Replication of avian influenza viruses in humans

Summary Volunteers inoculated with avian influenza viruses belonging to subtypes currently circulating in humans (H1N1 and H3N2) were largely refractory to infection. However 11 out of 40 volunteers inoculated with the avian subtypes, H4N8, H6N1, and H10N7, shed virus and had mild clinical symptoms: they did not produce a detectable antibody response. This was presumably because virus multiplication was limited and insufficient to stimulate a detectable primary immune response. Avian influenza viruses comprise hemagglutinin (HA) subtypes 1–14 and it is possible that HA genes not so far seen in humans could enter the human influenza virus gene pool through reassortment between avian and circulating human viruses.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses.

Comparative studies on naturally occurring avian influenza viruses have been carried out in order to investigate the determinant(s) for pathogenicity for chickens. At least one virus isolate from each of the nine different hemagglutinin (HA) subtypes was included. The polypeptides of these viruses were studied by analyzing infected cell extracts on SDS-polyacrylamide gels. Both viral glycoproteins, HA and neuraminidase, showed remarkable variation in their electrophoretic mobility even among serologically closely related viruses. Pulse-chase experiments revealed that most avian influenza virus strains had an HA which was not susceptible to proteolytic cleavage in MDCK, turkey (TEC), and chicken embryo cells (CEC). Only viruses belonging to the subtype Hav5 and some strains of the subtype Hav1 possessed a cleaved HA in these cells. Only the virus strains with cleaved HA were produced in infectious form in MDCK, CEC, TEC, as well as in duck embryo cells (DEC) and quail embryo cells (QEC). The other virus strains produced plaques in these cells only in the presence of trypsin. There was a strict correlation between the cleavability of the HA, the potential of the virus to be produced in infectious form in a wide range of host cells, and their pathogenicity for chickens. No evidence was obtained for an involvement of the neuraminidase in determining pathogenicity. For the nonpathogenic viruses it could be shown that they can replicate and produce infectious progeny in some organs of the chicken. The results obtained permit the conclusion that in naturally occurring avian influenza viruses the structure of the hemagglutinin, that is its susceptibility to proteolytic cleavage in a broad spectrum of host cells, is the determining factor for pathogenicity.     

Heterotypic interference between influenza viruses A/Aichi/2/68 and B/Massachusetts/1/71.

Virus particles produced by MDCK cells mixedly infected with 3 PFU/cell each of A/Aichi/2/68 (H3N2) (Aichi) and B/Massachusetts/1/71 (Mass) influenza viruses exclusively possessed haemagglutinin (HA) of Mass, although approximately one-fifth of the mixed yield had coding potential for Aichi serotype. Synthesis of major viral proteins of Aichi was markedly suppressed by co-infecting Mass. By increasing the multiplicity of co-infecting Aichi to 30 PFU/cell, interference became reciprocal. Aichi interfered with replication of Mass more severely than Mass did with replication of Aichi. All the major viral proteins of both Aichi and Mass were expressed within the infected cells.     

A common antigenic epitope in influenza A virus (H1, H2, H5, H6) hemagglutinin]

Avian influenza A viruses belonging to hemagglutinin (HA) subtypes H5 and H6 were studied in the infectivity neutralization test and radioimmunoprecipitation assay (RIPA) with monoclonal antibody MAb C179. This MAb recognizes a conformational antigenic epitope in the stem region of HA formed by two regions (amino acid positions 318-322 in HA1 subunit and 47-58 in HA2), conserved in all H1 and H2 influenza viruses. MAb C179 reacts with HA of H5 viruses in RIPA and neutralizes these strains as efficiently as H2 viruses. C179 precipitates H6 subtype HA but does not neutralize the infectivity of these viruses. Comparison of amino acid sequences of H2, H5, and H6 strains showed identical epitope recognized by MAb C179 in H5 and H6 HAs, which differs from epitopes of H1 and H2 by two amino acids in the HA2 subunit. Causes of disagreement between immunoprecipitation of H6 HA by MAb C179 and neutralization of this serosubtype by this MAb are discussed.     

Importance of cross-reacting antibodies in creating resistance to influenza

Hyperimmune rabbit serum to A/PR8/34 virus was analysed by selective adsorption of antibody by viruses belonging to hemagglutinin subtypes H0, H1 and Hsw1. The presence of three antigenic determinants was demonstrated in viral HA: one was common for HA of H0, H1, and Hsw1 viruses, the other was common for HA of H0, H1 and Hsw1 viruses, and the third was strain-specific for this virus. The protective effect of formalin-inactivated influenza vaccines prepared from virus variants with H0 and H1 hemagglutinins against challenge with A/PR8/34 virus was studied. The presence in the vaccine influenza virus of at least one antigenic determinant common with that of the challenge virus was shown to create a certain degree of protection in mice against influenza. The presence of two antigenic determinants in HA of vaccine and challenge virus increased considerably the protective effect of the vaccine.     

Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry

At present there is much interest in the cell tropism and host range of influenza viruses, especially those of the H5N1 subtype. We wished to develop a method that would enable investigation of attachment of infectious virus through the interaction of the hemagglutinin molecule and live mammalian and avian cells and the subsequent infection of these cells. To this end, influenza viruses of various HA subtypes were constructed that either carry the green fluorescent protein (GFP) instead of the neuraminidase protein, or that express GFP in the cytoplasm of infected cells. The HA genes were derived from influenza viruses A/PR/8/34 (H1N1), A/Netherlands/178/95 (H3N2) and A/Vietnam/1194/04 (H5N1). Using these pairs of viruses, attachment and post-attachment events in the virus replication cycle can be distinguished. In general, the expression of NeuAc(alpha2-3)Gal or NeuAc(alpha2-6)Gal receptors on the cells tested corresponded with the attachment of the viruses that were studied with respect to predicted receptor specificity. Virus attachment was not always predictive for efficient infection of the cells.     

Genetically destined potentials for N-linked glycosylation of influenza virus hemagglutinin

The addition of oligosaccharide side chains to influenza virus hemagglutinin (HA) is believed to facilitate viral escape from immune pressure in the human population. To determine the implicit potentials for acquisition of N-linked glycosylation, we analyzed the genetic background of 16 subtypes of avian influenza virus, some of which may be potential pandemic viruses in the future. We found a significant difference among HA subtypes in their genomic sequences to produce N-glycosylation sites. Notably, recently circulating avian influenza viruses of the H5 and H9 subtypes may have rather greater capacities to undergo mutations associated with glycosylation of HA than past pandemic viruses. We hypothesize that influenza viruses maintained in natural reservoirs could have different potentials for sustained circulation, depending on their HA subtypes, if introduced into the human population.     

Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method

This study presents an interconnected approach for circumventing two inherent limitations associated with studies defining the natural history of influenza A viruses in wild birds. The first limiting factor is the ability to maintain a cold chain from specimen collection to the laboratory when study sites are in more remote locations. The second limiting factor is the ability to identify all influenza A virus HA subtypes present in an original sample. We report a novel method for molecular subtyping of avian influenza A virus hemagglutinin genes using degenerate primers designed to amplify all known hemagglutinin subtypes. It was shown previously that templates larger than 200 bp were not consistently amplifiable from ethanol-fixed cloacal swabs. For this study, new primer sets were designed within these constraints. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating hemagglutinin subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different hemagglutinin subtypes. This study further demonstrates the complex ecobiology of avian influenza A viruses in wild birds.     

The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding

Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968, or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D.     

Mutations of two transmembrane cysteines of hemagglutinin (HA) from influenza A H3N2 virus affect HA thermal stability and fusion activity

Influenza A H3N2 virus caused 1968 Hong Kong influenza pandemic, and has since been one of the most prevalent seasonal influenza viruses in global populations, representing a credible pandemic candidate in future. Previous studies have established that the hemagglutinin (HA) protein is the predominant antigen and executes receptor binding and membrane fusion. Homologous sequence analysis of all HA subtypes of influenza viruses revealed that two cysteine residues (540 and 544) are uniquely present in the transmembrane domain (TM) of HA proteins from all influenza A H3N2 viruses. However, the functions of these two cysteines have not been fully studied. Here, we generated three mutants (C540S, C544L, and 2C/SL) to investigate the effects of the two TM cysteines on the biological functions of H3 HA. We herein presented evidences that the mutations of one or two of the cysteines did not affect the proper expressions of HA proteins in cells, and more importantly all mutant H3 HAs showed decreased thermal stability but increased fusion activity in comparison with wildtype HA. Our results taken together demonstrated that the two TM cysteines are important for the biological functions of H3 HA proteins.     

Antigenic determinants of human influenza viruses among the influenza viruses isolated from animals

Comparative studies of the antigenic properties of hemagglutinin (HA) of animal and human viruses revealed both similarities between them and complete differences in the composition of antigenic determinants. Avian influenza viruses A/chicken/Kamchatka/12/71, A/pintail/Primorie/730/76, and A/bat/Alma-Ata/73/77 were completely identical with human strains of influenza virus. Influenza A/horse/Miami/63 contains one antigenic determinant H3.1.HA of A/tern/Turkmenia/18/73 (Hav7) viruses has a peculiar set of antigens. Apart from two antigenic determinants H3.1 and H3.3 inherent in human virus strains, HA of A/tern/Turkmenia/18/73 virus contains an antigenic determinant the population of antibodies to which shows no relation to HA of subtypes Hav2-Hav9.     

