Reassortment and evolution of current human influenza A and B viruses

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.     

2004-2008年我国流行的A型流感病毒抗原性及HA1基因特性的研究

目的 了解我国2004-2008年A(H1N1、H3N2)型流感病毒流行情况、抗原性和基因特性变异关系,了解疫苗株与我国流行株之间抗原性变化情况.方法 选择2004年以来我国分离的A(H1N1、H3N2)型流感病毒进行抗原性及HA1区基因序列,通过比对HA1蛋白位点变异情况,分析我国流感病毒抗原性及基因特性变化情况.结果 A(H1N1)亚型流感毒株抗原性2004-2007年分离的A(H1N1)亚型流感病毒的抗原性与疫苗株A/New Caledonia/20/1999(H1N1)类似;2008年我国流行的A(H1N1)亚型毒株的抗原性与2008-2009年北半球的流感疫苗株A/Brisben/59/2007(H1N1)类似.2004-2005年分离的A(H3N2)亚型流感病毒的抗原性与疫苗株A/Fujian/411/12002(H3N2)比较发生了变异;2006-2007年我国流行的H3N2毒株与A/Wiscansin/67/2006(H3N2)类似,2008年我国流行的H3N2毒株与疫苗株A/Brisben/10/2006(H3N2)类似.结论 2004-2008年我国流行的A(H1N1、H3N2)亚型流感病毒的抗原性和基因特性发生了改变.     

武汉市2003年流感监测及当前优势流行毒株的抗原性和基因特性分析

目的 探讨2003年武汉市流感流行概况，分析优势流行毒株抗原性和基因特性变异特征。方法 每周在流感监测哨点医院统计流行病学资料，采样分离流感病毒标本，对其中3株H3病毒做血凝抑制试验并对其编码HA1区基因进行序列测定和分析。结果 从418份咽拭标本中分离到流感病毒58株，其中5，7株为H3亚型，1株为乙型；全年月分离率和门诊流感样病例数的变化有冬、夏两个高峰，最高峰均出现在7月；新分离到的3株H3病毒的抗原性与本年度疫苗组分株A／Panama／2007／99有较大差异，编码HA1区的氨基酸序列有14个位点发生了变异，基因种系发生树分析也证实他们的HA1基因存在较大差异。结论 2003年武汉市流感活动呈双峰型增强，H3病毒活动显著加强，其抗原性和基因特性与疫苗株相比均发生了较大的变异。     

Antigenic drift and variability of influenza viruses

Annual influenza epidemics are caused by rapid evolution of the viral genome. Continuous and extensive antigenic variation has been shown for hemagglutinin (HA), the principal immunizing antigen of the virus. Monitoring of the antigenicity of circulating influenza viruses is necessary for selection of the most suitable vaccine strains. In this study, characterization of influenza A/H3N2 and influenza B viruses recently circulating in Germany was performed by molecular and antigenic analysis. Sequencing and phylogenetic analysis of the HA1 gene revealed that two distinct groups of H3N2 viruses co-circulated during 1997/1998. The majority of isolates clustered with the new drift variant A/Sydney/5/97, as was also shown by antigenic characterization. A noteworthy genetic drift of H3N2 viruses was evident during the winter 1998/1999. However, serological characterization using hemagglutinin inhibition tests did not result in detection of viruses belonging to different groups as confirmed by molecular analysis. Influenza B viruses isolated during 1996/1997 were antigenically closely related to the prototype vaccine strains B/Beijing/184/93 or B/Harbin/7/94. Molecular analysis demonstrated that our German 1996/1997 isolates differed by nine amino acids from B/Harbin/7/94 and represented a group of viruses that was completely different from the Harbin strain. Retrospective studies revealed the circulation of B/Yamanshi/166/98-like viruses in Germany already during the 1996/1997 season. Our results suggest that molecular analysis of the HA gene is important to complement the antigenic characterization for a better selection of appropriate vaccine strains.     

