To elucidate the structure of the antigenic sites of avian H5 influenza virus haemagglutinin (HA) we analysed escape mutants of a mouse-adapted variant of the H5N2 strain A/Mallard/Pennsylvania/10218/84. A panel of five anti-H5 monoclonal antibodies (mAbs) was used to select 16 escape mutants. The mutants were tested by ELISA and haemagglutination inhibition with this panel of anti-H5 mAbs and the HA genes of the mutants were sequenced. The sequencing demonstrated that the amino acid changes were grouped in two antigenic sites. One corresponded to site A in the H3 HA. The other contained areas that are separated in the amino acid sequence but are topographically close in the three-dimensional structure and partially overlap in the reactions with mAbs. This site corresponds in part to site B in the H3 structure ; it also includes a region not involved in site B that partially overlaps site Sa in the H1 HA and an antigenic area in H2 HA. Mutants with the amino acid change K152N, as well as those with the change D126N, showed reduced lethality in mice. The substitution D126N, creating a new glycosylation site, was accompanied by an increase in the sensitivity of the mutants to normal mouse serum inhibitors. Several amino acid changes in the H5 escape mutants occurred at the positions of reported changes in H2 drift variants. This coincidence suggests that the antigenic sites described and analysed here may be important for drift variation if H5 influenza virus ever appears as a pathogen circulating in humans.

We found and genetically described two novel SARS-like coronaviruses in feces and oral swabs of the greater (R. ferrumequinum) and the lesser (R. hipposideros) horseshoe bats in southern regions of Russia. The viruses, named Khosta-1 and Khosta-2, together with related viruses from Bulgaria and Kenya, form a separate phylogenetic lineage. We found evidence of recombination events in the evolutionary history of Khosta-1, which involved the acquisition of the structural proteins S, E, and M, as well as the nonstructural genes ORF3, ORF6, ORF7a, and ORF7b, from a virus that is related to the Kenyan isolate BtKY72. The examination of bats by RT-PCR revealed that 62.5% of the greater horseshoe bats in one of the caves were positive for Khosta-1 virus, while its overall prevalence was 14%. The prevalence of Khosta-2 was 1.75%. Our results show that SARS-like coronaviruses circulate in horseshoe bats in the region, and we provide new data on their genetic diversity.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.

Summary. The effects of monoclonal antibody (MAb) C179 recognizing a conformational epitope in the middle of the hemagglutinine (HA) stem region were examined in a mouse model in the experiments of prevention and treatment of lethal bronchopneumonia caused by influenza A virus of H5 subtype. To model the lethal infection, avian nonpathogenic strain A/mallard duck/Pennsylvania/ 10218/84 (H5N2) was adapted to mice. This resulted in highly pathogenic pneumovirulent mouse-adapted (MA) variant, which was characterized. Three amino acid changes were found in the HA1 subunit of HA of MA virus. One of these was located inside the region of the conformational epitope recognized by MAb C179. However, this substitution was not significant for the recognition of HA and virus neutralization by MAb C179 in vitro and in vivo. Intraperitoneal administration of two different concentrations of MAb C179 one day before or two days after the virus challenge significantly decreased mortality rate. These results suggest that MAb C179 is efficient not only in the prevention and treatment of H1 and H2 influenza virus bronchopneumonia, as was reported previously, but also of H5-induced bronchopneumonia as well, and demonstrate in vivo the existence of a common neutralizing epitope in the HAs of these three subtypes.*

The family Bunyaviridae has more than 530 members that are distributed among five genera or remain to be classified. The genus Orthobunyavirus is the most diverse bunyaviral genus with more than 220 viruses that have been assigned to more than 18 serogroups based on serological cross-reactions and limited molecular-biological characterization. Sequence information for all three orthobunyaviral genome segments is only available for viruses belonging to the Bunyamwera, Bwamba/Pongola, California encephalitis, Gamboa, Group C, Mapputta, Nyando, and Simbu serogroups. Here we present coding-complete sequences for all three genome segments of 15 orthobunyaviruses belonging to the Anopheles A, Capim, Guamá, Kongool, Tete, and Turlock serogroups, and of two unclassified bunyaviruses previously not known to be orthobunyaviruses (Tataguine and Witwatersrand viruses). Using those sequence data, we established the most comprehensive phylogeny of the Orthobunyavirus genus to date, now covering 15 serogroups. Our results emphasize the high genetic diversity of orthobunyaviruses and reveal that the presence of the small nonstructural protein (NSs)-encoding open reading frame is not as common in orthobunyavirus genomes as previously thought.