[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.

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

The neuraminidase inhibitor GS4104 (oseltamivir phosphate) is efficacious against A/Hong Kong/156/97 (H5N1) and A/Hong Kong/1074/99 (H9N2) influenza viruses.

In 1997, an H5N1 avian influenza A/Hong Kong/156/97 virus transmitted directly to humans and killed six of the 18 people infected. In 1999, another avian A/Hong/1074/99 (H9N2) virus caused influenza in two children. In such cases in which vaccines are unavailable, antiviral drugs are crucial for prophylaxis and therapy. Here we demonstrate the efficacy of the neuraminidase inhibitor GS4104 (oseltamivir phosphate) against these H5N1 and H9N2 viruses. GS4071 (the active metabolite of oseltamivir) inhibited viral replication in MDCK cells (EC(50) values, 7.5-12 microM) and neuraminidase activity (IC(50) values, 7.0-15 nM). When orally administered at doses of 1 and 10 mg/kg per day, GS4104 prevented death of mice infected with A/Hong Kong/156/97 (H5N1), mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2), or human A/Hong Kong/1074/99 (H9N2) viruses and reduced virus titers in the lungs and prevented the spread of virus to the brain of mice infected with A/Hong Kong/156/97 (H5N1) and mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2) viruses. When therapy was delayed until 36 h after exposure to the H5N1 virus, GS4104 was still effective and significantly increased the number of survivors as compared with control. Oral administration of GS4104 (0.1 mg/kg per day) in combination with rimantadine (1 mg/kg per day) reduced the number of deaths of mice infected with 100 MLD(50) of H9N2 virus and prevented the deaths of mice infected with 5 MLD(50) of virus. Thus, GS4104 is efficacious in treating infections caused by H5N1 and H9N2 influenza viruses in mice.

Cross-protection of mice immunized with different influenza A (H2) strains and challenged with viruses of the same HA subtype.

Cross-protection of mice immunized with inactivated preparations of human and avian influenza A (H2) viruses was determined after lethal infection with mouse-adapted (MA) variants of human A/Jap x Bell/57 (H2N1) and avian A/NJers/78 (H2N3) viruses. The MA variants differed from the original strains by acquired virulence for mice and changes in the HA antigenicity. These studies indicated that mice vaccinated with human influenza A (H2) viruses were satisfactorily protected against challenge with A/Jap x Bell/57-MA variant; the survival rate was in the range of 61%-88.9%. Immunization of mice with the same viral preparations provided lower levels of protection against challenge with A/NJers/78-MA variant. Vaccination of mice with the avian influenza A (H2) viruses induced better protection than with human strains against challenge with both MA variants. Challenge with A/NJers/78-MA variant revealed that 76.2%-95.2% of animals were protected when vaccinated with avian influenza virus strains isolated before 1980, and that the protection reached only 52.4%-60.0% in animals vaccinated with strains isolated in 1980-1985. The present study revealed that cross-protection experiments in a mouse model could provide necessary information for the development of appropriate influenza A (H2) virus vaccines with a potential for these viruses to reappear in a human population.

Changes of morphological, biological and antigenic properties of avian influenza A virus haemagglutinin H2 in the course of adaptation to new host.

The alterations of avian influenza A virus haemagglutinin (HA) H2 as a result of adaptation to mice were first investigated in this study. HA of mouse-adapted (MA) variant was somewhat different from that of the original strain in electrophoretical mobility, antigenic structure and in haemagglutination activity with mouse red blood cells.

Amino acid changes in the hemagglutinin and matrix proteins of influenza a (H2) viruses adapted to mice.

Mouse-adapted (MA) variants of human and avian influenza A (H2) viruses were generated and characterized with respect to acquisition of virulence in mice. From the nucleotide sequence the amino acid sequence was deduced. The HA1 subunit of the hemagglutinin (HA) contained three amino acid substitutions in the A/black duck/New Jersey/1580/78-MA variant (Glu216-->Asp, Lys307-->Arg, and Thr318-->Ile) and two substitutions in the A/JapanxBellamy/57-MA variant (Lys25-->Thr and Ser203-->Phe). In the M1 protein, there were two substitutions in the A/black duck/New Jersey/1580/78-MA variant (Asn30-->Asp and Gln214-->His) and a single substitution in the A/JapanxBellamy/57-MA variant (Met179-->Lys). The M2 protein amino acid sequences of the parental virus and the MA variants differed by a single identical mutation (Asn93-->Ser). The localization and atomic distances of the observed mutations on the three-dimensional (3D) structure of the HA protein were analyzed for influenza H2 viruses. The obtained results were similar to those published earlier on H1, H3 and H5 subtypes. The amino acid changes in the HA protein could be divided into two groups. In one group the substitutions were situated at the top of the molecule, while in the other group they were clustered in the stem area at the interface region between three HA monomers. The analysis revealed that the substitutions observed in the MA variants probably increase the flexibility of the HA molecule and/or weaken the interactions between monomers or subunits in the HA trimer. The relationships of the observed amino acid changes in the HA and M proteins to the biological properties of the respective viruses and possible mechanisms involved in the acquisition of viral virulence are discussed.

The source of avian paramyxoviruses isolated during an outbreak of influenza among children.

In 1986 five avian paramyxovirus (PMV) strains were isolated in embryonated chicken eggs from sick children with influenza. The strains were identified as PMV-2 serotype due to the close antigenic relationships between their HN-proteins and of the reference PMV-2 strains isolated from different birds all over the world. No seroconversion to the isolates was found in the sick children, however, HI-antibodies were detected in hen's sera, eggs of which were used for the new strains isolation. The possible origin of isolated PMV-2 viruses is discussed.

[Selection of antigenic variants of influenza virus hemagglutinin by host cells and choice of the optimal system for culturing them]. / Selektsiia kletkami khoziana antigennykh variantov gemaggliutinina virusov grippa i vybor optimal'nykh sistem dlia kul'tivirovaniia.

Reviews published reports on the relationship between the culturing system and formation of antigenic variants of influenza virus. Analyzes molecular biological changes in the virus hemagglutinin and the main methods of selection. Discusses the significance of this phenomenon for sero-epidemiological studies, influenza surveillance, and production of anti-influenza vaccines and diagnostic agents.

[Molecular basis and mechanisms of Influenza viruses adaptation to reproduction in murine lung]. / Molekuliarnye osnovy i mekhanizmy adaptatsii virusov grippa k reproduktsii v legkikh myshe'i.

Analyzes modern data on the role of individual segments of genome in acquisition of virulence during influenza virus adaptation to reproduction in the lungs of mice. Discusses the significance of molecular mechanisms (loss of potential glycosylation site(s), optimum pH of HA-mediated fusion, beta-inhibitor sensitivity, pH-dependent association/dissociation of M1 protein with viral RNP, and host factors) involved in adaptation of influenza virus to a new host.