Molecular Epidemiology of Influenza A/H3N2 Viruses Circulating in Mexico from 2003 to 2012

In this work, nineteen influenza A/H3N2 viruses isolated in Mexico between 2003 and 2012 were studied. Our findings show that different human A/H3N2 viral lineages co-circulate within a same season and can also persist locally in between different influenza seasons, increasing the chance for genetic reassortment events. A novel minor cluster was also identified, named here as Korea, that circulated worldwide during 2003. Frequently, phylogenetic characterization did not correlate with the determined antigenic identity, supporting the need for the use of molecular evolutionary tools additionally to antigenic data for the surveillance and characterization of viral diversity during each flu season. This work represents the first long-term molecular epidemiology study of influenza A/H3N2 viruses in Mexico based on the complete genomic sequences and contributes to the monitoring of evolutionary trends of A/H3N2 influenza viruses within North and Central America.     

Isolation and genetic characterization of new reassortant H3N1 swine influenza virus from pigs in the midwestern United States

Since the introduction of H3N2 swine influenza viruses (SIVs) into U.S. swine in 1998, H1N2 and H1N1 reassortant viruses have emerged from reassortment between classical H1N1 and H3N2 viruses. In 2004, a new reassortant H3N1 virus (A/Swine/Minnesota/00395/2004) was identified from coughing pigs. Phylogenetic analyses revealed a hemagglutinin segment similar to those of contemporary cluster III H3N2 SIVs and a neuraminidase sequence of contemporary H1N1 origin. The internal genes were of swine, human, and avian influenza virus origin, similar to those of contemporary U.S. cluster III H3N2 SIVs. The recovery of H3N1 is further evidence of reassortment among SIVs and justifies continuous surveillance.     

Molecular epidemiology of A/H3N2 and A/H1N1 influenza virus during a single epidemic season in the United States

To determine the spatial and temporal dynamics of influenza A virus during a single epidemic, we examined whole-genome sequences of 284 A/H1N1 and 69 A/H3N2 viruses collected across the continental United States during the 2006-2007 influenza season, representing the largest study of its kind undertaken to date. A phylogenetic analysis revealed that multiple clades of both A/H1N1 and A/H3N2 entered and co-circulated in the United States during this season, even in localities that are distant from major metropolitan areas, and with no clear pattern of spatial spread. In addition, co-circulating clades of the same subtype exchanged genome segments through reassortment, producing both a minor clade of A/H3N2 viruses that appears to have re-acquired sensitivity to the adamantane class of antiviral drugs, as well as a likely antigenically distinct A/H1N1 clade that became globally dominant following this season. Overall, the co-circulation of multiple viral clades during the 2006-2007 epidemic season revealed patterns of spatial spread that are far more complex than observed previously, and suggests a major role for both migration and reassortment in shaping the epidemiological dynamics of human influenza A virus.     

Spatial dynamics of human-origin H1 influenza A virus in North American swine

The emergence and rapid global spread of the swine-origin H1N1/09 pandemic influenza A virus in humans underscores the importance of swine populations as reservoirs for genetically diverse influenza viruses with the potential to infect humans. However, despite their significance for animal and human health, relatively little is known about the phylogeography of swine influenza viruses in the United States. This study utilizes an expansive data set of hemagglutinin (HA1) sequences (n = 1516) from swine influenza viruses collected in North America during the period 2003-2010. With these data we investigate the spatial dissemination of a novel influenza virus of the H1 subtype that was introduced into the North American swine population via two separate human-to-swine transmission events around 2003. Bayesian phylogeographic analysis reveals that the spatial dissemination of this influenza virus in the US swine population follows long-distance swine movements from the Southern US to the Midwest, a corn-rich commercial center that imports millions of swine annually. Hence, multiple genetically diverse influenza viruses are introduced and co-circulate in the Midwest, providing the opportunity for genomic reassortment. Overall, the Midwest serves primarily as an ecological sink for swine influenza in the US, with sources of virus genetic diversity instead located in the Southeast (mainly North Carolina) and South-central (mainly Oklahoma) regions. Understanding the importance of long-distance pig transportation in the evolution and spatial dissemination of the influenza virus in swine may inform future strategies for the surveillance and control of influenza, and perhaps other swine pathogens.     

