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.     

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.     

Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.     

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 analysis of intraepidemic variants of influenza A (H3N2) viruses by hyperimmune rat antisera

Hyperimmune rat antisera prepared against 5 recent antigenic variants of influenza A (H3N2) viruses were studied for haemagglutination inhibiting (HI) antibodies to the homologous and the heterologous viruses. The ratios of homologous to heterologous reactions varied from one animal to another in immunizations with each of the immunogens. Some antisera exhibited a ratio high enough to allow differentiation of the epidemic variants and demonstration of an intraepidemic heterogeneity of field strains isolated during the outbreak of 1985/86. The variation of cross-reactions of polyclonal antisera may reflect differences in the range of specificities of anti-haemagglutinin antibodies produced by individual animals. The significance of this finding in the classification of influenza A (H3N2) viruses is discussed. Lack of nonspecific inhibitors interfering with the HI test is an additional advantage of hyperimmune rat antisera in typing influenza A and B virus isolates.     

Selection of receptor-binding variants of human influenza A and B viruses in baby hamster kidney cells.

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D --> G (H1N1 virus), 128T --> A and 226I --> V (H3N2), and 187N --> D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L --> H, 208R --> H, and 225D --> G (H1N1), 194L --> I (H3N2), and 137G --> R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3'-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6'-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6'-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.     

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

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

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.     

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial.     

1999-2007年佛山市流感病毒活动情况及甲3亚型病毒HA1基因分析

目的总结1999-2007年佛山市流感病毒流行情况,分析甲3亚型（H3N2）毒株流行与其HA1基因进化的关系。方法用MDCK细胞分离流感病毒,培养后血凝阳性者进行型别鉴定。随机抽取每年2～3株甲3亚型毒株的细胞培养物提取RNA后进行HA1基因的逆转录,对其核苷酸和氨基酸序列及亲缘关系进行分析。结果甲型和乙型流感病毒在佛山市人群中同时流行。甲型流感毒株是人群感染流感的主要型别。HA1区氨基酸序列与历年的流感疫苗推荐株相比,点突变率为0.3%～6.08%。发生替换的重要位点包括了抗原决定簇的17个位点、抗体结合部位的10个位点和1个糖基化位点。结论佛山市的甲1和甲3亚型流感毒株活动呈此起彼伏的优势株转换现象。甲3亚型新旧毒株交替迅速,大致按照毒株分离的年代聚类成进化树的不同小侧枝,表明新的流行株出现后可能迅速突破地域局限。提示地区实验室及时监测和研究流感毒株的发生和发展是流感监测网络建设的基础。     

Mismatch between the 1997/1998 influenza vaccine and the major epidemic A(H3N2) virus strain as the cause of an inadequate vaccine-induced antibody response to this strain in the elderly

The success of influenza vaccination depends largely on the antigenic match between the influenza vaccine strains and the virus strains actually circulating during the season. In the past, this match has proved to be satisfactory in most seasons. In the 1997/1998 season, however, hemagglutination inhibition (HI) assays with ferret antisera indicated a considerable mismatch between the H3N2 vaccine component and the most prevalent epidemic influenza A(H3N2) virus. The results from antigenic analyses using pre- and postvaccination serum samples from volunteers of various ages, including residents of nursing homes who were more than 60 years of age, were in good agreement with the results obtained with ferret antisera. Homologous serum antibody responses to the H3N2 vaccine component as well as the cross-reactivity of the induced antibodies to the epidemic H3N2 strain, declined with increasing age of the vaccinees. As a consequence of these two effects, 84% of the vaccinees over 75 years of age did not develop HI antibody titers >/= 40 against the major H3N2 virus variant of 1997/1998, suggesting that they were not protected against infection with this virus variant. These findings support the current policy of the World Health Organization (WHO), which is to base worldwide influenza virus surveillance on results predominantly obtained by antigenic analyses of influenza virus isolates with ferret antisera in HI tests. If an antigenic mismatch is observed, the protective efficacy of the vaccine, especially for the elderly, may be insufficient. The observations also support the current policy to include the elderly in serologic efficacy trials.     

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.     

High Non‐Specific T Lymphocyte Response to the Adjuvanted H1N1 Vaccine in Comparison with the H1N1/H3N2/B‐Brisbane Vaccine without Adjuvant

Shortly after the report of pandemic 2009 influenza A (H1N1), vaccine manufacturers, in conjunction with public agencies, started developing a H1N1 vaccine. In 2009, various approaches were implemented around the globe. The United States and Australia finally approved only non‐adjuvanted H1N1 influenza vaccines, whereas Canada and the EU also approved adjuvanted vaccines. In 2010, seasonal influenza vaccine without adjuvant was again widely accepted in both hemispheres. The addition of adjuvant to the vaccine enhances the immunogenity of the vaccine in the presence of a relatively low amount of antigen. However, it might also induce undesirable non‐specific immune response. For this reason, we conducted a prospective observational study to monitor T cell absolute count and H1N1‐specific immunogenicity after 2009 and 2010 immunization. Fourteen healthy volunteers received the monovalent H1N1 AS03 adjuvanted influenza vaccine (3.5 μg of H1N1 and squalene‐based adjuvant) in October 2009. The immunization was associated with a significant increase in T lymphocyte absolute count (P < 0.0001), reaching abnormal values in 57% of subjects. During this period, none of the subject showed any manifestation of severe viral infection or inflammation. Acute infection by CMV or EBV viruses was also excluded. In October 2010, the same subjects received a seasonal non‐adjuvanted influenza vaccine (15 μg of each: H1N1, H3N2, and B‐Brisbane). However, after 2010 immunization, no change in T lymphocyte absolute count was observed. H1N1‐induced immunogenicity was good for both vaccines. Our results suggest a pronounced non‐specific T cell response after AS03‐adjuvanted 2009 H1N1 vaccination.     

Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A(H1N1) virus

Antigenic changes are often induced in the haemagglutinin (HA) of human isolates of influenza A(H1N1) and B viruses during their isolation and propagation in embryonated hens' eggs. In this report we describe molecular changes in the HA of an A(H1N1) virus which accompany egg adaptation. The HA1 amino acid substitutions of egg-adapted variants are located in the vicinity of the receptor binding site. This suggests that egg-adapted variants are selected on the basis of altered receptor site specificity with concomitant effects on the antigenic configuration of the HA molecule. In this study, at least three antigenically distinct groups of egg-adapted variants were observed. These observations have implications for the indiscriminate use of egg-adapted viruses in sero-epidemiological studies and vaccine production.     

Extent of antigenic cross-reactivity among highly pathogenic H5N1 influenza viruses

Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization (MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics. Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the best candidate diagnostic reagent for broad detection of H5N1 strains.     

Influenza virus infections from 0 to 2 years of age: A birth cohort study

Background/purposeInfluenza vaccine has been recommended in Finland since 2007 for all children of 6–35 months of age and in 2009 for those ≥6 months against pandemic influenza. We investigated the incidence of influenza and vaccine effectiveness in a birth cohort of children in 2008–2011.MethodsWe followed 923 children from birth to 2 years of age for respiratory tract infections. A nasal swab sample for PCR for influenza A and B viruses was taken at the onset of acute respiratory infections. Samples were collected either at the study clinic or at home by parents. Vaccination data was retrieved from the health registries.ResultsVaccination coverage of children aged 6–23 months was 22–47% against seasonal influenza and 80% against the A(H1N1)pdm09 virus in the pandemic season 2009–2010. During 3 influenza seasons, 1607 nasal swab samples were collected. Influenza was confirmed in 56 (6.1%) of 923 children (16 A(H1N1), 14 A(H3N2), and 26 B viruses). The incidence of influenza was 5.1% in 2008–2009, 2.7% in 2009–2010, and 5.0% in 2010–2011. Effectiveness of the adjuvanted vaccine against the pandemic influenza A(H1N1)pdm09 was 97% (95% confidence interval, 76–100%). Three children with influenza were hospitalized.ConclusionThe yearly incidence of seasonal influenza was 5% in this cohort of very young children with variable influenza vaccine coverage. Adjuvanted vaccine against the pandemic influenza was highly effective. Both seasonal and pandemic influenza cases were mostly non-severe.     

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.     

Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses.

Propagation of human influenza viruses in embryonated chicken eggs (CE) results in the selection of variants with amino acid substitutions near the receptor-binding site of the hemagglutinin (HA) molecule. To evaluate the mechanisms by which these substitutions enable human virus growth in CE, we studied the binding of 10 human influenza A (H1N1, H3N2) and B strains, isolated and propagated solely in MDCK cells, and of their egg-adapted counterparts to preparations of cellular membranes, gangliosides, sialylglycoproteins, and sialyloligosaccharides. All egg-adapted variants differed from nonadapted strains by increased binding to the plasma membranes of chorio-allantoic (CAM) cells of CE and by the ability to bind to CAM gangliosides. In addition, there was no decrease in affinity for inhibitors within allantoic fluid. These findings indicate that growth of human influenza viruses in CE is restricted because of their inefficient binding to receptors on CAM cells and that gangliosides can play an important role in virus binding and/or penetration. The effects of the egg-adaptation substitutions on the receptor-binding properties of the viruses include (i) enhancement of virus binding to the terminal Sia(alpha2-3)Gal determinant (substitutions in HA positions 190, 225 of H1N1 strains and in position 186 of H3N2 strains); (ii) a decrease of steric interference with more distant parts of the Sia(alpha2-3Gal)-containing receptors (a loss of glycosylation sites in positions 163 of H1 HA and 187 of type B HA); and (iii) enhanced ionic interactions with the negatively charged molecules due to charged substitutions at the tip of the HA [187, 189, 190 (H1), and 145, 156 (H3)]. Concomitantly with enhanced binding to Sia(alpha2-3)Gal-terminated receptors, all egg-adapted variants decreased their affinity for equine macroglobulin, a glycoprotein bearing terminal 6'-sialyl(N-acetyllactosamine)-moieties.     

Further studies of the antigenic properties of H3N2 strains of influenza A isolated from swine in South East Asia.

H3N2 strains of influenza A isolated from swine in Hong Kong were compared with human strains of H3N2 influenza A variants in reciprocal HI tests using ferret sera. One isolate from swine was indistinguishable from A/Hong Kong/68, one set of viruses isolated in 1976 and 1977 was most related to A/Hong Kong/68 but was not identical to it, two isolates from 1976 were 'bridging strains' that cross-reacted equally with the contemporary variants A/Victoria/3/75 and A/Texas/1/77, similarly to a small number of recent human isolates, and two isolates from 1977 were similar to A/Victoria/3/75. These general relationships were supported by neuraminidase inhibition tests. The findings confirm and extend previous results indicating that swine may be a reservoir of old and novel variants of influenza A H3N2 strains related to those that infect man.     

Persistence of Q strain of H2N2 influenza virus in avian species: Antigenic,biological and genetic analysis of avian and human H2N2 viruses

Summary The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5^–/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific -inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42° C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42° C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957.With 2 Figures