Phylogenetic analysis of H7 haemagglutinin subtype influenza A viruses

Summary.  A 945 nucleotide region (bases 76–1020) of the HA1 part of the HA gene was obtained for 31 influenza viruses of H7 subtype isolated primarily from Europe, Asia and Australia over the last 20 years. These were analysed phylogenetically and compared with sequences of the same region from 23 H7 subtype viruses available in Genbank. The overall results showed two geographically distinct lineages of North American and Eurasian viruses with major sublineages of Australian, historical European and equine viruses. Genetically related sublineages and clades within these major groups appeared to reflect geographical and temporal parameters rather than being defined by host avian species. Viruses of high and low virulence shared the same phylogenetic branches, supporting the theory that virulent viruses are not maintained as a separate entity in waterfowl. Received October 25, 1999/Accepted November 18, 1999     

Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype

A 380 nucleotide region (bases 613 to 992) of the HA1 part of the haemagglutinin (H) gene was obtained for 35 influenza viruses of H9 subtype isolated from around the world over the past 33 years. These were analyzed phylogenetically and compared with sequences from 19 H9 subtype viruses available in the Genbank database. These viruses do not show such clear geographical lineages as other subtypes (i.e. H5 or H7) and there is a high degree of variation at the cleavage site of the haemagglutinin. Genetically distinct lineages of H9 viruses have circulated contemporaneously in different locations. Thus, it is likely that the numerous infections of poultry and other birds with H9 subtype influenza viruses during the 1990s originate from separate introductions from feral birds. The observed heterogeneity of these viruses may reflect the gene pool for H9 viruses, which is maintained in shorebirds and gulls (Charadriiformes).     

Use of Staphylococcus aureus for rapid radioimmunoassay of influenza A virus haemagglutinin.

In a rapid method for the radioimmunoassay (RIA) of influenza A virus haemagglutinin, Staphylococcus aureus (strain Cowan I, Czechoslovak State Collection No Mau 55/64) was used for separation of bound and free antigens. With rabbit and human immune sera, the binding of antigen-antibody complexes to heat-killed, formalin-fixed staphylocci was comparable to the double antibody technique. The time required for the completion of binding reaction was about 10 min compared to 18--24 hr required for double antibody precipitation. S. aureus did not bind directly (i.e. in the absence of specific antibody) a significant amount of radiolabelled antigen.     

The variable codons of H3 influenza A virus haemagglutinin genes

Summary. We have analyzed several sets of well-studied haemagglutinin (HA) gene sequences of H3 subtype influenza A viruses to identify codons that are unusually variable, using a simple pairwise sliding window method, DnDscanning. For two of the sets there were results of detailed phylogenetic modeling studies of selection already published. A third set had been the subject of an antigen mapping study, the results of which provide a completely independent benchmark of selected changes in H3 HA genes. Our analyses show that the codons with greatest DnDscan scores (i.e. the most variable) were mostly those reported in the published studies as being positively selected; indeed the DnDscan results matched the antigenic mapping results more closely than did those of the phylogenetic modeling methods. These results suggest that codons under selection can be found even when, as with some sets of virus sequences, a phylogeny is uncertain or cannot be obtained because, for example, the sequences are recombinants, or when selection is not necessarily linked with phylogeny, as in host-switching events. The program DnDscan is available at .     

Antigenic and molecular characterization of subtype H13 hemagglutinin of influenza virus

Influenza A viruses with subtype H13 hemagglutinin display an unusual host range. Although common in shorebirds, they are very rare or absent in wild ducks; additionally, H13 viruses have been isolated from a whale. To study the molecular basis for this host range, we have determined the complete nucleotide sequences of the hemagglutinin genes of three H13 influenza viruses from different species or geographical areas: A/gull/Maryland/77, A/gull/Astrachan (USSR)/84, and A/pilot whale/Maine/84. Based on the deduced amino acid sequences, H13 hemagglutinin shares the basic structure of other type A hemagglutinin subtypes such as H3, but has clearly diverged from other completely sequenced subtypes. Unique features of H13 hemagglutinin include the occurrence, near the receptor binding pocket, of residues Arg/Lys-227 and Trp-229 (H3 numbering); the significance of these are unknown. The sequence of the HA1-HA2 cleavage site resembles those of avirulent avian influenza viruses. The whale H13 hemagglutinin is similar to those from gulls, supporting the hypothesis that influenza viruses from avian sources can enter marine mammal populations but are probably not permanently maintained there. Antigenic analysis using a panel of monoclonal antibodies suggests that, like other subtypes, H13 viruses are heterogeneous, with different antigenic variants predominating in the eastern versus the western hemispheres.     