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.     

Frequency of influenza H3N2 intra-subtype reassortment: attributes and implications of reassortant spread

Background

Increasing evidence suggests that influenza reassortment not only contributes to the emergence of new human pandemics but also plays an important role in seasonal influenza epidemics, disease severity, evolution, and vaccine efficacy. We studied this process within 2091 H3N2 full genomes utilizing a combination of the latest reassortment detection tools and more conventional phylogenetic analyses.

Results

We found that the amount of H3N2 intra-subtype reassortment depended on the number of sampled genomes, occurred with a steady frequency of 3.35%, and was not affected by the geographical origins, evolutionary patterns, or previous reassortment history of the virus. We identified both single reassortant genomes and reassortant clades, each clade representing one reassortment event followed by successful spread of the reassorted variant in the human population. It was this spread that was mainly responsible for the observed high presence of H3N2 intra-subtype reassortant genomes. The successfully spread variants were generally sampled within one year of their formation, highlighting the risk of their rapid spread but also presenting an opportunity for their rapid detection. Simultaneous spread of several different reassortant lineages was observed, and despite their limited average lifetime, second and third generation reassortment was detected, as well as reassortment between viruses belonging to different vaccine-associated clades, likely displaying differing antigenic properties. Some of the spreading reassortants remained confined to certain geographical regions, while others, sharing common properties in amino acid positions of the HA, NA, and PB2 segments, were found throughout the world.

Conclusions

Detailed surveillance of seasonal influenza reassortment patterns and variant properties may provide unique information needed for prediction of spread and construction of future influenza vaccines.

     

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.     

Stochastic processes are key determinants of short-term evolution in influenza a virus

Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

Molecular Epidemiology and Phylogenetic Analyses of Influenza B Virus in Thailand during 2010 to 2014

Influenza B virus remains a major contributor to the seasonal influenza outbreak and its prevalence has increased worldwide. We investigated the epidemiology and analyzed the full genome sequences of influenza B virus strains in Thailand between 2010 and 2014. Samples from the upper respiratory tract were collected from patients diagnosed with influenza like-illness. All samples were screened for influenza A/B viruses by one-step multiplex real-time RT-PCR. The whole genome of 53 influenza B isolates were amplified, sequenced, and analyzed. From 14,418 respiratory samples collected during 2010 to 2014, a total of 3,050 tested positive for influenza virus. Approximately 3.27% (471/14,418) were influenza B virus samples. Fifty three isolates of influenza B virus were randomly chosen for detailed whole genome analysis. Phylogenetic analysis of the HA gene showed clusters in Victoria clades 1A, 1B, 3, 5 and Yamagata clades 2 and 3. Both B/Victoria and B/Yamagata lineages were found to co-circulate during this time. The NA sequences of all isolates belonged to lineage II and consisted of viruses from both HA Victoria and Yamagata lineages, reflecting possible reassortment of the HA and NA genes. No significant changes were seen in the NA protein. The phylogenetic trees generated through the analysis of the PB1 and PB2 genes closely resembled that of the HA gene, while trees generated from the analysis of the PA, NP, and M genes showed similar topology. The NS gene exhibited the pattern of genetic reassortment distinct from those of the PA, NP or M genes. Thus, antigenic drift and genetic reassortment among the influenza B virus strains were observed in the isolates examined. Our findings indicate that the co-circulation of two distinct lineages of influenza B viruses and the limitation of cross-protection of the current vaccine formulation provide support for quadrivalent influenza vaccine in this region.     

