Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

Neutralizing Epitopes of Influenza Virus Hemagglutinin: Target for the Development of a Universal Vaccine against H5N1 Lineages

The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection. Particularly, variations within the amino acid sequences of major neutralizing epitopes of influenza virus hemagglutinin (HA) hindered the development of universal vaccines against H5N1 lineages. Based on distribution analyses of the identified major neutralizing epitopes of hemagglutinin, we selected three vaccine strains that cover the entire variants in the neutralizing epitopes among the H5N1 lineages. HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the preclinical efficacy of the vaccine formulations was evaluated in a mouse model. The combination of three selected vaccine strains could effectively neutralize viruses from clades 1, 2.1, 2.2, 4, 7, and 8 of influenza H5N1 viruses. In contrast, a vaccine formulation containing only adjuvanted monovalent BacHA (mono-BacHA) or a single strain of inactivated whole viral vaccine was able to neutralize only clade 1 (homologous), clade 2.1, and clade 8.0 viruses. Also, the trivalent BacHA vaccine was able to protect 100% of the mice against challenge with three different clades (clade 1.0, clade 2.1, and clade 7.0) of H5N1 strains compared to mono-BacHA or inactivated whole viral vaccine. The present findings provide a rationale for the development of a universal vaccine against H5N1 lineages. Furthermore, baculoviruses displaying HA will serve as an ideal choice for a vaccine in prepandemic or pandemic situations and expedite vaccine technology without the requirement of high-level-biocontainment facilities or tedious protein purification processes.The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection (20). This has been evidently recognized by the recent outbreaks of H5N1 avian influenza virus infection and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV). In fact, it has been well documented in the literature that H5N1 had acquired the ability to infect human tissues due mainly to the occurrence of mutation events (1). Highly pathogenic avian influenza (HPAI) H5N1 viruses are antigenically distinguishable owing to differences in hemagglutinin (HA) sequences, the principal determinant of immunity to influenza virus, resulting in different lineages or clades of H5N1 (13, 33). The control of infection with current H5N1 vaccines does not appear to be effective against heterologous strains or phylogenetically variant clades of H5N1 in part due to variations in the HA sequences, particularly within the neutralizing epitope region. Since present vaccines are based solely on the induction of neutralizing antibodies against these epitopes, differences in these sequences may render current vaccines unqualified for the prevention of influenza globally (15, 28, 31). To overcome such limitations and to completely realize the potential of vaccines worldwide, the concept of universal vaccines based on conserved viral proteins has recently been proposed. The highly conserved ion channel protein (M2) and the nucleoprotein (NP) of influenza virus have been evaluated for the induction of cross-protective cellular immunity and viral clearance (2, 35). Antibodies generated against these conserved proteins may reduce viral spread and accelerate recovery from influenza (14). However, antibodies specific to these proteins are poorly immunogenic and were found previously to be infection permissive (5-7, 13). Thus, the development of a vaccine based on influenza virus hemagglutinin appears to be the only viable option to prevent infections by HPAI viruses such as H5N1 viruses. Nevertheless, amino acid variations within the major antigenic neutralizing epitope regions among H5 subtypes restrict the development of such universal vaccines against different H5N1 lineages.The development of a universal vaccine based entirely on HA of influenza virus is still feasible, if the variation or conservation of neutralizing epitopes among the several HPAI H5N1 virus clades can be identified. An understanding of the distribution pattern of such neutralizing epitopes could help in the design of future vaccines by incorporating two or more ideal H5N1 strains in the vaccine composition. The neutralizing epitopes of the selected viral strains should cover the variations among most H5 subtypes in order to acquire broad-range protective immunity against most H5N1 subtypes. Previous attempts to identify amino acid substitutions within HA sequences of variants that escaped from neutralization by monoclonal antibodies (MAbs) revealed the neutralizing epitope sites of HA (9, 10). Along with previous findings, we report here the identification of other major neutralizing epitopes of H5N1 by mapping their amino acid sequences using neutralizing monoclonal antibodies (n-MAbs). Analysis of the distribution of all identified neutralizing epitopes among H5 subtypes revealed variations within the antigenic determinants of H5N1 subtypes from both human and avian sources. Based on these results, we have selected three vaccine strains comprising the major neutralizing epitopes of HA to cover the entire variants within H5N1 lineages. In order to test our hypothesis in vivo, HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the efficacy of the vaccine formulations was evaluated with a mouse model challenged with phylogenetically variant H5N1 strains.     