Influenza A/Fujian/411/02(H3N2)-lineage viruses in Finland: genetic diversity, epidemic activity and vaccination-induced antibody response

Summary. The first sporadic cases of Fujian/411/02-lineage viruses were recorded in Finland in winter 2001–2002. The first protracted but low-intensity outbreak occurred here during the first half of 2003, and the second outbreak early in autumn 2003, after detection of sporadic influenza A cases in the summer. The calculated incidence of influenza A in the Finnish army was 515/10000 during the first outbreak and 2066/10000 during the second outbreak. During the 2003–2004 epidemic season, the isolates fell into three groups for their haemagglutinin (HA1) sequences. In groups I and II, the strains were reassortants which differed for their neuraminidase (NA) sequences from the viruses of the previous spring. Group II viruses, which predominated in Finland during the 2003–2004 season, were characterized by loss of the glycosylation site at position 126 in HA1. The relevance of this loss to the epidemiology is discussed, as well as the frequent appearance of codominant amino acid mixtures at position 151 lining the catalytic cavity of the NA. Group III viruses, genetically related to Wellington/1/2004, the drift variant predominant in 2004 in the southern hemisphere, caused some localized outbreaks in Finland towards the end of the 2003–2004 epidemic. The antigenic match between the vaccine virus (Panama/2007/99) and the Fujian-lineage epidemic viruses in winter 2003–2004 was far from optimal. Nevertheless, high levels of prevaccination and postvaccination antibodies to the predomi- nant group II virus were recorded. Lower antibody levels were detected to the group III virus, which turned out to be a herald strain that reappeared in Finland during the following epidemic season.     

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005

Summary. Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003–04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.     

2004年中国甲3亚型流感病毒(H3N2)抗原性及基因特性研究

目的阐明2004年中国流行的甲3亚型流感病毒血凝素抗原性及其基因变异情况。方法对2004年分离的甲3亚型毒株先进行单向血凝抑制试验及交叉血凝抑制试验;在此基础上选取不同时间、地点的甲3亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析。结果单向血凝抑制实验结果表明,2004年共有52.3%毒株与A/Fujian/411/2002(H3N2)(20042005毒株)有4倍或以上的血凝抑制滴度差异,交叉血凝抑制实验结果表明,它们间的抗原比为4。HA1区核苷酸序列和氨基酸序列分析表明,我国从2004年2月分离的甲3亚型毒株开始出现了与A/Fujian/411/2002(H3N2)和A/Wellington/1/2004(H3N2)(2005年国际代表株)相比较,在其HA1蛋白分子上存在有4个氨基酸位点(159位Y>F,189位S>N,145位K>N,226位V>I)发生了替换。此类毒株首发于我国南方,然后到我国北方。结论我国2004年2月份以后所分离的甲3亚型流感毒株已经发生抗原性及基因特性的改变。     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

Antigenic and genetic variation in the hemagglutinins of H1N1 and H3N2 human influenza a viruses in the Shanghai area from 2005 to 2008

Continued rapid evolution of the influenza A virus is responsible for annual epidemics and occasional pandemics in the Shanghai area. In the present study, the representative strains of A/H1N1 and A/H3N2 influenza viruses isolated in the Shanghai area from 2005 to 2008 were antigenically and genetically characterized. The antigenic cartography method was carried out to visualize the hemagglutination-inhibition data. Antigenic differences were detected between circulating A/H1N1 strains isolated from 2005 to 2006 and the epidemic A/H1N1 strains isolated in 2008, which were found to be associated with the amino acid substitution K140E in HA1. The present vaccine strain A/Brisbane/59/2007 is considered to be capable of providing sufficient immunity against most of the circulating A/H1N1 viruses isolated in 2008 from the Shanghai population. The study showed that there were significant antigenic differences between the epidemic A/H3N2 strains isolated in 2007 and 2008, suggesting that antigenic drift had occurred in the A/H3N2 strains isolated in 2008. The P194L mutation was thought to be responsible for the antigenic evolution of influenza A/H3N2 viruses isolated from Shanghai in 2008. Evidence of antigenic drift suggests that the influenza A/H3N2 vaccine component needs to be updated.     