Evolution of novel reassortant A/H3N2 influenza viruses in North American swine and humans, 2009-2011

Novel H3N2 influenza viruses (H3N2v) containing seven genome segments from swine lineage triple-reassortant H3N2 viruses and a 2009 pandemic H1N1 (H1N1pdm09) matrix protein segment (pM) were isolated from 12 humans in the United States between August and December 2011. To understand the evolution of these novel H3N2 viruses in swine and humans, we undertook a phylogenetic analysis of 674 M sequences and 388 HA and NA sequences from influenza viruses isolated from North American swine during 2009-2011, as well as HA, NA, and M sequences from eight H3N2v viruses isolated from humans. We identified 34 swine influenza viruses (termed rH3N2p) with the same combination of H3, N2, and pM segments as the H3N2v viruses isolated from humans. Notably, these rH3N2p viruses were generated in swine via reassortment events between H3N2 viruses and the pM segment approximately 4 to 10 times since 2009. The pM segment has also reassorted with multiple distinct lineages of H1 virus, especially H1δ viruses. Importantly, the N2 segment of all H3N2v viruses isolated from humans is derived from a genetically distinct N2 lineage that has circulated in swine since being acquired by reassortment with seasonal human H3N2 viruses in 2001-2002, rather than from the N2 that is associated with the 1998 H3N2 swine lineage. The identification of this N2 variant may have implications for influenza vaccine design and the potential pandemic threat of H3N2v to human age groups with differing levels of prior exposure and immunity.     

The genesis and spread of reassortment human influenza A/H3N2 viruses conferring adamantane resistance

A dramatic rise in the frequency of resistance to adamantane drugs by influenza A (H3N2) viruses has occurred in recent years -- from approximately 2% to approximately 90% in multiple countries worldwide-and associated with a single S31N amino acid replacement in the viral matrix M2 protein. To explore the emergence and spread of these adamantane resistant viruses we performed a phylogenetic analysis of recently sampled complete A/H3N2 genome sequences. Strikingly, all adamantane resistant viruses belonged to a single lineage (the "N-lineage") characterized by 17 amino acid replacements across the viral genome. Further, our analysis revealed that the genesis of the N-lineage was due to a 4+4 segment reassortment event involving 2 distinct lineages of influenza A/H3N2 virus. A subsequent study of hemagglutinin HA1 sequences suggested that the N-lineage was circulating widely in Asia during 2005, and then dominated the Northern hemisphere 2005-2006 season in Japan and the USA. Given the infrequent use of adamantane drugs in many countries, as well as the decades of use in the US associated with little drug resistance, we propose that the globally increasing frequency of adamantane resistance is more likely attributable to its interaction with fitness-enhancing mutations at other genomic sites rather than to direct drug selection pressure. This implies that adamantanes may not be useful for treatment and prophylaxis against influenza viruses in the long term. More generally, these findings illustrate that drug selection pressure is not the sole factor determining the evolution and maintenance of drug resistance in human pathogens.     

Establishment of influenza A virus (H6N1) in minor poultry species in southern China

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of those viruses had a common source. Continuing surveillance in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants in 1999; however, the further development of H6N1 viruses has not been investigated since 2001. Here we report influenza virus surveillance data collected in southern China from 2000 to 2005 that show that H6N1 viruses have become established and endemic in minor poultry species and replicate mainly in the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had W312-like hemagglutinin and neuraminidase genes. However, reassortment of internal genes between different subtype virus lineages, including H5N1, H9N2, and other avian viruses, generated multiple novel H6N1 genotypes in different types of poultry. These novel H6N1/N2 viruses are double, triple, or even quadruple reassortants. Reassortment between a W312-like H6N1 virus and an A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest that W312-like viruses may not be the precursors of HK/97 virus but reassortants from an HK/97-like virus and another unidentified H6 subtype virus. These results provide further evidence of the pivotal role of the live poultry market system of southern China in generating increased genetic diversity in influenza viruses in this region.     