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.     

Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus

Two influenza A viruses whose hemagglutinin (HA) did not react with any of the reference antisera for the 13 recognized HA subtypes were isolated from mallard ducks in the USSR. Antigenic analysis by hemagglutination inhibition and double immunodiffusion tests showed that the HAs of these viruses are similar to each other but distinct from the HAs of other influenza A viruses. Nucleotide sequence analysis showed that these HA genes differ from each other by only 21 nucleotides. However, they differ from all other HA subtypes at the amino acid level by at least 31% in HAI. Thus, we propose that the HAs of these viruses [A/Mallard/Gurjev/263/82 (H14N5) and A/Mallard/Gurjev/244/82 (H14N6) belong to a previously unrecognized subtype, and are designated H14. Unlike any other HAs of influenza viruses, the H14 HAs contained lysine at the cleavage site between HA1 and HA2 instead of arginine. Experimental infection of domestic poultry and ferrets with A/Mallard/Gurjev/263/82 (H14N5) showed that the virus is avirulent for these animals. Based on comparative sequence analysis of different HA genes, it is suggested that differences of 30% or more at the amino acid level in HA1 constitute separate subtypes. Phylogenetic analysis of representatives of each HA subtype showed that H14 is one of the most recently diverged lineages while H8 and H12 branched off early during the evolution of the HA subtypes. These latter two subtypes (H8 and H12) have been isolated very infrequently in recent years, suggesting that these old subtypes may be disappearing from the influenza reservoirs in nature.     

Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses

The structural basis for the different proteolytic cleavability of influenza virus hemagglutinin (HA) was investigated with a group of pathogenic and nonpathogenic avian influenza viruses belonging to the antigenic subtype H7 (Hav1). Infected cel lysates or lystates of purified virus particles were subjected to two-dimensional gel electrophoresis. The first dimension, isoelectric focusing,- was done under nonreducing conditions, the second dimension, SDS-PAGE, under reducing conditions. The results obtained permit the following conclusions: The amino acid sequence of the connecting peptide between HA₁ and HA₂ determines proteolytic cleavability by a trypsin-like cellular enzyme. Upon proteolytic cleavage of HA of pathogenic strains, peptides of differing positive charge were eliminated. These HAs have, however, significantly more basic connecting peptides than HAs of nonpathogenic viruses. HAs of nonpathogenic H7 strains appear to have a connecting peptide similar to the human influenza viruses, since treatment of these viruses with trypsin results in a similar small charge shift which probably corresponds to the elimination of one basic amino acid. Thus, the primary structure of the connecting peptide determines biological activation and thereby pathogenicity of these viruses.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

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.     

Extensive heterogeneity in the hemagglutinin of egg-grown influenza viruses from different patients

We establish that the cultivation of influenza (H3N2) virus from any infected individual in chicken embryos (eggs) can result in the isolation of viruses with antigenic and/or structural heterogeneity in the hemagglutinin (HA) molecule. This variability contrasted sharply with the apparent lack of antigenic alterations in the HA of influenza viruses isolated from patients in Madin Darby canine kidney (MDCK) cells. The most common subpopulation of egg-grown influenza viruses had the same phenotype as MDCK cell-grown virus and may best represent the virus circulating in humans. It should be considered the optimal strain for use in vaccine and epidemiologic studies.     

HA2-specific monoclonal antibodies as tools for differential recognition of influenza A virus antigenic subtypes

Antigenic reactivity of a set of monoclonal antibodies (MAb) raised against the HA2 subunit of hemagglutinin of H3 subtype was characterized in a rapid culture assay. MAbs FC12 and FE1, known to recognize the same antigenic site (IV), cross-reacted with influenza viruses of H3 and H4 subtypes, regardless of their host origin. No cross-reactivity was detected with other antigenic subtypes tested (H1-H13). The involvement of conserved residues D160, N168, and F171 in the differential recognition of H3 and H4 subtypes is proposed. In contrast, MAb IIF4 that recognizes antigenic site II exhibited a broader inter-subtype reactivity including subtypes H3, H4, H5, H8 and some viruses of H2, H6 and H13 subtypes. The ability of HA2-specific antibodies to differentially react with distinct antigenic subtypes can be utilized in development of diagnostics and in the influenza virus surveillance.