An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses

A newly developed lectin neuraminidase test (LNT) and a panel of mouse monoclonal and post-infection ferret antibodies have been used to analyse antigenic drift in N1 neuraminidases of influenza A viruses isolated between 1933 and 1957 and also between 1977 and 1980. Significant antigenic differences were detected among the 'early' (1933-57) viruses since the NA of viruses isolated one year apart could be distinguished serologically. The NA of the 're-emerged' virus A/USSR/92/77 (H1N1) was antigenically related but not identical to influenza A viruses isolated in 1949 (A/Paris/49 (H1N1), A/Geneva/49 (H1N1) which thus predates the previously observed antigenic similarity of A/USSR/77 with A/FW/50 (H1N1) virus.     

Laboratory-based surveillance of influenza virus in the United States during the winter of 1977--1978. II. Isolation of a mixture of A/Victoria- and A/USSR-like viruses from a single person during an epidemic in Wyoming, USA, January 1978

At a time when outbreaks and sporadic cases of influenza caused a A/Victoria/3/75-like and A/Texas/1/77-like H3N2 strain of influenza were occurring in the Rocky Mountain region of the USA, about 60% of the students of a high school in Cheyenne, Wyoming, were involved in an outbreak of influenza-like illness. Six influenza A(H1N1) virus isolates were obtained from throat swabs collected from 12 of these students. Virus isolated from a seventh student, however, contained a mixture of H1 and H3 (A/Victoria/3/75-like) hemagglutinins and N1 and N2 neuraminidases, as shown by the ability to clone from the mixture viruses with antigenic components H1N1, H3N1, and H3N2. An antigenic hybrid virus with H3N1 composition was re-isolated from the original throat swab. The results show that one student was shedding a mixture of A/Victoria/3/75(H3N2)-like and A/USSR/90/77(H1N1)-like viruses at the time his throat swab was taken.     

Antigenic and genetic characterization of influenza viruses circulating in Bulgaria during the 2015/2016 season

Influenza virological surveillance is an essential tool for early detection of novel genetic variants of epidemiologic and clinical significance. The aim of this study was to determine the antigenic and molecular characteristics of influenza viruses circulating in Bulgaria during the 2015/2016 season. The season was characterized by dominant circulation of A(H1N1)pdm09 viruses, accounting for 66% of detected influenza viruses, followed by B/Victoria-lineage viruses (24%) and A(H3N2) viruses (10%). All sequenced influenza A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses belonged to the 6B.1, 3C.2a and 1A genetic groups, respectively. Amino acid analysis of 57 A(H1N1)pdm09 isolates revealed the presence of 16 changes in hemagglutinin (HA) compared to the vaccine virus, five of which occurred in four antigenic sites, together with 16 changes in neuraminidase (NA) and a number of substitutions in proteins MP, NP, NS and PB2. Despite the many amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically closely related to A/California/7/2009 vaccine virus. Bulgarian A(H3N2) strains (subclade 3C.2a) showed changes at 11 HA positions four of which were located in antigenic sites A and B, together with 6 positions in NA, compared to the subclade 3C.3a vaccine virus. They contained unique HA1 substitutions N171K, S312R and HA2 substitutions I77V and G155E compared to Bulgarian 3C.2a viruses of the previous season. All 20 B/Victoria-lineage viruses sequenced harboured two substitutions in the antigenic 120-loop region of HA, and 5 changes in NA, compared to the B/Brisbane/60/2008 vaccine virus. The results of this study reaffirm the continuous genetic variability of circulating seasonal influenza viruses and the need for continued systematic antigenic and molecular surveillance.     

2012-2018年青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征

目的 了解2012-2018年青岛市人群A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征。方法 收集2012年4月-2018年3月间青岛市A(H1N1)pdm09毒株397份,逆转录聚合酶链反应(RT-PCR)扩增神经氨酸酶(Neuraminidase,NA)和血凝素(Hemagglutinin,HA)基因全长,序列测定后进行耐药位点和氨基酸变异及进化分析。结果 5株发生了H275Y突变,为奥司他韦耐药株;另有4株S247N突变,可能为奥司他韦耐药株。2012-2018年H275Y突变株检出率依次为2.8 %、2.0 %、0.0 %、1.1 %、0.0 %和0.7 %。NA和HA进化树显示,2012-2013年青岛H275Y突变株与美国耐药株A/Tennessee/03/2013更接近,2013-2014年青岛H275Y突变株与国内株和日本札幌耐药株更接近,这两个年度耐药株的毒株起源可能有所不同。突变株在酶活性位点、抗原决定簇、受体结合位点及其他功能位点(如HA位点D222、Q223和NA位点V241I、N369K和N386K)的转变与野生敏感株一致。结论 青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株明显增加且流行起源不同,但并未取得比野生株更强的流行能力。奥司他韦仍可作为流感预防和治疗的有效手段。     

广州市甲型H1N1流感病毒的持续进化变异监测

目的 通过对H1N1流感病毒进行基因进化变异监测,为H1N1流感的科学防控提供研究数据.方法 对广州市2017-2019年132株H1N1流感病毒进行血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)基因测序,分析不同流行年度病毒的分子变异特点.结果2017-2019年广州市H1...     

Antibody status to influenza A/Singapore/1/57(H2N2) in Finland during a period of outbreaks caused by H3N2 and H1N1 subtype viruses.

The incidence of haemagglutination inhibition (HI) antibody (titre greater than or equal to 12) to influenza A/Singapore/1/57(H2N2) in sera collected from a Finnish population in the summer of 1981 was 58%. Subjects born after 1968 were essentially seronegative, and a comparable low HI antibody status was also recorded among the elderly, the lowest being in people born during the period 1901-10. A small increase in antibody titre to the H2N2 virus was observed in the different age groups after infections with the H3N2, but not the H1N1, subtype influenza A viruses. The heterotypic response, which could be due to HI or NA antibodies, was restricted almost exclusively to subjects already exhibiting this antibody in acute phase sera. Moreover, the anamnestic boosting was not as strong as that described in earlier studies from samples collected at the beginning of the present era of H3N2 viruses. At population level, the HI antibody status to H2N2 was about the same at the beginning and end of the follow-up period which covered eight epidemic seasons. The results are discussed with respect to the doctrine of ''original antigenic sin'' and to the threat of re-emergence of the H2N2 viruses.     

Comparison of influenza viruses isolated from man and from whales.

Four isolates of influenza virus strains from Moscow and Habarovsk that caused outbreaks of influenza in November and December 1977 in several cities of the USSR were studied and their haemagglutinins and neuraminidases were compared with those of other human and animal influenza viruses including A/whale/Pacific Ocean/76. In H1 tests these isolates, designated A/USSR/77, reacted with immune serum against A/FM/1/47 (H1N1) to the homologous titre, and with antiserum against A/whale/PO/19/76 virus to 1/8 of the homologous titre. In neuraminidase inhibition tests all A/USSR/77 isolates showed the presence of human N1 type neuraminidase, more closely related to A/sw/New Jersey/76 (Hsw1N1) than to A/FM/1/47 (H1N1) virus. The haemagglutinin of A/whale/Pacific Ocean/19/76 virus occupies an intermediate position between H0 and H1, but its neuraminidase is close to Nav2. The virus from whales multiplies better at low (28°C) and at high (40°C) temperatures than do the viruses of human origin that were tested.     

甲型H1N1流感病毒基因组序列分析及其特性研究

目的 分析甲型H1N1流感病毒的基因组序列特征,阐明该毒株的遗传变异及分子特性.方法 GenBank中获取流感病毒全序列,对各段基因与已知序列进行分析比较,绘制进化树,并分析和预测甲型毒株的致病性、药物敏感性和现有疫苗的预防保护作用.结果 甲型H1N1病毒的HA、PB2、PB1、PA、NP、NS基因与美国本土的猪流感病毒序列具有高度同源性,NA和M基因具有典型的欧亚株系猪流感病毒特征.该病毒具有人传人的分子基础,HA上HA1和HA2裂解位点序列为PSIQSR↓+GLFGAI,尚不具备高致病性流感病毒的特征.病毒对金刚烷胺类药物耐药,而对达菲和扎那米韦敏感.HA片段5个抗原决定区氨基酸序列与人用流感疫苗具有较大差异,推测现有疫苗对预防本次疫情基本无效.结论 甲型H1N1是一种北美和欧亚两种猪流感病毒的混合体,开发针对本病毒的流感疫苗有助于进一步控制疫情蔓延.     

Recent zoonoses caused by influenza A viruses

Influenza is a highly contagious, acute illness which has afflicted humans and animals since ancient times. Influenza viruses are part of the Orthomyxoviridae family and are grouped into types A, B and C according to antigenic characteristics of the core proteins. Influenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals and birds, occasionally producing devastating pandemics in humans, such as in 1918, when over twenty million deaths occurred world-wide. The two surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host and therefore show the greatest variation. For influenza A viruses, fifteen antigenically distinct HA subtypes and nine NA subtypes are recognised at present; a virus possesses one HA and one NA subtype, apparently in any combination. Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. In the 20th Century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these epidemics is most effectively measured by monitoring excess mortality due to pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be the source of all influenza A viruses in other species. Human pandemic strains are thought to have emerged through one of the following three mechanisms: genetic reassortment (occurring as a result of the segmented genome of the virus) of avian and human influenza A viruses infecting the same host direct transfer of whole virus from another species the re-emergence of a virus which may have caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and H9N2 have been transmitted from birds to humans but have apparently failed to spread in the human population. Such incidents are rare, but transmission between humans and other animals has also been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian viruses occurs in an intermediate animal with subsequent transference to the human population. Pigs have been considered the leading contender for the role of intermediary because these animals may serve as hosts for productive infections of both avian and human viruses and, in addition, the evidence strongly suggests that pigs have been involved in interspecies transmission of influenza viruses, particularly the spread of H1N1 viruses to humans. Global surveillance of influenza is maintained by a network of laboratories sponsored by the World Health Organization. The main control measure for influenza in human populations is immunoprophylaxis, aimed at the epidemics occurring between pandemics.     

Molekulare Analyse humaner Influenzaviren

The evolution of influenza viruses is increasingly pursued by molecular analyses that complement classical methods. The analyses focus on the two surface proteins hemagglutinin (HA) and neuraminidase (NA) which determine the viral antigenic profile. Influenza A(H3N2) viruses are exceptionally variable, so that usually at least two virus variants cocirculate at the same time. Together with influenza B viruses they caused approximately 90% of influenza virus infections in Germany during the last 12 seasons, while influenza A(H1N1) viruses only played a subordinate part. Unexpectedly, reassorted viruses of subtype A(H1N2) appeared during the seasons 2001/02 and 2002/03, but were isolated only rarely and gained no epidemiological significance. Furthermore, during the season 2001/02 influenza B viruses of the Victoria-lineage reappeared in Germany and other countries of the northern hemisphere after 10 years of absence. These viruses reassorted with the cocirculating Yamagata-like influenza B viruses, as could be seen by the appearance of viruses with a Victoria-like HA and a Yamagata-like NA.     

Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy

In order to explore the occurrence of antigenic drift in swine influenza A(H1N1) viruses and the match between epidemic and vaccine strains, 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and reference strains from earlier outbreaks were examined using serological and molecular methods. In contrast to swine H3N2 viruses, no significant antigenic drift was observed in swine H1N1 viruses isolated from the late 1980s up to 1996 inclusive. However, a marked antigenic and genetic heterogeneity in haemagglutination inhibition tests and nucleotide sequence analyses was detected among the 26 recent swine H1N1 virus strains. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 virus variants at the various farms of the study and points to a marked difference between the epidemiologies of human and swine influenza viruses. The observed heterogeneity may hamper the control of swine influenza by vaccination and indicates that the efficacy of current swine influenza vaccines requires re-evaluation and that the antigenic reactivity of swine influenza viruses should be monitored on a regular basis.     