Isolation and identification of swine influenza recombinant A/Swine/Shandong/1/2003(H9N2) virus

Ten influenza virus isolates were obtained from infected pigs from different places in Shandong province showing clinical symptoms from October 2002 to January 2003. All 10 isolates were identified in China's National Influenza Research Center as influenza A virus of H9N2 subtype. The complete genome of one isolate, designated A/Swine/Shandong/1/2003(H9N2), was sequenced and compared with sequences available in GenBank. The results of analyses indicated that the sequence of A/Swine/Shandong/1/2003(H9N2) was similar to those of several chicken influenza viruses and duck influenza viruses recently prevalent in South China. According to phylogenetic analysis of the complete gene sequences, A/Swine/Shandong/1/2003(H9N2) possibly originated from the reassortment of chicken influenza viruses and duck influenza viruses. It was found that the amino acid sequence at the HA cleavage site in Sw/SD/1/2003 is R-S-L-R-G, differing clearly from that of other H9N2 subtype isolates of swine influenza and avian influenza, which is R-S-S-R-G.     

1968−2014年中国甲型H3N2流感病毒PB1基因进化分析

【目的】分析季节性H3N2流感病毒PB1基因序列的变异情况,揭示H3N2流感病毒PB1基因的分子特征与进化趋势。【方法】对1968?2014年中国地区82株人H3N2毒株、2012?2014年江苏省分离的81株甲型H3N2流感病毒、6株SIV和4株AIV H3N2亚型PB1、PB1-F2基因进行分子进化分析。【结果】1968?2014年中国H3N2流感毒株PB1核苷酸和氨基酸相似性分别为90.91%?100%和96.91%?100%。系统进化树分析,1968?2014年共173株H3N2流感病毒总体上分为4个分支,2002?2014年分离毒株位于第IV分支上,1968?1994年分离毒株位于第II和III分支;猪源H3N2亚型分布于第I、II、IV分支上;分子特征显示PB1氨基酸52、113、179、216、576、586、619、621、709位在2002年以后发生适应性改变,替换了原来的氨基酸;PB1-F2基因编码截断型蛋白长度有52、34、25、24、11 aa (猪源),PB1-F2蛋白毒力关键位点上未出现高致病性特征突变。【结论】自1968年起H3N2亚型PB1基因变异逐步趋于稳定,且PB1-F2截断型毒株正逐渐成为一类新的进化特征,但PB1基因与其他亚型之间发生重配以及关键毒力位点的变异仍应是监测的重点。     

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.     

Identification of the progenitors of Indonesian and Vietnamese avian influenza A (H5N1) viruses from southern China

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.     

Expanded Cocirculation of Stable Subtypes,Emerging Lineages,and New Sporadic Reassortants of Porcine Influenza Viruses in Swine Populations in Northwest Germany

The emergence of the human 2009 pandemic H1N1 (H1N1pdm) virus from swine populations refocused public and scientific attention on swine as an important source of influenza A viruses bearing zoonotic potential. Widespread and year-round circulation of at least four stable lineages of porcine influenza viruses between 2009 and 2012 in a region of Germany with a high-density swine population is documented here. European avian influenza virus-derived H1N1 (H1N1av) viruses dominated the epidemiology, followed by human-derived subtypes H1N2 and H3N2. H1N1pdm viruses and, in particular, recently emerging reassortants between H1N1pdm and porcine HxN2 viruses (H1pdmN2) were detected in about 8% of cases. Further reassortants between these main lineages were diagnosed sporadically. Ongoing diversification both at the phylogenetic and at the antigenic level was evident for the H1N1av lineage and for some of its reassortants. The H1avN2 reassortant R1931/11 displayed conspicuously distinct genetic and antigenic features and was easily transmitted from pig to pig in an experimental infection. Continuing diverging evolution was also observed in the H1pdmN2 lineage. These viruses carry seven genome segments of the H1N1pdm virus, including a hemagglutinin gene that encodes a markedly antigenically altered protein. The zoonotic potential of this lineage remains to be determined. The results highlight the relevance of surveillance and control of porcine influenza virus infections. This is important for the health status of swine herds. In addition, a more exhaustive tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required.     

Comparative analysis of evolutionary mechanisms of the hemagglutinin and three internal protein genes of influenza B virus: multiple cocirculating lineages and frequent reassortment of the NP, M, and NS genes

Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged.