Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice

Irina A. Rudneva1, Natalia A. Ilyushina1,2, Tatiana A. Timofeeva1, Robert G. Webster2 and Nikolai V. Kaverin1

1 The D. I. Ivanovsky Institute of Virology, 16 Gamaleya Street, 123098 Moscow, Russia
2 Division of Virology, Department of Infectious Diseases, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA

Correspondence
Nikolai V. Kaverin
kaverin{at}online.ru


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such ‘compensating’ mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The identity of the future pandemic influenza virus is not known. In 1997, the avian H5N1 virus was transmitted from poultry to humans in Hong Kong and caused 18 human cases of influenza with a high mortality rate (CDC, 1998). In February 2003, two cases of H5N1 infection in humans occurred in Hong Kong, one of them fatal (Wuethrich, 2003). In 2004–2005, during an extensive outbreak of H5N1 infection in poultry in eight countries of Southeast Asia, over 100 human cases occurred in Vietnam, Thailand and Cambodia, 50 % of them fatal (WHO, 2005). Human infection with H9N2 virus has also been reported (Guo et al., 1999; Peiris et al., 1999).

The occasional infection of humans by the recent Asian H5 and H9 viruses and the high virulence of H5 viruses in mammals depend on features of several viral components. The virulence of the new H5 isolates in mammals depends, as does the pathogenicity of H5 viruses in birds, on the presence of additional basic amino acid residues at the cleavage site of the haemagglutinin (HA) molecule (Webby et al., 2004). The role of the other structural features of the HA in virulence and other biological characteristics of H5 and H9 viruses is less clear. Changes in the HA receptor-binding site of H9 isolates allow adaptation to mammalian-type sialic receptors (Matrosovich et al., 2001), and a change at position 227 of the HA1 subunit of an H5 virus has a role in the virulence of the virus in mice (Hatta et al., 2001). Several features distinguishing the HAs of H5 viruses isolated from humans and chickens from those isolated from aquatic birds have also been described (Matrosovich et al., 1999). However, the basis for the relationship between the characteristics of the HA and the biological features of H5 and H9 viruses is far from understood. The problem is especially important in connection with the recently reported tendency of avian H5 viruses to widen their host range and produce systemic infection in ducks (Sturm-Ramirez et al., 2004).

Antigenic analysis of new H5 isolates has shown a change in reactivity pattern of the HA with polyclonal sera and monoclonal antibodies (mAbs) comparable to the anti-genic drift in human influenza viruses (Guan et al., 2004). It has been suggested that this kind of evolution may be explained by immune pressure caused by the use of a vaccine, which could have created a survival advantage for those H5 viruses that undergo antigenic variation. It is not known whether the antigenic variation is connected with the changes in pathogenicity exhibited by recent avian H5 isolates.

In the course of our previous studies of H5 (Kaverin et al., 2002) and H9 (Kaverin et al., 2004) influenza viruses, immunological characterization and sequencing of the HA molecules of escape mutants allowed us to map antigenic sites in the three-dimensional structure of the HA molecule. Use of mouse-adapted variants of non-pathogenic avian H5 and swine H9 influenza viruses allowed us to register changes in virulence associated with amino acid substitutions that resulted in resistance to mAbs.

Antibody-selected mutations, either in escape mutants or in drift variants, are sometimes associated with phenotypic changes, including those that alter the virus's affinity for cell receptors or virulence. For example, the acquisition of glycosylation sites by escape mutants of an H2 virus is associated with decreased cell fusion and receptor-binding activity of HA (Tsuchiya et al., 2002). An H3 drift variant is reported to have a decreased capacity to reproduce in mouse lungs (Reading et al., 1997), and a change in the receptor-binding ability of HA has been described for H3 escape mutants (Daniels et al., 1987). An escape mutant of a virulent avian H5 virus has been shown to have decreased virulence for birds (Philpott et al., 1990). An amino acid change acquired by a human H1N1 virus in the course of adaptation to mice was shown to induce a change in the reaction with a mAb (Gitelman et al., 1986). In our studies on the HA of H5 and H9 escape mutants, specific amino acid changes were associated with a decrease in virulence for mice (Kaverin et al., 2002, 2004). This decreased virulence often (for H9) coincided with reduced affinity of the mutant HA for sialyl substrates (Kaverin et al., 2004; Ilyushina et al., 2004b, c). We believe that such phenotypic changes resulted from HA mutations selected by the mAbs rather than from mutations elsewhere that were randomly co-selected, because we saw several cases in which a loss of virulence was associated with a specific amino acid change in two or more escape mutants independently selected with different mAbs.

The purpose of the current study was to gain further insight into the connection between antibody-selected mutations in HA and virulence. To do so, we attempted to restore the pathogenicity of low-virulence H5 and H9 escape mutants by lung-to-lung passage in mice. Our goal was to assess whether the reacquisition of virulence was associated with the exact restoration of the wild-type sequence of HA or whether the initial virulence could be restored by other mutations. The results of our analysis of the readapted variants are reported here.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses.
The avian non-pathogenic influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) and the swine influenza virus A/Swine/Hong Kong/9/98 (H9N2), both taken from the virus repository of the Virology Department of St Jude Children's Research Hospital (Memphis, TN, USA), were adapted to mice by lung-to-lung passage (Smirnov et al., 2000; Kaverin et al., 2004). The mouse-adapted variants were designated Mld/PA/84-MA and Sw/HK/9/98-MA, respectively (GenBank accession nos AF512925 and AY428485). Escape mutants of the mouse-adapted variants were selected with panels of anti-H5 HA and anti-H9 HA mAbs (Kaverin et al., 2002, 2004). Of 16 H5 escape mutants (Kaverin et al., 2002), we used three in the present study that had decreased virulence for mice: m55(2), m58(1) and m176/26 (GenBank accession nos AF512927–AF512929). Of 18 H9 escape mutants (Kaverin et al., 2004), we used two low-virulence mutants: m8C4 and m18B10 (GenBank accession nos AY428501 and AY428491). The viruses were propagated in 10-day-old embryonated chicken eggs. The virus-containing allantoic fluid was stored at 4 °C or –70 °C. For studies on virus binding to substrates containing sialic acid, the allantoic fluids were clarified by low-speed centrifugation and used without further purification. For use in ELISAs, virus was concentrated and partially purified by layering on 20 % sucrose and pelleting by centrifugation at 23 000 r.p.m. in an SW27.1 rotor at 4 °C for 90 min.

mAbs.
mAbs cp46, cp55 and cp58 to A/Chicken/Pennsylvania/1370/83 (H5N2) virus, mAbs 176/26 and 364/1 to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and mAbs 7B10, 8C4, 18B10, 18G4, 3D11, 4G3, 19A10, 2F4, 18B1 and 15F1 to A/Duck/Hong Kong/Y280/97 (H9N2) virus were produced in the Virology Department and the Department of Infectious Diseases, St Jude Children's Research Hospital. Ascites fluids containing the antibodies were prepared by the method described by Kohler & Milstein (1976) with modifications as described previously (Kaverin et al., 2004).

Infection of mice.
White outbred female mice weighing 6–8 g (purchased from the Laboratory Animal Breeding Institution of the Russian Academy of Medical Sciences, Andreevka, Moscow Region, Russia) were lightly anaesthetized with diethyl ether and inoculated intranasally. For the assessment of virulence, mice were inoculated with 100 µl of serial 10-fold dilutions of virus-containing allantoic fluid (six mice per dilution). Mouse deaths were registered during the 10 days after inoculation and the mortality rate was expressed in terms of 50 % mouse lethal dose (MLD50), as calculated by the method of Reed & Muench (1938). In experiments on the readaptation of low-virulence escape mutants to mice, lung-to-lung passages were used. Infected mice were sacrificed 2 days after inoculation. Homogenates of lung tissue were prepared in PBS, clarified by low-speed centrifugation and used for the passage. At each passage, two mice were kept for observation to register mortality.