Epidemiologic Study of Human Influenza Virus Infection in South Korea from 1999 to 2007: Origin and Evolution of A/Fujian/411/2002-Like Strains

Influenza epidemics arise through the accumulation of viral genetic changes, culminating in a novel antigenic type that is able to escape host immunity. Following an outbreak of the A/Fujian/411/2002-like strains in Asia, including China, Japan, and South Korea, in 2002, Australia and New Zealand experienced substantial outbreaks of the same strains in 2003, and subsequently worldwide outbreaks occurred in the 2003-2004 season. The emergence of A/Fujian/411/2002-like strains coincided with a higher level of influenza-like illness in South Korea than what is seen at the peak of a normal season, and there was at least a year''s difference between South Korea and the United States. Genetic evolution of human influenza A/H3N2 viruses was monitored by sequence analysis of hemagglutinin (HA) genes collected in Asia, including 269 (164 new) HA genes isolated in South Korea from 1999 to 2007. The Fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the HA1 domain, which sharply distinguished between the A/Moscow/10/1999-like and A/Fujian/411/2002-like strains. This fast variation, equivalent to approximately 10 amino acid changes within a year, occurred in Asia and would be the main cause of the disappearance of the reassortants, although the reassortant and nonreassortant Fujian-like strains circulated simultaneously in Asia.Influenza is an important respiratory infectious disease causing seasonal epidemics or occasional pandemics across the world, with considerable morbidity and mortality. The influenza outbreaks are associated with antigenic variation of influenza viruses. Annual influenza epidemics typically occur during the winter season in temperate regions, whereas tropical regions may function as permanent mixing pools for viruses, providing a ready source of extended viral transmission (7, 26). Antigenic and genetic analyses revealed that there was a continuous circulation of human influenza A/H3N2 viruses in East and Southeast Asia via a regional network from which epidemics in the temperate regions were seeded (22). In particular, southern China was considered a potential epicenter for the emergence of novel influenza virus strains (23).The enveloped influenza virus contains eight segments of negative-sense single-stranded RNA, each of which codes for a particular viral protein(s). The gene segment coding for a surface glycoprotein hemagglutinin (HA) is of major importance because HA is the primary target of immune response and the primary component of influenza vaccine. HA is a homotrimeric protein synthesized as a single polypeptide, HA0, that is cleaved into two subunits, HA1 and HA2, for receptor binding and cell entry (18). Antibodies against HA are elicited during virus infection to inhibit binding with receptor effectively (27). Accumulation of amino acid variation in HA is clustered in variable antigenic sites around the receptor binding site, which leads to gradual antigenic drift in the influenza viruses. It was previously proposed that an antigenic drift variant of epidemiological importance usually requires changes of at least four amino acids across two or more antigenic sites, but a single amino acid substitution at one antigenic site can cause sufficient antigenic change (9, 10). The influenza virus can also acquire a new subtype by reassortment of one or more gene segments, which, combined with antigenic drift, provides the basis for the remarkable antigenic variability in viral populations (12).A/Moscow/10/1999-like or antigenically equivalent A/Panama/2007/1999-like strains of H3N2 have been circulating worldwide since 1999. The emergence of A/Fujian/411/2002-like strains caused an epidemic in China, Japan, and South Korea in 2002 (3, 13, 21). It was shown that a two-amino-acid substitution was critical for antigenicity distinct from that of A/Panama/2007/1999 (14). Interestingly, a descendant of the Fujian strain reassorted, which caused an unusually severe influenza season in Australia and New Zealand in 2003 and in North America and Europe and worldwide in the 2003-2004 season (2, 4, 13). This reassortment caused a minor clade to provide a HA gene that later became part of the dominant strain in the same season (4, 13), reaching South America after an additional 6 to 9 months (22). The appearance of the Fujian strains thus prompted a change in the selection of vaccine components in 2004. However, nonreassortant strains were the dominant strains in Asia in the 2002-2003 season and thereafter.The elucidation of how and when a new influenza virus emerges as an epidemic strain requires a deeper understanding of the mechanisms that underlie viral evolution. We have determined the nucleotide sequence of the HA gene segments of influenza viruses in nasal swabs collected from infected patients aged 6 months and older during the 1999-2007 influenza seasons in South Korea. At the same time, influenza-like illness was monitored during each season, and the phylogeny of HA sequences available worldwide was analyzed to investigate the origin and evolution of the H3N2 Fujian strains.     

