR-portable CE system should provide an improved, fast on-site molecular genetic diagnostic method.

Glycosylation on Hemagglutinin Affects the Virulence and Pathogenicity of Pandemic H1N1/2009 Influenza A Virus in Mice

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.

A randomized clinical trial of an inactivated avian influenza A (H7N7) vaccine.

BackgroundConcern for a pandemic caused by a newly emerged avian influenza A virus has led to clinical trials with candidate vaccines as preparation for such an event. Most trials have involved vaccines for influenza A (H5N1), A (H7N7) or A (H9N2).ObjectiveTo evaluate dosage-related safety and immunogenicity of an inactivated influenza A (H7N7) vaccine in humans.DesignOne hundred twenty-five healthy young adults were randomized to receive two doses intramuscularly of placebo or 7.5, 15, 45 or 90 µg of HA of an inactivated subunit influenza A (H7N7) vaccine (25 per group), four weeks apart. Reactogenicity was evaluated closely for one week and for any adverse effect for six months after each dose. Serum hemagglutination-inhibiting and neutralizing antibody responses were determined four weeks after each dose and at six months.ResultsReactogenicity evaluations indicated the vaccinations were well tolerated. Only one subject developed a ≥4-fold serum hemagglutination-inhibition (HAI) antibody response and a final titer of ≥1∶40 four weeks after dose two and only five subjects developed a neutralizing antibody rise and a final titer of ≥1∶40 in tests performed at a central laboratory. Four of the five were given the 45 or 90 µg HA dosage. A more sensitive HAI assay at the study site revealed a dose-response with increasing HA dosage but only 36% in the 90 µg HA group developed a ≥4-fold rise in antibody in this test and only one of these achieved a titer of ≥1∶32.ConclusionThis inactivated subunit influenza A (H7N7) vaccine was safe but poorly immunogenic in humans.Trials RegistrationClinicalTrials.gov NCT00546585

The neuraminidase and matrix genes of the 2009 pandemic influenza H1N1 virus cooperate functionally to facilitate efficient replication and transmissibility in pigs

The 2009 pandemic H1N1 virus (pH1N1) contains neuraminidase (NA) and matrix (M) genes from Eurasian avian-like swine influenza viruses (SIVs), with the remaining six genes from North American triple-reassortant SIVs. To characterize the role of the pH1N1 NA and M genes in pathogenesis and transmission, their impact was evaluated in the background of an H1N1 triple-reassortant (tr1930) SIV in which the HA (H3) and NA (N2) of influenza A/swine/Texas/4199-2/98 virus were replaced with those from the classical H1N1 A/swine/Iowa/15/30 (1930) virus. The laboratory-adapted 1930 virus did not shed nor transmit in pigs, but tr1930 was able to shed in infected pigs. The NA, M or both genes of the tr1930 virus were then substituted by those of pH1N1. The resulting virus with both NA and M from pH1N1 grew to significantly higher titre in cell cultures than the viruses with single NA or M from pH1N1. In a pig model, only the virus containing both NA and M from pH1N1 was transmitted to and infected sentinels, whereas the viruses with single NA or M from pH1N1 did not. These results demonstrate that the right combination of NA and M genes is critical for the replication and transmissibility of influenza viruses in pigs.

Viral genetic sequence variations in pandemic H1N1/2009 and seasonal H3N2 influenza viruses within an individual, a household and a community

Background There are few data in the literature on viral sequence variation between host generations/successive transmission events. Relatively little is known about the sequence heterogeneity of the influenza viruses transmitted within families. Objectives To study the molecular epidemiology of influenza virus and to determine the sequence variation within an individual, a household and a community during the first wave of influenza pandemic in 2009. Study design A prospective study of household transmission of influenza A in Hong Kong was conducted during the pandemic in 2009. The HA and NA sequences of pandemic and seasonal influenza A viral isolates identified in this household transmission study were sequences and analyzed. Results Our results indicated that there were multiple introductions of influenza viruses into Hong Kong. Sequence analysis of these isolates suggested that members of these family clusters acquired the infection by household transmissions. Interestingly, unlike those concluded from previous household transmission studies, we observed sequence variations between sequential samples from the same person and also within the same household. Conclusions Family clusters of influenza A viral infection are predominantly the result of secondary transmission within a household. Our results also suggested that the intra-host viral sequence variation might be more common that than previously thought.

Glycosylation Site Alteration in the Evolution of Influenza A (H1N1) Viruses

Influenza virus typically alters protein glycosylation in order to escape immune pressure from hosts and hence to facilitate survival in different host environments. In this study, the patterns and conservation of glycosylation sites on HA and NA of influenza A/H1N1 viruses isolated from various hosts at different time periods were systematically analyzed, by employing a new strategy combining genome-based glycosylation site prediction and 3D modeling of glycoprotein structures, for elucidation of the modes and laws of glycosylation site alteration in the evolution of influenza A/H1N1 viruses. The results showed that influenza H1N1 viruses underwent different alterations of protein glycosylation in different hosts. Two alternative modes of glycosylation site alteration were involved in the evolution of human influenza virus: One was an increase in glycosylation site numbers, which mainly occurred with high frequency in the early stages of evolution. The other was a change in the positional conversion of the glycosylation sites, which was the dominating mode with relatively low frequency in the later evolutionary stages. The mechanisms and possibly biological functions of glycosylation site alteration for the evolution of influenza A/H1N1 viruses were also discussed. Importantly, the significant role of positional alteration of glycosylation sites in the host adaptation of influenza virus was elucidated. Although the results still need to be supported by experimental data, the information here may provide some constructive suggestions for research into the glycosylation of influenza viruses as well as even the design of surveillance and the production of viral vaccines.

Molecular epidemiology and antigenic analyses of influenza A viruses H3N2 in Taiwan

The severity of an influenza epidemic season may be influenced not only by variability in the surface glycoproteins, but also by differences in the internal proteins of circulating influenza viruses. To better understand viral antigenic evolution, all eight gene segments from 44 human H3N2 epidemic strains isolated during 2004–2008 in Taiwan were analyzed to provide a profile of protein variability. Comparison of the evolutionary profiles of the HA, NA and PB2 genes of influenza A (H3N2) viruses indicated that they were derived from a group of H3N2 isolates first seen in 2004. However, the PA, M and PB1 genes were derived from a different group of H3N2 isolates from 2004. Tree topology revealed the NP and NS genes could each be segregated into two groups similar to those for the polymerase genes. In addition, new genetic variants occurred during the non-epidemic period and become the dominant strain after one or two seasons. Comparison of evolutionary patterns in consecutive years is necessary to correlate viral genetic changes with antigenic changes as multiple lineages co-circulate.

Preclinical evaluation of a modified vaccinia virus Ankara (MVA)-based vaccine against influenza A/H5N1 viruses

Highly pathogenic avian influenza viruses of the H5N1 subtype are responsible for an increasing number of infections in humans since 2003. More than 60% of the infections is lethal and new infections are reported frequently. In the light of the pandemic threat caused by these events the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the HA gene of an influenza A/H5N1 virus is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous influenza A/H5N1 viruses in mice. We also evaluated the recombinant MVA vector expressing the HA of influenza A/H5N1 virus A/Vietnam/1194/04 (MVA-HA-VN/04) in non-human primates. Cynomolgus macaques were immunized twice and then challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity. Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia. Therefore MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic avian influenza A/H5N1 viruses.

High genetic and antigenic similarity between a swine H3N2 influenza A virus and a prior human influenza vaccine virus: A possible immune pressure-driven cross-species transmission

In late April of 2009, a global outbreak of human influenza was reported. The causative agent is a highly unusual reassortant H1N1 influenza virus carrying genetic segments derived from swine, human and avian influenza viruses. In this study, we compared the HA, NA and other gene segments of a swine H3N2 influenza A virus, A/Swine/Guangdong/z5/2003, which was isolated from pigs in 2003 in Guangdong Province, China, to the predominant human and swine H3N2 viruses. We found that the similarity of gene segments of A/Swine/Guangdong/z5/2003 was closer to Moscow/99-like human H3N2 virus than Europe swine H3N2 viruses during 1999–2002. These results suggest that A/Swine/Guangdong/z5/2003 may be porcine in origin, possibly being driven by human immune pressure induced by either natural H3N2 virus infection or use of A/Moscow/10/99 (H3N2)-based human influenza vaccine. The results further confirm that swine may play a dual role as a “shelter” for hosting influenza virus from humans or birds and as a “mixing vessel” for generating reassortant influenza viruses, such as the one causing current influenza pandemic.

Type and subtype distribution of influenza virus and genetic evolution of hemagglutinin in Shanghai area in duration of 2004 - 2008

To analyze the type and subtype distribution of influenza virus and the genetic evolution of hemagglutinin (HA) in Shanghai area during 2004 to 2008. All 962 throat swabs were collected from influenza-like patients in 5 influenza sentry hospitals and influenza outbreaks. Influenza viruses were isolated in MDCK cell lines, and then viral types and subtypes were identified. The HA of influenza A isolates selected by outbreak or sporadic patients in different areas and epidemic seasons were sequenced and analyzed by phylogenetic trees. A/H3N2, accounting for 54.9% (162/295), was the dominate subtype in recent years, but less popular in the end of 2005 to the middle of 2006 with 0% (0/16)and 23.5% (8/34) of positive specimen, respectively. There were more A/H1N1 isolates in 2005 - 2006 with 21.4% (12/56), 43.8% (7/16) and 76.5% (26/34) of positive specimen, respectively, but declined obviously in 2007 - 2008 accounting for only 0% (0/44) and 5.0% (7/139). Influenza B virus was more popular in 2004 to 2005 with 42.9% (24/56) and 56.2% (9/16), respectively, and not isolated from 2006 to 2007, then increased in 2008 accounting for 34.5% (48/139). Phylogenetic tree of HA showed that A/H1N1 isolates in the same year clustered from 2005 to 2008, and most A/H3N2 isolated were homologous in the same year during 2004 - 2008 while some were inserted to the clusters of near years and more distinguished sequences appeared. A/H1N1 and A/H3N2 isolates were all similar to the vaccine strains recommended by WHO. The distribution of influenza type and subtype kept on changing each year, but A/H3N2 dominated in most years. A/H1N1 and A/H3N2 in the same year clustered, but some A/H3N2 of near years were and evolved faster with more distinguished strains appeared in same interval. Generally, HA of influenza A isolates in Shanghai during 2004 to 2008 were similar to the WHO reference strains.

An Efficient RNA Aptamer against Human Influenza B Virus Hemagglutinin

Aptamers are known for their higher discriminating ability between closely related molecules and their requirement for only a small region for binding, as compared to an antibody. In the present studies, we have isolated a specific RNA aptamer against the influenza virus B/Johannesburg/05/1999 by an in vitro selection procedure. The aptamer bound efficiently to the HA of influenza B and required 5 mM MgCl(2) ion for its recognition. The aptamer not only distinguished HA derived from the influenza A virus, but also inhibited HA-mediated membrane fusion.

Cellular virology

Cellular virology

Recent changes among human influenza viruses

Recurrent epidemics of influenza are due to the frequent emergence of antigenic variants. With co-circulation of two influenza A subtypes and two antigenically distinct lineages of B viruses, genetic reassortment also has an important role in antigenic drift, as illustrated by recent changes in both A and B viruses. The H1N2 subtype viruses, which emerged during 2001, possessed a H1 HA similar to those of contemporary A/New Caledonia/20/99 (H1N1)-like viruses and seven genes closely related to those of recent H3N2 viruses, and did not represent a significant increase in the antigenic diversity of circulating viruses. The re-emergence of B/Victoria/2/87-lineage viruses, previously prevalent during the 1980s, in 2000 has been followed by the predominant circulation of reassortant B viruses possessing a B/Victoria-lineage HA and a B/Yamagata-lineage NA similar in sequence to those of recent B/Sichuan/379/99-like viruses. These events emphasize not only the lack of divergence in the complementary functional characteristics of the HA and NA of divergent influenza B lineages, but also the apparent convergence in compatibility between the H1 and N2 components of the two influenza A subtypes.