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.     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. II. Reactivity with rabbit sera against intact virus and purified undissociated haemagglutinin.

Rabbit sera produced against either intact virus or purified undissociated haemagglutinin were examined for reactivity with highly purified haemagglutinin glycopolypeptides. Sensitive radioimmunoassay for 125I-labelled glycopolypeptides revealed antibody reactive with either glycopolypeptide HA1, or glycopolypeptide HA2. Antibodies against the carbohydrate moiety were responsible only for a part of the binding activity. Under the conditions employed, the binding activity for glycopolypeptide HA2 was much stronger than for glycopolypeptide HA1. Competition assays suggested that immune reactivities were due to distinct antibody populations (i.e. with a specifity for glycopolypeptide HA1 and glycopolypeptide HA2, respectively). The immune reactivity to both haemagglutinin constituents, glycopolypeptides HA1 and HA2, was also shown by gel double diffusion. The precipitin line(s) corresponding to glycopolypeptide HA1 was (were) usually more distinct than precipitin line(s) corresponding to glycopolypeptide HA2. The glycopolypeptides HA1 and HA2 showed the reaction of nonidentity in immunodiffusion analysis.     

Assessment of the antigenic relatedness among H3 hemagglutinins of type A influenza viruses by competition radioimmunoassay.

Competition radioimmunoprecipitation assays were used to assess the antigenic relatedness of the hemagglutinins of four of the major variants of type A influenza viruses of the H3N2 subtype. Purified whole virus preparations representative of A/Hong Kong/68 (A/HK/68), A/England/72 (A/Eng/72), A/Port Chalmers/73 (A/PC/73), and A/Victoria/75 strains were used as competing antigens. These antigens were used to inhibit the binding of iodinated preparations of purified HA obtained from each of the variants to their homologous antiserum. These analyses indicated that the degree of cross-reactivity of the three later variants with A/HK/68 was inversely related to the sequence of their isolation from human populations and that each of these variants differed antigenically from the immediately preceding and/or succeeding variants by approximately 50%. A fifth strain A/Scotland/74 (A/Scot/74) was also used as a competing antigen and it did not follow the chronological pattern observed with the four major variants. The major proportion of the cross-reactivity observed between these strains appears to be mediated through antibodies with lower avidity for heterologous HAs. However, heterologous competition assays identified two identical antigenic determinants; one was expressed on the A/HK/68, A/Eng/72, A/PC/73, and A/Scot/74 variants and the other was found on only the latter three.     

Antigenic drift of epidemic strains of influenza A virus (H3N2) in 1985

The study included 230 strains of influenza A (H3N2) virus isolated in the epidemic of 1985. A high degree of heterogeneity of the virus population was established with polyclonal sera, monoclonal antibodies and the method of kRNA-bRNA hybridization for the determination of the genome composition. Among the strains of one epidemic (1985) three antigenically heterogenous groups of strains were detected similar with reference A/Philippines/2/82, A/Ken/1/84, and A/Mississippi/1/85 strains. It was shown that in the process of antigenic drift in some strains there occurred a reversion of the antigenic determinants similar to those of previously circulating epidemic A/Texas/1/77 strain.     

Characterization of a new avian influenza virus subtype and proposed designation of this haemagglutinin as Hav10.

The haemagglutinin of A/Dk/alb/60/76, an influenza A virus isolated from feral ducks in Canada, possesses no antigenic relatedness to any of the 16 reference haemagglutinin subtypes. Results of serological tests (HI and double immunodiffusion) with monospecific antisera to the haemagglutinin of this virus indicate that it represents a new avian haemagglutinin subtype. We propose that this haemagglutinin be designated as Hav10 under the current system of nomenclature.     