Divergent genetic evolution of hemagglutinin in influenza A H1N1 and A H1N2 subtypes isolated in the south-France since the winter of 2001-2002.

BACKGROUND: Influenza A viruses are divided into subtypes based on their hemagglutinin (H1 to H15) and neuraminidase (N1 to N9) glycoproteins. Of these, three A subtypes H1N1, H3N2 and H1N2 circulate in the human population. Influenza A viruses display a high antigenic variability called "antigenic drift" which allows the virus to escape antibody neutralization. OBJECTIVES: Evaluate the mutations apparition that might predict a divergent antigenic evolution of hemagglutinin in influenza A H1N1 and A H1N2 viruses. STUDY DESIGN: During the three winters of 2001-2002 to 2003-2004, 58 A H1N1 and 23 A H1N2 subtypes have been isolated from patients with influenza-like illness in the south of France. The HA1 region was analyzed by RT-PCR and subsequently sequenced to compare the HA1 genetic evolution of influenza A H1N1 and A H1N2 subtypes. RESULTS: Our results showed that 28 amino acid substitutions have accumulated in the HA1 region since the circulation of A/New Caledonia/20/99-like viruses in France. Of these, fifteen were located in four antigenic sites (B, C, D and E). Six of them were observed only in the A H1N2 isolates, six only in the A H1N1 isolates and three in both subtypes. Furthermore, nine of twenty two A H1N2 isolates from the winter of 2002-2003 shared a T90A amino acid change which has not been observed in any A H1N1 isolate; resulting in the introduction of a new glycosylation site close to the antigenic site E. This might mask some antigenic E determinants and therefore, modify the A H1N2 antigenicity. CONCLUSIONS: The divergent genetic evolution of hemagglutinin may ultimately lead to a significant different antigenicity between A H1N1 and A H1N2 subtypes that would require the introduction of a new subtype in the vaccine batches.     

Molecular evolution of human influenza A/H3N2 virus in Asia and Europe from 2001 to 2003

Hemagglutinin sequences of 146 human influenza A/H3N2 strains identified in respiratory specimens from Asia and Europe during the 2001-2003 influenza seasons were analyzed by DNA sequencing. Our results suggest that four amino acid substitutions, L25I, H75Q, H155T, and Q156H, led to the antigenic conversion of the previously predominant A/Panama/2007/99-like strains to the more recent A/Fujian/411/2002-like strains.     

2006年广东省流感病毒流行的分子基础

目的了解广东省2006年流行性感冒（流感）流行情况的分子基础。方法选择广东省流感监测网络实验室分离的11株流感毒株,对血凝素基因进行基因和抗原性的分析。此外,还检测人群抗体水平。结果2006年流行的H1N1亚型病毒血凝素蛋白重链（HA1）区氨基酸序列与A/New Caledonia/20/99（H1N1）相比,同源性为95.7%～96.3%,有2个抗原决定簇的氨基酸发生了替换。Victoria系的HA1区基因与B/HongKong/330/2001相比,同源性为97.2%～97.5%,有6个位于抗原决定簇的氨基酸发生了替换,这种变化和B/Malaysia/2506/2004相一致,这在B型的基因种系发生树也得到证实。同时发现人群针对H1N1亚型和B/Victoria的抗体水平较低。结论2006年广东H1N1亚型和B/Victoria系病毒株发生了一定程度的变异,以及人群保护性抗体水平较低,共同造成其在本地的流行。     