Serological methods.
ELISA was performed essentially as described by Philpott et al. (1989) with modifications as described previously (Kaverin et al., 2002). The haemagglutination inhibition (HI) test was performed by a standard method (Palmer et al., 1975).

Assay of virus binding to sialic acid-containing substrates.
The binding of the viruses to fetuin was performed in a direct solid-phase assay using immobilized virus and biotinylated polyvalent synthetic sialoglycoconjugates (Sug-PAA-biot), synthesized as described earlier (Bovin et al., 1993; Tuzikov et al., 2000; Mochalova et al., 2003). According to monosaccharide analysis data, the molar content of Sug in all conjugates was 20 % and the molar content of biotin in Sug-PAA-biot was 5 %. The affinity of the virus for Sug-PAA-biot was measured essentially as described previously (Ilyushina et al., 2004c). Briefly, 96-well PVC microtitre plates were coated with virus at a titre of 4–8 haemagglutination units (50 µl per well) at 4 °C for 16 h, followed by washing with 0·05 % Tween 20 in PBS (PBS-T). After adding 30 µl Sug-PAA-biot in working buffer (PBS supplemented with 0·02 % Tween 20, 0·02 % BSA and 3 µM of the neuraminidase inhibitor 2,3-didehydro-2,4-dideoxy-4-amino-N-acetyl-D-neuraminic acid) to each well, plates were incubated at 4 °C for 1 h. The starting concentration of Sug-PAA-biot was 20 µM on sialic acid; twofold serial dilutions were used. Plates were washed with cold PBS-T and incubated with horseradish peroxidase–streptavidin conjugate in the working buffer at 4 °C for 1 h. After washing, 100 µl of substrate solution (0·1 M sodium acetate, pH 5·0, containing 4 mM o-phenylenediamine and 0·004 % H2O2) was added to each well and the reaction was stopped with 2 M H2SO4. Absorbance was determined at 492 nm using a Multiscan plate reader (Labsystems). Dissociation constants (Kd) were determined as N-acetylneuraminic acid concentration at the point 0·5xAmax of Scatchard plots. Data are reported as the mean of at least four individual experiments for each virus.

PCR amplification and sequencing.
Viral RNA was isolated from virus-containing allantoic fluid using the RNeasy Mini kit (Qiagen) as specified by the manufacturer. Reverse transcription of viral RNA and subsequent PCR was performed using primers specific for the HA gene segment, as described previously (Hoffmann et al., 2001). PCR products were purified with the QIAquick PCR purification kit (Qiagen). The sequencing reaction was performed by the Inter-institute Center ‘Genome’ (Institute of Molecular Biology, Russian Academy of Sciences). The DNA template was sequenced using a DNA ABI PRISM 3100-Avant sequencer and BigDye Terminator v3.1 kit; DNA sequences were completed and edited using the Lasergene sequence analysis software package (DNASTAR).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Readaptation of low-virulence H5 and H9 escape mutants to mice
Five low-virulence escape mutants (selected with five different mAbs) were chosen for the readaptation to mice, including two H9 mutants selected from Sw/HK/9/98-MA virus and three H5 mutants selected from Mld/PA/84-MA virus. The low-virulence H9 mutants, m8C4 and m18B10, had the same amino acid change in the HA1 subunit, T198N (H3 numbering, here and elsewhere), which resulted in a potential glycosylation site (Kaverin et al., 2004). Of the three H5 mutants, the mutants m58(1) and m176/26 had the same amino acid change, D131N, which resulted in a new glycosylation site. The mutant m176/26 had an additional mutation, K82R, in the HA2 subunit. The mutant m55(2) had the amino acid change K156N. It was the only low-virulence escape mutant used in the present studies that had no new glycosylation sites acquired during the selection with mAbs (Kaverin et al., 2002).

