Readaptation of a low-virulence influenza H9 escape mutant in mice: the role of changes in hemagglutinin as revealed by site-specific mutagenesis.

In our earlier studies, we showed that an escape mutant of mouse-adapted H9N2 influenza virus carrying a T198N amino acid change in heamagglutinin (HA) has a lowered virulence for mice. The readaptation of this mutant to mice was associated with N198S or N198D reverse mutations. In this study, single-gene reassortants having HA gene of the wild-type virus, its low-virulence escape mutant, or a readapted variant were generated by site-specific mutagenesis and assayed for virulence. The results showed that antibody-selected mutations in the HA of H9 influenza virus can decrease mortality and virus accumulation in mouse lungs, though not in nasal turbinates, and the effect may be compensated by reverse mutations in the course of passaging.

[Impact of mutations in the hemagglutinin gene of H5N1 influenza virus on antigenicity and virulence as revealed by site-specific mutagenesis].

In our earlier studies, we have shown that amino acid changes in the hemagglutinin (HA) of influenza H5N1 virus escape mutants conferring resistance to monoclonal antibodies (MAbs) may correlate with a decrease of virus virulence for mice and that the virulence can be restored to the initial level by serial passages. In the present study, the mutations identical to those observed in the HA of a low-virulent escape mutant and its readapted variant were introduced into the HA gene by site-specific mutagenesis. The viruses produced by plasmid transfection and containing the HA gene either of A/Vietnam/1203/2004 (H5N1) virus with a deletion at the cleavage site, or of a low-virulent escape mutants, or of its readapted variant, in the presence of 6 genome segments of A/Puerto Rico/8/34 (H1N1) virus and the NA gene of A/Vietnam/1203/2004 (H5N1) virus, were assayed for virulence. Determination of virulence for mice indicated that amino acid substitution in the HA gene of a low-virulent escape mutant produced a decrease of virulence whereas the additional mutation identical to that acquired by the escape mutant in the course of readaptation restored the virulence to initial level. The findings are the first strong evidence for lower H5N1 virus virulence resulting from the amino acid substitution changing the antigenic specificity of HA and for restored virulence arising from compensating mutation in the HA gene.

[Amino acid substitutions in the hemagglutinin of H5 influenza virus changing the antigenic specificity and virus virulence].

In our earlier studies, we mapped the hemagglutinin antigenic epitopes of H5 influenza virus by selecting mutants resistant to the neutralizing effect of the antibody (escape mutants). Several escape mutants were shown to have a lowered virulence for mice. The readaptation of low-virulent escape mutants to mice resulted in the restoration of virulence. In the present communication. We present data on the assay of virulence of single-gene reassortants containing HA genes of the wild-type virus, low-virulent escape mutant, or re-adapted variant, and the other genes of a mouse-adapted H9N2 Influenza virus. The results demonstrate that the amino acid change S145F (H3 numbering) in the hemagglutinin ensuring the resistance to a monoclonal antibody can be deleterious to virulence, and that the damaging effect on virulence may be compensated for by additional amino acid changes in position 186 in the hemagglutinin arising in the course of virus passaging in mice. The data indicate that the compensational mutations restoring the pathogenic potential of antigenic variants may be regarded as an additional factor in the evolution of influenza virus hemagglutinin.

[Optimization of the gene composition of influenza H5 virus hemagglutinin-containing reassortants and their efficacy in immune cross-protection experiments].

The reassortant described in the authors' previous paper contained 6 genes originating from the high-yield virus A/Puerto Rico/8/34 (H1N1) and the genes of hemagglutinin (HA) and neuraminidase (NA) of the low-pathogenic avian influenza A/Duck/Primorie/2621/2001 (H5N2) (6:2 reassortant). The reassortant was used for the backcrossing with the parent avian virus in order to optimize the gene composition. Genotyping of the highest-yield second-generation reassortment indicated that it had obtained the PB1, HA, and NA genes from the virus A/Duck/Primorie/ 2621/2001 and the other genes received the genes from the virus A/Puerto Rico/8/34 (5:3 reassortant). The yield produced in the embryonated chicken eggs by the 5:3 reassortant was higher than that produced by the 6:2 reassortant although it did not achieve the reproduction of the parent virus A/Puerto Rico/8/34. Murine immunization with the inactivated reassortant containing the HA and NA genes of the virus A/Duck/Primorie/2621/2001 (H5N2) provided an efficient protection against the virus containing HA and NA of a recent H5N1 strain.

Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants.

Reassortants between a low-pathogenic avian influenza virus strain A/Duck/Primorie/2621/2001 (H5N2) and a high-yield human influenza virus strain A/Puerto Rico/8/34 (H1N1) were generated, genotyped and analyzed with respect to their yield in embryonated chicken eggs, pathogenicity for mice, and immunogenicity. A reassortant having HA and NA genes from A/Duck/Primorie/2621/2001 virus and 6 genes from A/Puerto Rico/8/34 virus (6:2 reassortant) replicated efficiently in embryonated chicken eggs, the yields being intermediate between the yields of the avian parent virus and those of the A/Puerto Rico/8/34 parent strain. The reassortant having the HA gene from A/Duck/Primorie/2621/2001 virus and 7 genes from A/Puerto Rico/8/34 virus (7:1 reassortant) produced low yields. A variant of the 7:1 reassortant selected by serial passages in eggs had an amino acid substitution in the hemagglutinin (N244D, H3 numbering). The variant produced yields similar to those of the 6:2 reassortant. A 5:3 reassortant generated by a back-cross of the 6:2 reassortant with the avian parent and having PB1, HA and NA genes of A/Duck/Primorie/2621/2001 virus produced higher yields than the 7:1 or 6:2 reassortants, although still lower than the yields of A/Puerto Rico/8/34 virus. The 7:1, 6:2 and 5:3 reassortants were pathogenic for mice, with the level of virulence close to A/Puerto Rico/8/34 virus, in contrast to the extremely low pathogenicity of the A/Duck/Primorie/2621/2001 parent strain. Immunization of mice with an inactivated 6:2 H5N2 reassortant provided efficient immune protection against a reassortant virus containing the HA and NA genes of a recent H5N1 isolate. The results are discussed in connection with the problem of the improvement of vaccine strains against the threatening H5N1 pandemic.