Antibodies to new variants of subtype A(H3 N2) influenza virus in pigs

Following an explosive epidemic of A(H3N2) influenza among the human population of Czechoslovakia in 1983, haemagglutination-inhibiting antibodies (titre range 10-640) against strains A/Texas/77, A/Bangkok/79 and A/Philipines 2/83 were detected in 93% of sera collected from 135 pigs on three farms. Only 6.6% of sera were negative. Anti-neuraminidase antibodies were detected at rates of 81% and 23% in two and one of the herds, respectively. Antibodies against A/RNP were demonstrated by the immunodiffusion test in only one of the herds in 10 out of 45 sera tested. This herd was also found to possess antibodies against both envelope antigens of a human A(H1N1) subtype strain. Haemagglutination-inhibition tests with strains A/Hong Kong/68 (H3 N2), A/sw/Shope 15/31, A/sw/Bavaria 2/77 and A/New Jersey 8/76 (H1 N1) were negative in the sera from all three herds.     

The value of complement fixation and haemagglutination inhibition tests in the diagnosis of influenza A.

Antibody response of 133 influenza A patients from three outbreaks since 1972-73 was analyzed by complement fixation (CF) and haemagglutination inhibition (HI) methods. During the first outbreak, a significant response was more often measured by CF than by HI. During the last outbreak HI appeared more useful than CF for routine serological diagnosis; 23% of cases verified by HI were missed by CF. The poor response of CF antibodies was associated with the high level of pre-infection antibodies.     

Temperature-sensitive mutants of influenza virus. VI. Transfer of TS lesions from the Asian subtype of influenza A virus (H2N2) to the Hong Kong subtype (H3N2).

Temperature-sensitive genetic lesions were transferred from the ts-1 (H2N2) and ts-2 (H2N2) mutants of influenza A virus to wild-type influenza A (H3N2) virus by genetic reassortment. The ts-2 (H3N2) recombinants appeared to be homogeneous and did not undergo recombination with one another, suggesting that the original ts-2 mutant of influenza A (H2N2) contained a ts lesion(s) on only one RNA segment of its genome. In contrast the ts-1 (H3N2) recombinants fell into three phenotypic subsets which differed in degree of temperature sensitivity. Initially, the three subsets of ts-1 (H3N2) recombinants were thought to represent distinct complementation-recombination groups. However, complementation-recombination between the three subsets of ts-1 (H3N2) recombinants subsets was variable. Subsequent study indicated that mutants in each of the three subsets shared one ts lesion and two of the subsets shared an additional ts lesion. The mechanism whereby viruses of the three subsets which share one or two ts lesions, and nevertheless undergo apparent complementation-recombination on occasion is not understood.     

Type a influenza viruses in birds in southern Spain: serological survey by enzyme-linked immunosorbent assay and haemagglutination inhibition tests

Of 927 sera taken from poultry from 84 flocks, 33% proved seropositive. Sixty-three per cent of flocks were found to be seropositive to ELISA (almost all situated within areas where there were waterfowl). The comparable figures, using an HI test, were 16% and 47%, respectively. These data suggest that the influenza A viruses may be enzootic in this area. Of 331 wild birds tested, belonging to 18 species of nine families, 40% proved seropositive to ELISA. Notable high infection rates were found among Anatidae (43%), flamingoes (43%) and sparrows (31%); the latter species may play an important role in carrying the disease from its natural reservoirs to domestic farms. Antibody titres found in wild birds were considerably higher than those found in poultry.     

Genetic and antigenic variation in the haemagglutinin of recently circulating human influenza A (H3N2) viruses in the United Kingdom

Summary Variation in the haemagglutinin (HA) gene of influenza A (H₃N₂) viruses isolated in the U. K. and abroad from 1992–1994, was determined by nucleotide sequencing of the HA1 domain of the HA gene. Viruses isolated in the U.K. early in the 1992–93 season were from the A/Beijing/353/89 lineage and were replaced later that season by viruses from the A/Beijing/32/92 lineage. Viruses from the new lineage continued to be isolated during the 1993–94 season, but were heterogeneous. Most of these isolates were more closely related to an A/Beijing/32/92 variant, A Hong Kong/23/92, but could be distinguished into three groups by serology (of which one group was circulating during the previous season) and four groups based on sequence variation in the HA gene. However, phylogenetic analysis of antigenically-distinct isolates showed that the HA gene is evolving along one lineage. Sequence analysis identified mainstream, subgroup and strain specific amino acid substitutions. There was a broad correlation between the observed amino acid changes and the antigenic sites of the HA. The results of this study highlight the value of regular molecular analysis of circulating viruses.     