Influenza activity in China: 1998-1999

During 1989-1999, influenza A H3N2 and H1N1 subtypes and B type viruses were still co-circulating in human population in China, while influenza A (H3N2) virus was predominant strain. The two antigenically and genetically distinguishable strains of influenza B virus were also still co-circulating in men in southern China. The antigenic analysis indicated that most of the H3N2 viruses were A/Panama/2007/99 (H3N2)-like strain, the most of the H1N1 viruses were antigenically similar to A/Beijing/262/95 (H1N1) virus. However, most of the influenza B viruses were B/Beijing/184/93-like strain, but few of them were antigenically similar to B/Shandong/7/97 virus. In the summer of 1998, the influenza outbreaks caused by H3N2 subtype of influenza A virus occurred widely in southern China. Afterwards, during 1998-1999 influenza season, a severe influenza epidemic caused by H3N2 virus emerged in northern China. The morbidity was reached as high as 10% in Beijing area. It was interesting that during influenza, surveillance from 1998 to 1999, five strains of avian influenza A (H9N2) virus were isolated from outpatients with influenza-like illness in July-August of 1998, and another one was repeatedly isolated from a child suffering from influenza-like disease in November of 1999 in Guangdong province. The genetic analysis revealed that the five strains isolated in 1998 were genetically closely related to H9N2 viruses being isolated from chickens (G9 lineage virus), whereas, A/Guangzhou/333/99 (H9N2) virus was a reassortant derived from reassortment between G9 and G1 lineage of avian influenza A (H9N2) viruses due to its genes encoding the HA, NA, NP and NS proteins, closely related to G9 lineage virus, the rest of the genes encoding the M and three polymerase (PB2, PB1 and PA) were closely related to G1 lineage strain of H9N2 virus. However, no avian influenza A (H5N1) virus has so far been isolated neither from in or outpatients with influenza-like disease in mainland China. Unfortunately, where did the reassortment occur and how did the reassortant transmit to men? These questions are still unknown.     

The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.     

Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province,China

From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been performed.Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade 2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6 vaccine strain).     

郴州市2009年流感病原学监测及A(H1N1)NA基因特征研究

[目的]了解2009年郴州市流感病毒流行株的病原学特征,为流感防控提供科学依据。[方法]采集哨点医院ILI咽拭子标本,用MDCK细胞进行病毒分离,采用HA和HI试验对流感病毒进行分型鉴定;采集暴发疫情ILI咽拭子用PCR法检测流感病毒核酸;随机抽取4、10月份的季节性A(H1N1)流感毒株进行NA基因测序,测序结果与国内各省市流行株及WHO推荐的北半球疫苗株作同源性比对,绘制种系进化树。[结果]分离哨点医院监测标本2284份,阳性254份:其中季节性A(H1N1)109株、A(H3N2)97株、甲型H1N130株、B型18株;PCR检测3024份标本,甲型H1N1核酸阳性1002份,阳性率33.13%;BLAST比对结果显示,4、10月份的季节性A(H1N1)毒株与2009年WHO推荐的北半球疫苗毒株A/Brisbane/59/2007的核酸同源性分别为99%和98%;种系进化分析结果表明4月份分离株与疫苗株亲缘关系最近,而10月份分离株较4月份分离株已明显发生变异。[结论]2009年郴州市甲型流感病毒异常活跃,其中1～6月季节性A(H1N1)为优势毒株,7～9月A(H3N2)为优势毒株,10～12月甲型H1N1为优势毒株,且出现甲型H1N1流感大流行。季节性A(H1N1)病毒在流行过程中NA基因已经发生了一定的变异,有必要持续跟踪和监测其变异情况。     

河南省甲型H1N1流感病毒神经氨酸酶基因进化分析

目的分析河南省甲型H1N1流感病毒神经氨酸酶(NA)基因的变异情况,了解其变异现状及特点。方法对35株甲型H1N1流感病毒毒株提取病毒总RNA,设计引物运用RT-PCR技术扩增编码NA蛋白的基因序列,测序分析核酸序列并用MEGA4.1软件构建进化树,用BLAST进行比对分析。结果分离株NA基因316位G/A和742位A/G(N端106位V/I和248位N/D)发生变异,2个突变位点同时存在;同时,通过BLAST分析,发现我国多个省份和亚洲其它多个地区病毒株NA基因存在945位A/G和1 338位T/C突变,进化树分析发现,这两个位点的突变可能来自于中国猪-人之间相互感染。结论甲型H1N1流感病毒神经氨酸酶(NA)基因在中国内地传播期间个别核酸位点发生了突变,其氨基酸抗原区有一位点发生变异(106 V/I)。     