The low-virulence escape mutants were lung-to-lung passaged in mice as described in Methods. At the tenth passage, the lung tissue homogenate was clarified by low-speed centrifugation, diluted 1 : 10–4 and used for the inoculation of embryonated chicken eggs. The allantoic fluid was collected after incubation for 48 h at 37 °C and used for limiting-dilution cloning. Two to three virus clones were obtained for each passage series. The clones were designated RA (readapted), followed by the designation of the respective mutant and the number of the clone. The virulence of the clones was assessed by parallel titration in mice and in embryonated chicken eggs and expressed as 50 % egg infectious dose (EID50)/MLD50. The virulence of all readapted H9 variants (readaptants) (Table 1) and all H5 readaptants except RAm58(11) (see Table 4) was restored to that of the respective mouse-adapted wild-type virus. The differences between the EID50/MLD50 values of the low-virulence mutants and those of the readapted clones were statistically significant (P<0·01 for H9 clones and P<0·05 for H5 clones) except for RAm58(11).


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Table 1. Effect of readaptation of escape mutants of Sw/HK/9/98-MA influenza virus (H9N2) on virulence in mice

 

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Table 4. Effect of readaptation of escape mutants of Mld/PA/84-MA influenza virus (H5N2) on virulence in mice

 
Sequence analysis and phenotypic characterization of the H9 readapted variants
We compared the nucleotide sequences and the encoded amino acid sequences of the readapted variants with those of the respective escape mutants and wild-type mouse-adapted viruses (see Tables 1 and 4). All H9 readaptants had a mutation leading to the loss of the potential glycosylation site at position 198; i.e. the site acquired by the escape mutants m8C4 and m18B10 in the course of selection. However, the wild-type sequence was not restored in any of the readaptants: in neither case did the reverse change (N198T) occur. Instead, asparagine 198 was changed in one readaptant to serine and in three others to aspartic acid (Table 1). The readapted variants of the mutant m18B10 acquired an additional amino acid change at position 95.

For serological characterization of the readapted variants, we used anti-H9 HA mAbs. The anti-H9 HA mAbs that were unable to discern the wild-type virus from mutants m8C4 and m18B10 did not reveal any differences in reactions with the readaptants compared to the wild-type virus and mutant viruses (not shown). The results of reactions of wild-type Sw/HK/9/98-MA, escape mutants m8C4 and m18B10, and four readaptants in ELISA and HI with three mAbs discerning the escape mutants from the wild-type mouse-adapted virus (Kaverin et al., 2004) are presented in Table 2. The results of ELISA revealed that all four readaptants reacted with the mAbs to the same extent as the wild-type virus. However, the restoration of HI to wild-type level was complete only for the readaptant RAm8C4(1), which had acquired the change N198S, i.e. a conservative amino acid substitution with respect to the wild-type sequence. The other three readaptants, RAm8C4(2), RAm18B10(1) and RAm18B10(2), which had acquired the non-conservative change N198D, had fully restored HI reactions with two mAbs, 18B10 and 15F1, but their reaction with mAb 8C4 was only partially restored.


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Table 2. Reactions of the H9 escape mutants and their readapted variants with anti-HA mAbs

ELISA results are expressed as a percentage of the amount of mAb binding to the wild-type virus, which is designated 100 %. The HI titre is the dilution of antibody that inhibits 8 haemagglutination units of virus.

 
Our previous studies (Kaverin et al., 2004) revealed a correlation between decreased virulence of H9 escape mutants and reduced affinity of their HA for high-molecular-mass sialic acid-containing substrates. The low-virulence mutants we chose for readaptation in the present study, m8C4 and m18B10, exhibited a sharp decrease in their affinity for fetuin, as measured in a direct solid-phase assay, as well as for 6'-sialylglycopolymers as revealed by a competitive assay based on the inhibition of peroxidase-labelled fetuin. This finding prompted us to measure the affinity of the readapted variants of H9 escape mutants for sialic acid-containing substrates. We used a direct test with the biotinylated polyvalent synthetic 6'-sialylglycopolymers obtained by conjugation of a 1-N-glycyl derivative of 6'-sialyllactose or 6'-sialyllactosamine to poly(4-phenylacrylate) (designated 6'SL-PAA-biot and 6'SLN-PAA-biot, respectively). The assay revealed (Table 3) that the affinity for the synthetic substrates was restored in all H9 readaptants to the level shown by the wild-type mouse-adapted virus.


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Table 3. Affinity of H9 escape mutants and their readapted variants for sialyl substrates

{kappa}d=mean±SExt{alpha},n–1 (µM sialic acid), where t{alpha} is Student's coefficient with probability {alpha}=0·95, from four independent experiments. ND, Not determined.

 
The results obtained in the course of the sequence analysis and phenotypic characterization of H9 readapted variants strongly suggested that the reacquisition of virulence does not need the exact restoration of the wild-type HA sequence. Instead, the removal of the glycosylation site, acquired previously by the escape mutant, seems to be important. The removal of the glycosylation site was accompanied by an increase in the binding of some sialic acid-containing substrates, which may be regarded as a possible mechanism involved in the restoration of virulence. Interestingly, in this particular case we never observed the elimination of the glycosylation site by a true reverse mutation, i.e. by restoration of the initial wild-type amino acid and nucleotide sequence. The possible cause of this lack of true reverse mutations in the analysed H9 readaptants is discussed below.

Sequence analysis and immunological characterization of the H5 readapted variants
The passage in mice of those H5 escape mutants that had acquired a new glycosylation site at position 131 (Kaverin et al., 2002), i.e. mutants m58(1) and m176/26, resulted in the selection of five virus clones (Table 4). Among them, four variants exhibited a statistically significant increase in virulence. Like the H9 readaptants, they lost the glycosylation site acquired by the escape mutants. However, in this case, the readapted variants, unlike those of the H9 subtype, regained the exact wild-type sequence at the site of the mutation. Some of them also had additional mutations. The only clone that exhibited no increase in virulence, RAm58(11), retained the glycosylation site present in the low-virulence escape mutant m58(1) (Table 4).

The readaptation of the mutant m55(2), the only low-virulence mutant that had no additional glycosylation sites, produced two readapted variants, RAm55(13) and RAm55(14). Both variants retained the mutation at position 156 that was associated with decreased virulence in the escape mutant m55(2) and acquired an additional mutation, T113I. Although these readaptants preserved the amino acid change K156N, they both exhibited increased virulence, to a level close to that of the wild-type virus (Table 4).

The H5 readaptants that had lost the glycosylation site at position 131 through restoration of the wild-type sequence at this position, i.e. RAm58(12), RAm176/26(11), RAm176/26(12) and RAm176/26(21), restored the reaction with mAbs to the wild-type pattern (Table 5). As might be expected, the mAb reaction pattern of the clone RAm58(11), which had retained the glycosylation site and did not show restored mouse virulence, was the same as that of the low-virulence mutant m58(1). The readapted clones RAm55(13) and RAm55(14), which retained the mutation at position 156, also retained the mutant reaction pattern with the mAbs, although their virulence was restored to wild-type levels (Table 5).


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Table 5. Reactions of the H5 escape mutants and their readapted variants with anti-HA mAbs

ELISA results are expressed as a percentage of the amount of mAb binding to the wild-type virus, which is designated 100 %. The HI titre is the dilution of antibody that inhibits 8 haemagglutination units of virus.

 
As our previous studies (Ilyushina et al., 2004a) had not revealed any definite correlation between the level of virulence and the affinity to sialic substrates for H5 escape mutants (unlike H9 mutants), we did not perform measurement of the binding of H5 readaptants to sialic acid-containing substrates.