Host cell-mediated variation in H3N2 influenza viruses

The influence of the host cell on the selection of antigenic variants of influenza A H3N2 viruses and the relevance of host cell selection to the induction of immunity by these viruses have been investigated. Influenza viruses were isolated from human clinical samples during a single epidemic, were passaged in mammalian Madin-Darby Canine Kidney (MDCK) cells or in embryonated hens eggs, and were tested for antigenic variability in the hemagglutinin (HA) molecule with a panel of monoclonal antibodies. In many cases, the HA of virus cultivated in eggs was antigenically distinct from the HA of virus from the same individual grown in mammalian cells. Viruses recovered from different individuals were antigenically similar to each other when grown in mammalian cell lines yet were antigenically heterogeneous when cultivated in eggs. The HA genes of viruses isolated from different individuals during the epidemic were shown, by sequence analysis, to differ from each other by five or six amino acid residues. Sequence analyses of the HA genes of MDCK cell-grown and egg-grown virus obtained from the same individual demonstrated that the molecular changes between antigenically distinct HAs of MDCK cell- and egg-grown A/Mem/12/85 virus involved a single amino acid substitution at residue 156 in HA1, which lies at the tip of the HA molecule and immediately adjacent to the receptor-binding site. However, the amino acid sequences of HAs from MDCK-grown and egg-grown viruses (A/Mem/2/85) isolated from a second individual were identical although these viruses exhibited antigenic differences when examined with anti-HA monoclonal antibodies. Therefore, single amino acid changes in the HA molecule may not be the sole cause of antigenic changes in the HA observed between pairs of MDCK cell-grown and egg-grown viruses and genes other than that encoding the HA may contribute to the host cell-mediated antigenic variation of these viruses. Nevertheless, antigenic differences between viruses grown in eggs and MDCK cells did not influence their ability to protect, since ferrets infected with either live egg-grown or MDCK-grown virus were protected equally well from challenge with virus grown in either host cell type.     

Antigenic and Genetic Evolution of Low-Pathogenicity Avian Influenza Viruses of Subtype H7N3 following Heterologous Vaccination

Outbreaks of low-pathogenicity avian influenza (LPAI) viruses of the H7N3 subtype were first detected in Italy in October 2002, and the virus continued to circulate between 2002 and 2004 in a densely populated poultry area in the northeast portion of that country. This virus circulated in unvaccinated and vaccinated poultry farms, and the infection was controlled in August 2003 by culling, control of movements, improved biosecurity, and heterologous vaccination. In 2004, H7N3 reoccurred in vaccinated poultry farms in which infection had been successfully controlled by the vaccination program. To shed light on this occurrence and the temporal pattern and genetic basis of antigenic drift for avian influenza viruses (AIVs) in the absence and presence of heterologous vaccination, a collection of H7N3 viruses isolated in 2002 and 2004 were characterized genetically and antigenically. Molecular analysis showed that viruses isolated in the 2004 outbreaks after the implementation of vaccination had acquired specific amino acid signatures, most of which were located at reported antibody binding sites of the hemagglutinin (HA) protein. Antigenic characterization of these 2004 isolates showed that they were antigenically different from those isolated prior to the implementation of vaccination. This is the first report on antigenic and genetic evolution of H7 LPAI viruses following the application of heterologous vaccination in poultry. These findings may have an impact on control strategies to combat AI infections in poultry based on vaccination.     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.     

Molecular and antigenic evolution of human influenza A/H3N2 viruses in Quebec, Canada, 2009-2011

Background

A/H3N2 variability leads to poor vaccine effectiveness when the vaccine strain is not well matched to the circulating virus.

Objectives

We aim to describe the molecular and antigenic evolution of A/H3N2 viruses recovered during the last 3 influenza seasons in Quebec, Canada.

Study design

Clinical samples from 33 patients with culture-confirmed A/H3N2 infections were collected over 3 consecutive seasons (March 2009-2011). The isolates’ HA gene was amplified and sequenced; phylogenetic analyses of the HA1 region were conducted. To characterize A/H3N2 antigenic properties, standard hemagglutination inhibition (HI) and microneutralization (MN) assays were performed.