深圳市2005—2007年H1N1亚型流感病毒HAl基因分子流行病学研究

目的 探讨2005-2007年深圳市H1N1流感病毒HA1基因变异特征。方法 选取深圳市2005-2007年分离的H1N1流感毒株，提取病毒RNA，用RT-PCR扩增HA1区基因片段，产物纯化后测序并进行基因序列分析。结果 2005-2007年流感病毒分离率平均为7.16%，H1N1流感病毒在2005年和2006年的分离数占总分离数的比例分别为56.14%和66.03%，而2007年仅占3.61%。核苷酸同源性和基因进化树结果一致，2005年4月份之前分离株与A/New Caledonia/20/1999为同一分支，2005年5月份之后的分离株与A/Solomon Island/3/2006为一支，而2006-2007年分离株又与国家代表株A/GDLH/219/2006在一个分支。氨基酸序列分析显示，绝大多数的毒株均在第130位点缺失一个赖氨酸；2005年5月以后的大部分毒株出现以下氨基酸变异：T82K、Y94H、R146K、R209K、T267N，2006年5月份之后的毒株在抗原决定簇B区发生了A190T、H193Y、E195D氨基酸变异，同时也发生A区R146K的置换。但所有毒株的潜在糖基化和受体结合位点均比较保守。发现1株病毒A/SZ/68/2007具特殊性，经与参照毒株比较，其326个氨基酸中有50个发生变化，其中有11个位于抗原决定簇位点、6个位于受体结合位点，且有4个氨基酸变化导致糖基化位点丢失。结论 2005-2007年深圳市人群中至少有3个类型HA1基因不同的H1N1流感病毒株；由于氨基酸变异引起病毒发生抗原漂移，其代表株为A/GDLH/219/2006；发现的A/SZ/68/2007病毒毒株具有特殊性，其抗原特性和流行病学意义还有待探讨。     

北京市2010年-2011年儿童甲型H3N2流感病毒神经氨酸酶基因进化和耐药性位点分析

目的:了解2010年9月1日-2011年8月31日期间北京市儿童甲型H3N2病毒的神经氨酸酶基因进化和耐药性位点情况。方法:采集儿童流感样病例标本,分离与鉴定流感病毒。选取14株甲型H3N2流感毒株进行神经氨酸酶(NA)基因的扩增和序列测定。并对NA的基因变异、进化特点和耐药性位点进行分析。结果:共分离64株甲型H3N2毒株。序列分析发现2010年-2011年毒株NA抗原决定簇氨基酸位点发生变异。进化树分析结果显示NA基因进化为不同的两支。毒株NA耐药性位点无变异。结论:北京市2010年-2011年儿童甲型H3N2流感毒株NA基因呈现两个不同的进化分支,基因特性有明显改变,尚未有耐药位点变异。     

天津地区2005年甲1亚型流感病毒HA1区基因特性分析

目的 了解天津地区夏季一起小学校流感样暴发疫情中分离出2株甲1亚型流感病毒株HA1基因变异情况。方法MDCK细胞和鸡胚双腔法分离流感病毒，收获病毒液提取病毒的RNA，进行RT—PCR，扩增产物纯化后测序，用DNASTAR软件进行序列分析。结果新分离株HA1基因长度为325个氨基酸，与国际疫苗株A／NewCaledonia／20／99相比氨基酸同源性高于98％，氨基酸替换位点有5个，其中165位位于抗原决定簇Cal区。结论天津地区新分离甲1亚型流感病毒抗原性发生进一步漂移，但变异程度不大。     

The haemagglutinins of influenza A (H1N1) viruses in the 'O' or 'D' phases exhibit biological and antigenic differences.

Influenza A (H1N1) viruses when initially isolated in mammalian cell cultures (MDCK cells) had different agglutination reactions with chicken and guinea-pig erythrocytes compared to the same viruses after passage. On first isolation the virus HA resembled the ''O'' phase viruses described originally by Burnet and Bull and agglutinated mammalian but not avian erythrocytes. After passage, the virus HA resembled a classical ''D'' phase virus and agglutinated both avian and mammalian erythrocytes. Monoclonal and polyclonal antisera detected antigenic differences between the HAs of the viruses in the ''O'' and ''D'' phases. The ''O'' phase virus HA reacted preferentially with antibodies in post infection human antisera. Viruses in the ''O'' phase replicated poorly in the allantoic cavity of embryonated hens'' eggs whilst ''D'' phase virus replicated in both MDCK cells and in embryonated hens'' eggs. At least three distinguishable subpopulations of influenza A (H1N1) viruses may co-exist in clinical throat swab material, including viruses possessing HAs in the ''O'' and ''D'' phases and other ''D'' phase viruses cultivable in embryonated hens'' eggs but antigenically distinguishable from the corresponding ''D'' phase virus in MDCK cells.