Impact of prior seasonal influenza vaccination and infection on pandemic A (H1N1) influenza virus replication in ferrets.

Early epidemiologic and serologic studies have suggested pre-existing immunity to the pandemic A (H1N1) 2009 influenza virus (H1N1pdm) may be altering its morbidity and mortality in humans. To determine the role that contemporary seasonal H1N1 virus infection or trivalent inactivated vaccine (TIV) might be playing in this immunity we conducted a vaccination-challenge study in ferrets. Vaccination with TIV was unable to alter subsequent morbidity or contact transmission in ferrets following challenge with H1N1pdm. Conversely, prior infection with the contemporary seasonal H1N1 strain altered morbidity, but not transmission, of H1N1pdm despite the detection of only minimal levels of cross reactive antibodies.

[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.

Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia?

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia.

Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the "Trojan horse" of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

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.

Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses.

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.

[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.

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 and reassortment studies with avian H2 influenza viruses.

In order to assess the degree of immune cross-protection among avian H2 influenza virus strains, mice were immunised with beta-propiolactone-inactivated virus preparations and infected intranasally with mouse-adapted variant of A/Black Duck/New Jersey/1580/78 (H2N3) strain. The experiments with 11 avian H2 strains revealed that both Eurasian and American H2 avian influenza viruses exhibit either high or moderate degree of cross-protection. The grouping of the strains in accordance with their cross-protection efficiency does not coincide with H2 phylogenetic branches. Several reassortant clones were obtained with the use of A/Pintail Duck/Primorie/695/76 (H2N3) strain and high-yield X-67 reassortant as parent viruses, among them a high-yield H2N3 reassortant. Taking into account the data on cross-protection among avian H2 strains, the high-yield H2N3 reassortant may be regarded as a prototype strain to be used for the preparation of killed vaccines in the case of a new appearance of avian H2 haemagglutinin in circulation in humans.

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.

[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.

Selection of receptor-binding variants of human influenza A and B viruses in baby hamster kidney cells.

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D --> G (H1N1 virus), 128T --> A and 226I --> V (H3N2), and 187N --> D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L --> H, 208R --> H, and 225D --> G (H1N1), 194L --> I (H3N2), and 137G --> R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3'-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6'-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6'-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.

Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.

Vero cells, MDCK cells and embryonated chicken eggs (eggs) were used to evaluate influenza virus growth characteristics and immunogenicity induced by inactivated influenza B vaccines. Both cell lines produced comparable quantities of total viral and haemagglutinin (HA) proteins. Sequence analysis indicated genetic identity of the HA of Vero- and MDCK-grown virus counterparts with maintenance of antigenic characteristics of viruses derived from humans. The egg-grown influenza B/Memphis/1/93 variant differed from cell-grown counterparts at amino acid position 198 (Pro-Thr) and lost a glycosylation site. The level of neuraminidase (NA) activity was the highest in egg-grown virus, while MDCK- and Vero cell-grown viruses possessed 70% and 90% less NA activity respectively when fetuin was used as a substrate. Although each of the vaccines induced high and comparable levels of serum antibodies, mammalian cell-derived vaccines induced antibodies that were more cross reactive, and those antibodies induced by egg-derived vaccine were more specific to the homologous antigen. ELISPOT analysis indicated that the mammalian cell-grown vaccines induced high frequencies of IgG-producing cells directed against both cell- and egg-grown antigens, while egg-grown vaccine induced high frequencies of IgG and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown virus antigen. Taken together, our results suggest that mammalian cells are a viable option for the production of influenza virus vaccines.

Immunogenicity and protective efficacy in mice of influenza B virus vaccines grown in mammalian cells or embryonated chicken eggs.

The immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated chicken eggs (hereafter referred to as eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.

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.

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.

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.

[Changes in biological and physico-chemical properties of avian influenza virus A hemagglutinin H2 during adaptation to a new host]. / Izmeneniia biologicheskikh i fiziko-khimicheskikh svoistv gemaggliutinina H2 ptich'ego virusa grippa A v protsesse adaptatsii k novomu khozainu.

Avian influenza A virus with H2 hemagglutinin has been adapted to mice for the first time. Alterations in the hemagglutinin of adapted variants of the virus as a result of adaptation to a new host are described. Hemagglutinin of a highly virulent adapted variant differed from the parental avirulent strain by antigenic structure, electrophoretic mobility, and receptor activity during interactions with murine red cells.

Replication of influenza A viruses in a green monkey kidney continuous cell line (Vero).

A Vero cell line was investigated as a suitable host system for primary isolation and cultivation of influenza A viruses. The efficiency of primary isolation for currently circulating (H3N2) strains was similar in Vero and MDCK cells. Of 72 egg-adapted strains investigated, 90.3% were detectable hy hemagglutinin (HA) titration in Vero cells after the first passage and 51.4% after the second. The amino acid sequences of the HA1 region of influenza A viruses isolated and passaged in Vero cells were identical to those of their MDCK-grown counterparts. At low MOI, high yields of influenza virus were achieved in Vero cells by multiple additions of trypsin to the medium. After 20 passages of A/England/1/53 (H1N1) in Vero cells, the titer of infectious virus was 8.37 log10 TCID50/mL, and virus protein yields were as high as in MDCK cells.