双重荧光定量RT-PCR快速检测流感病毒H1、H3亚型的研究

目的利用荧光定量RT-PCR技术建立一种快速检测流感病毒H1、H3亚型的方法。方法根据H1、H3亚型流感病毒HA基因的相对保守序列,设计两对引物及其相应的Taqman探针,利用一步法RT-PCR试剂盒建立优化反应体系后,将荧光定量RT-PCR的产物采用10倍稀释法,即107～100copies/μl,再次用荧光定量PCR方法检验建立体系的灵敏度和重复性,并建立相对定量标准曲线;利用多种流感病毒和具有相似临床症状的呼吸道病毒检验建立体系的特异性。结果 H1和H3亚型流感病毒的检测灵敏度为102copies/μl,扩增效率分别为101.35%和113.28%,标准曲线相关系数大于99%,重复性良好,特异度实验未发现有非特异性扩增。结论本研究建立的双重荧光定量RT-PCR技术可以快速、准确地检测H1、H3亚型流感病毒。     

Antigenic and genetic conservation of H3 influenza virus in wild ducks

The hemagglutinins of H3 influenza viruses isolated from migratory ducks on the Pacific flyway in Japan during the period 1977 to 1985 were analyzed antigenically and genetically. Antigenic analysis using monoclonal antibodies to the hemagglutinins of A/Aichi/2/68 (H3N2) and A/duck/Hokkaido/8/80 (H3N8) viruses showed that antigenic drift occurred extensively in human strains, whereas the hemagglutinins of duck viruses were highly conserved. It was also found that the hemagglutinins of duck viruses were antigenically closely related to that of human 1968 H3 prototype strains. Nucleotide sequence analysis of seven duck H3 hemagglutinin genes showed a limited number of changes among the six duck isolates and between these duck isolates and Aichi/68. The deduced amino acid sequence revealed amino acid changes randomly distributed throughout the molecule and not confined to antigenic sites. These findings indicate that the duck virus hemagglutinin genes are conserved in nature and that viruses of different lineages cocirculate.     

Simultaneous detection of influenza A, influenza B, and respiratory syncytial viruses and subtyping of influenza A H3N2 virus and H1N1 (2009) virus by multiplex real-time PCR

A multiplex real-time PCR assay was developed to simultaneously detect and discriminate influenza A virus subtypes, including novel H1N1 (2009) and seasonal H3N2 virus, influenza B virus, and respiratory syncytial virus (RSV) in a single test tube, with detection sensitivity and specificity of 99% and 100%, respectively, for the four pathogens.     

Etiological diagnosis of influenza A virus by enzymatic radioimmunoassay.

An enzymatic radioimmunoassay for influenza A virus was developed by using polystyrene beads coated with rabbit immunoglobulin G to capture viral hemagglutinins (H1 and H3). Captured hemagglutinin was detected with goat immunoglobulin G followed by affinity-purified rabbit anti-goat immunoglobulin G labeled with alkaline phosphatase. [3H]AMP was added to quantify alkaline phosphatase activity, and free [3H]adenosine was measured with a scintillation counter. The assay detected as little as 0.1 ng of purified hemagglutinin. It was specific for hemagglutinin subtype and, depending on the source of the goat immunoglobulin G used, detected either H1 or H3. There was no reaction with neuraminidase or core antigens of influenza strain WSN-33. The clinical efficacy of the assay was evaluated with sequential nasal washes from 33 patients with naturally acquired H1N1 influenza. In the first 3 days of infection, the assay was consistently less sensitive than the viral culture, although detectable antigen persisted in secretions longer than did the infectious virus. Testing of multiple samples greatly increased the number of individuals in whom an etiological diagnosis could be made by immunoassay (81% of patients were positive for viral antigens at some point in their illness), and such testing was necessary to achieve the sensitivity of a single culture. Mean antigen levels were highest in nasal washes with the highest titers of infectious virus.