The results obtained with the H5 readapted variants confirmed the observation on the role of the loss of glycosylation site made during the studies on the H9 readaptants. In the case of H5 readaptants, the loss was achieved by restoration of the wild-type nucleotide and amino acid sequence. Moreover, analysis of the H5 readaptants RAm55(13) and RAm55(14) revealed another kind of change associated with the restoration of virulence, i.e. an additional amino acid change in a remote part of the HA molecule.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many studies have registered and analysed the association between immunologically relevant changes in influenza virus HA and alterations of virulence and other phenotypic features connected with virus–host interactions. In our recent studies, we registered a decrease in virulence of some of the H5 (Kaverin et al., 2002) and H9 (Kaverin et al., 2004) escape mutants.

A decrease in virulence of an antibody-selected variant may result from a randomly co-selected mutation in a gene other than that encoding the antibody target, in this case HA. However, this explanation seems unlikely for the low-virulence H5 and H9 escape mutants selected with mAbs in our present and previous studies. In both H5 and H9 escape mutants, the loss of virulence associated with the acquisition of a new glycosylation site occurred in two or more mutants carrying the same amino acid change (D131N in H5 and T198N in H9). The identical mutations were selected with different mAbs, which reacted with partially overlapping epitopes, such as mAbs cp58 and 176/26 for H5 and 8C4 and 18B10 for H9. The H5 mutants with a mutation at position 156, m55(1) and m55(2), were previously selected with the same mAb in independent selection experiments (Kaverin et al., 2002, 2004). The mutation at position 156 has been shown to be responsible for loss of virulence for birds of a pathogenic H5 strain (Philpott et al., 1990), and the mutation at position 131 in H3 HA correlates with both a change in virulence for mice and the acquisition of resistance to a mAb (Gitelman et al., 1986). For all low-virulence H9 mutants except one, we registered an association of the decrease in virulence with the decrease in the affinity for high-molecular-weight sialic acid-containing substrates (Kaverin et al., 2004).

These considerations argue against a random co-selection of mutations in viral genes other than the HA gene as the cause of the reduction in virulence of the H5 and H9 escape mutants. However, the readaptation raises the problem anew. The association of an amino acid change in HA with the restoration of pathogenicity upon readaptation cannot be regarded as proof of a cause-and-effect relationship between structural changes in HA and an increase in virulence. Virulence-enhancing mutations in different viral genes are likely to be selected during lung-to-lung passage. However, for several reasons, discussed below, it seems likely that the amino acid changes in HA at least contribute to the observed restoration of virulence.

First, the loss of glycosylation sites (in positions where such sites had been acquired during selection of mutants with mAbs) was invariably associated with the restoration of virulence in both H5 and H9 readapted variants. In all four H9 readaptants, the potential glycosylation site at position 198 was lost, although in neither readaptant was the initial amino acid sequence of the virulent wild-type strain restored. The H5 escape mutants m58(1) and m176/26 also lost the glycosylation site during readaptation (if the passaging resulted in the restoration of virulence), whereas the only clone in which virulence was not restored, RAm58(11), retained the glycosylation site. For both H5 and H9 mutants, readaptants with identical amino acid changes were produced during different readaptation experiments. Such repeated association of a certain type of structural change with the regaining of virulence argues for a cause-and-effect relationship between mutations leading to loss of the glycosylation site at position 131 in H5 HA and at position 198 in H9 HA and the restoration of virulence. Secondly, the positions where the amino acid changes occurred during readaptation were previously identified as those involved in the variation of virulence. This observation concerns not only the role of position 131 (Gitelman et al., 1986; Tsuchiya et al., 2002), but also the mutation T113I in the readaptants RAm55(13) and RAm55(14). In both readaptants the attenuating mutation K156N was preserved, yet the viruses regained virulence. It was recently reported that the change T113I in recent H5N1 isolates leads to increased virulence (Hulse et al., 2004). This seems to be a strong, although indirect, indication of a role for the change T113I in increasing virulence during readaptation.