Identification of mutations at the antigenic and glycosylation sites in hemagglutinin protein of H5N1 strain

Hemagglutinin (HA) is the principal antigen, present on the viral surface. It is the primary target for neutralizing antibodies. In this paper, we have carried out studies on human hemagglutinin protein from H5N1 strain with homologous hemagglutinin from non-human sources of H5N1 strains. In all strains, part of the antigenic site (128-141) predicted by computer program “Antigenic”, corresponds to immunodominant site Sa of H1 subtype. In {"type":"entrez-protein","attrs":{"text":"AAF02304","term_id":"6048753","term_text":"AAF02304"}}AAF02304 strain, A156→S156 mutation lies at the antigenic subsite of site 2 that corresponds to site B in the H3 subtype. In some strains of non-human origins, there are mutations at the antigenic sites. Interestingly, in {"type":"entrez-protein","attrs":{"text":"AAY56367","term_id":"66775625","term_text":"AAY56367"}}AAY56367 strain mutation L138→H138 lies at the receptor binding site, which also overlaps the antigenic site. Therefore, this amino acid substitution may influence both the specificity of receptor recognition and antibody binding. Seven potential glycosylation sites in human HA and in some strains of non-human sources have been predicted by computer program, Scan Prosite. In some strains of HA from non-human sources because of mutation, an additional glycosylation site appeared at the antigenic site. Therefore in these strains the oligosaccharides will mask the surface of HA as well as antigenic site. Hence these strains will not be recognized by host immune system.     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

Genetic mutations in influenza H3N2 viruses from a 2012 epidemic in Southern China

Background

An influenza H3N2 epidemic occurred throughout Southern China in 2012.

Methods

We analyzed the hemagglutinin (HA) and neuraminidase (NA) genes of influenza H3N2 strains isolated between 2011–2012 from Guangdong. Mutation sites, evolutionary selection, antigenic sites, and N-glycosylation within these strains were analyzed.

Results

The 2011–2012 Guangdong strains contained the HA-A214S, HA-V239I, HA-N328S, NA-L81P, and NA-D93G mutations, similar to those seen in the A/ Perth/16/2009 influenza strain. The HA-NSS_061–063 and NNS_160–162 glycosylation sites were prevalent among the 2011–2012 Guangdong strains but the NA-NRS_402–404 site was deleted. Antigenically, there was a four-fold difference between A/Perth/16/2009 -like strains and the 2011–2012 Guangdong strains.

Conclusion

Antigenic drift of the H3N2 subtype contributed to the occurrence of the Southern China influenza epidemic of 2012.

     

Nucleotide sequence of the influenza virus A/USSR/90/77 hemagglutinin gene

The complete nucleotide sequence of the hemagglutinin gene of influenza virus A/USSR/90/77 was determined. Comparison of hemagglutinin amino acid sequences from H1 field strains revealed five potential antigenic sites. Four of these sites correspond to those observed for H3 hemagglutinins, whereas the fifth apparently derives from differences in the glycosylation patterns between subtypes.     

Conservation and variation in the hemagglutinins of Hong Kong subtype influenza viruses during antigenic drift.

The nucleotide sequence was determined for the hemagglutinin gene of the Hong Kong subtype influenza strain A/Bangkok/1/79. The amino acid sequence predicted from these data shows a total of 36 amino acid changes as compared with hemagglutinin for a 1968 Hong Kong strain, 11 more than had occurred in a 1975 strain. The distribution of these changes confirmed that there are conserved and highly variable regions in hemagglutinin as the viral gene evolves during antigenic drift in the Hong Kong subtype. Of the four variable regions found in this study, only two have been seen previously. Correlation of highly variable areas in the hemagglutinins of Hong Kong subtype field strains with sites of amino acid changes in antigenically distinct influenza variants enabled us to predict likely antigenic regions of the protein. The results support and extend similar predictions made recently, based on the three-dimensional arrangement of hemagglutinin from a 1968 influenza strain.     

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background

The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.

Methodology/Principal Findings

HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.

Conclusions/Significance

Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.     

Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.     

Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence

Background

The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents.

Methodology/Principal Findings

To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (d_A) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function.

Conclusions/Significance

Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics.     

Mechanism of antigenic drift in influenza virus. Amino acid sequence changes in an antigenically active region of Hong Kong (H3N2) influenza virus hemagglutinin