Results

In 2009, we observed an antigenic drift from A/Brisbane/10/2007 (vaccine strain used in 2008-2009 and 2009-2010) to A/Perth/16/2009 (vaccine strain used in 2010-2011). Antigenic analysis of clinical influenza strains recovered in Quebec during 2009-2010 also illustrated antigenic drift from the previously prevalent A/Brisbane/10/2007-like (March 2009) to A/Perth/16/2009-like (December 2009) strains. In 2010-2011, the emergence of >4 substitutions in 4 different H3 antigenic sites suggested a genetic drift. However, HI and MN results confirmed the emergence of a drift in only 1 strain (8-fold difference in titers), while 19 others remained antigenically similar to A/Perth/16/2009 but exhibited titer differences (2-4-fold) just inferior to the standard definition of a drift.

Conclusion

Antigenic and molecular characterization of H3N2 viruses over three seasons revealed that not only is the number of HA mutations important, but the nature and location of key mutations may play a significant role in antigenic drift.     

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.     

Characterization of influenza A/Fujian/411/2002(H3N2)-like viruses isolated in Portugal between 2003 and 2005

In Portugal, influenza surveillance is achieved through the National Influenza Surveillance Programme (NISP), in close collaboration with other European and global surveillance networks. The NISP integrates epidemiological, clinical and virological data based on the information collected by a Network of Sentinel Medical Practitioners and by a network of Emergency Units of Hospitals and Health Care Centres. In this study, genetic and antigenic characterization of influenza A viruses of the A/Fujian/411/2002 lineage, isolated during the 2003/2004 and 2004/2005 influenza winter seasons, in the context of the NISP, are described. Antigenic analysis of A/Fujian/411/2002-like viruses, first detected and isolated during the 2003/2004 winter season, revealed a close similarity with the reference strains A/Kumamoto/102/2002 and A/Wyoming/3/2003. Genetic analysis confirmed this similarity and revealed two different phylogenetic branches. The 2004/2005 influenza A(H3) isolates formed, both antigenic and genetically, a more homogeneous group and were closely related to A/Oslo/807/2004 and A/California/7/2004. During this season, the characterization of the influenza viral strains has shown continuous evolution to variants close related to A/Oslo/807/2004. The majority of amino acid substitutions detected in the haemagglutinin occurred at antigenic sites. This study reflects the contribution of individual countries for the surveillance and knowledge of the molecular epidemiology of the infection, essential for a concerted action towards the global monitoring of the disease. It also reflects the importance of constant monitoring of genetic and antigenic characteristics of circulating influenza strains, which will certainly be a major contribution to the formulation of influenza vaccines.     

Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies

Antigenic variation among influenza B viruses is different from that of influenza A in several ways. Antigenic shift has not been observed, distinct antigenic variants of influenza B cocirculate, and antigenically similar viruses have been isolated many years apart. To study the mechanism of antigenic drift in influenza B viruses, monoclonal antibodies were used to select antigenic variants of B/Hong Kong/8/73 virus hemagglutinin (HA). Analyses of the nucleotide sequences of the HA gene of B/Hong Kong/8/73 and the eight variants identified specific regions of the influenza B HA molecule involved in antigenicity, and enabled antigenic mapping data to be correlated with the structure of the protein. The altered amino acids in the variants, when compared to the HA of A/Aichi/2/68, were found in two of the four antigenic regions previously identified for type A viruses. In addition, four of the eight variants showed multiple nucleotide changes some of which gave rise to double amino acid changes. In addition, in the present study monoclonal antibodies which belong to the same antigenic group recognize amino acid changes in regions corresponding to antigenic sites A and B of the H3 HA. These results are in contrast to those obtained with HA variants of A/Memphis/1/71 virus. In the influenza A studies only single amino acid changes were found and these correlated well with the three-dimensional structure as determined by D. C. Wiley, I. A. Wilson, and J. J. Skehel, (1981, Nature (London) 289, 366-373); monoclonal antibodies which recognized one region did not recognize any of the other antigenic sites. Our results suggest that although the basic three-dimensional structure of the influenza B HA may be similar to that of A viruses, the B HA molecule may be folded in a more compact manner so that antigenic sites A and B are in closer proximity to each other than in the H3 structure.