The amino acid positions involved in virulence in the present studies were earlier shown to be in the vicinity of the receptor-binding site of HA (Weis et al., 1988) and thus are likely to be involved in receptor recognition.

These considerations do not rule out the role of mutations in other genes. Several genes have been shown to be involved in the virulence of influenza virus for mice, among them PB2 (Hatta et al., 2001), NP, M and NS (Brown, 1990). To determine whether the amino acid substitutions in HA are alone sufficient for the restoration of virulence, one would have to measure the virulence of reassortants having the HA gene of a mutant or a readaptant, with the other genes of the wild-type virus. Such reassortants can be obtained by plasmid transfection (Hoffmann et al., 2001). We intend to use this approach in our future studies.

Notably, the loss of a glycosylation site at position 131 in four H5 readaptants resulted from a reverse mutation and restoration of the wild-type sequence, whereas the removal of the potential glycosylation site in all four H9 readaptants resulted from mutations that did not restore the initial amino acid sequence of the virulent wild-type virus. Why was the initial H9 sequence not restored? It is not known, but examination of the nucleotide changes (Table 1) reveals that the creation of a new glycosylation site at position 198 requires a transversion, i.e. a pyrimidine to purine change. The mutations removing this site in the readapted variants are all transitions (purine to purine). Transitions occur much more easily than transversions. This general rule was recently shown to apply to the replication of several RNA-containing viruses (Kuge et al., 1989; Speller et al., 1993; Sanchez et al., 2003) and one may surmise that influenza virus is no exception. The loss of the glycosylation site is much more likely to result from a transition than a transversion. Therefore, it seems likely that for this reason the readaptation of the H9 escape mutants involved pseudoreversions, which are easily achieved by a transition, rather than true reversion to the wild-type sequence, which requires a transversion. On the other hand, the H5 escape mutants m58(1) and m176/26 acquired a glycosylation site at position 131 as a result of a transition (Table 4), which made a true reversion much more likely in H5 than in H9 mutants.

Avian influenza viruses are evolving rapidly. It is not known whether antigenic variation is connected with the changes in pathogenicity exhibited by recent avian H5 isolates, but such a connection cannot be excluded. Our data may be regarded as an indication of a possible mechanism underlying such a connection. Our previous studies (Kaverin et al., 2002, 2004) have shown that antibody-selected mutations frequently have additional effects, sometimes deleterious to virulence. It seems legitimate to suggest that such effects may occur in nature during antigenic drift or drift-like evolution such as that described recently for the new H5 strains. In this aspect, the occurrence of the amino acid change T113I in one of the H5 readaptants (Table 4), identical to the change associated with an increase in virulence in a natural H5 isolate (Hulse et al., 2004), is especially noteworthy. The results reported in the present communication suggest that, if the antibody-selected mutation is not too damaging, it may be compensated for by new mutations. This means that the amino acid changes selected by antibodies may have sequels in the course of further virus dissemination, i.e. additional mutations can compensate for the damaging effects of the initial antibody-selected changes. Such compensating effects may represent a hitherto-unrecognized factor in influenza virus evolution.


   ACKNOWLEDGEMENTS
 
The research described in this publication was made possible in part by NATO collaborative linkage grant 979155, and by grants 03-04-48353 and 04-04-48819 from the Russian Foundation for Basic Research (RFBR) and the American Lebanese Syrian Associated Charities (ALSAC).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bovin, N. V., Korchagina, E. Yu., Zemlyanukhina, T. V., Byramova, N. E., Galanina, O. E., Zemlyakov, A. E., Ivanov, A. E., Zubov, V. P. & Mochalova, L. V. (1993). Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides. Glycoconj J 10, 142–151.[CrossRef][Medline]

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Received 16 May 2005; accepted 21 July 2005.



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