During antigenic drift in influenza viruses, changes in antigenicity are associated with changes in amino acid sequence of the large hemagglutinin polypeptide, HA1. In ten variants of Hong Kong (H3N2) influenza virus selected with monoclonal antibodies, the proline residue at position 143 in HA1 changed to serine, threonine, leucine or histidine. In other variants, asparagine 133 changed to lysine, glycine 144 to aspartic acid and serine 145 to lysine. All these changes are possible by single base changes in the RNA except the last, which requires a double base change. Residues 142 to 146 also changed in field strains of Hong Kong influenza isolated between 1968 and 1977 (Laver et al., 1980). The single amino acid sequence changes in HA1 of the monoclonal variants were detected by comparing the compositions of the soluble tryptic peptides from the variants with the known sequences of these peptides from wild-type virus. Two insoluble tryptic peptides, comprising residues 110 to 140 and 230 to 255 in the HA1 molecule, were not examined and we do not know if additional changes occurred in these regions.In order to determine whether sequential changes at the same position occurred during antigenic drift, antibody prepared against the new antigenic site on the variants in which proline 143 changed to histidine or threonine was used to select second generation variants of these variants. In the first case, the glycine residue (144) next to the histidine changed to aspartic acid, and in the second, the threonine residue at position 143 reverted to proline and the virus regained the antigenicity of wild-type.Although monoclonal antibodies revealed dramatic antigenic differences between the variants and wild-type virus, only those variants with changes at position 144 of glycine to aspartic acid or at position 145 of serine to lysine could be distinguished from wild-type virus using heterogeneous rabbit or ferret antisera. The other variants, including those which showed sequence changes in widely separated positions of HA1, could not be distinguished from wild-type with heterogeneous antisera.These findings suggest that sequence changes in the region comprising residues 142 to 146 of HA1 affect an important antigenic site on the hemagglutinin molecule, but how these changes affect the antigenic properties, or whether this region actually forms part of the antigenic site is not known.     

The nature of amino acid substitution in antigenic drift of hemagglutinin N3 and neuraminidase N2 from the influenza virus

The nature of amino acid replacements in 16 drift variants of hemagglutinin H3 subtype and 5 drift variants of neuraminidase N2 subtype of the influenza A virus were studied. The dependences of relative replacement frequencies and relative quantities of frequent replacements upon differences of properties of substituted residues are plotted. In contrast to most of the known proteins, amino acid replacements in hemagglutinin and neuraminidase depend weakly on the physico-chemical parameters of amino acids. For the antigenic determinants studied the replacement frequencies were compared to those calculated according to two models: one for conservative replacements and the other for accidental mutation of the genetic code. The differences in the nature of amino acid replacements are found in four antigenic determinants of hemagglutinin. The replacements in experimentally selected proteins are shown to go beyond limitations of natural variants. The explanations of the reasons of low epidemicity of some strains and ineffective attempt to imitate the natural antigenic drift of viruses by using experimental selection are proposed. The causes of time-limited circulation of H3N2 influenza virus subtype are discussed.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong

The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of "internal" virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.     

Coat proteins of strains of two RNA viruses: comparison of their amino acid sequences

Summary The amino acid sequences of four strains of tobacco mosaic virus isolated in different parts of the world are compared. The differences between the strains are discussed with respect to special proteinchemical features (such as beginning of the chain, deletion of amino acids, number of different amino acids, sizes and distribution of regions with invariable amino acids) and with respect to the possibility of deducing the most probable nucleotide sequence for the coat protein cistron of tobacco mosaic virus.The complete amino acid sequences of the two RNA bacteriophage strains fr and f₂ are compared. According to their coat proteins three groups of phages can be formed: 1) MS 2, f₂ M 12 and R 17, 2) fr and 3) Q.     

Evolution of the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus in Morocco During Two Influenza Seasons 2009–2011

To study genetic evolution of Moroccan influenza A(H1N1)pdm09 virus strains, we conducted a molecular characterization of the hemagglutinin gene subunit 1 (HA1) of 36 influenza A(H1N1)pdm09 virus strains. The stains were collected from patients in Rabat and Casablanca during two influenza seasons 2009–2010 and 2010–2011. Nucleotide and amino acid sequences of 14 influenza A(H1N1)pdm09 virus strains from 2009 to 2010 were ~97 and 99 %, respectively, similar to the reference strain A/California/07/2009 (H1N1). Phylogenetic analysis of 22 influenza A(H1N1)pdm09 virus strains from 2010 to 2011 revealed a co-circulation of three well-described different genetic groups. Most important, none of the identified groups showed significant changes at the antigenic site of the virus HA1 subunit which may alter the efficacy of California/07/2009 (H1N1) vaccine.     

A transposable epitope of HLA-137, B40 molecules

The monoclonal antibody M1340.2 defines a novel subtype of HLA-1340 that is expressed by the Sweig cell line. This molecule, called HLA-B40^*, lacks an antigenic determinant that is common to HLA-137 and the HLA-Bw60 subtype of HLA-1340. Genes encoding HLA-B40^* and HLA-BW60 have now been isolated and the amino acid sequences of these proteins compared with other HLA-13 locus molecules. These results show that HLA-B40^* is a unique protein which differs from HLA-BW60 by eight amino acid substitutions. Comparison of the sequences for HLA-B40^*, -Bw60, and -B7 localizes the MB40.2 epitope to a cluster of three substitutions at positions 177, 178, and 180 at the end of the ₂ domain. Gene conversion or reciprocal recombination are postulated to have transferred this cluster of substitutions, and their associated epitope, during the evolution of HLA-B locus genes. The epitope may consist of an a helical segment which is exclusively found on MB40.2-positive molecules.     

Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland

Background

In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48.

Methods/Results

The nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule.

Conclusions

The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.