H5N1 influenza--continuing evolution and spread.
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Animals↗
Biomedical subjects
Elena A Govorkova
Publications and source records attributed to Elena A Govorkova.
Immunization with reverse-genetics-produced H5N1 influenza vaccine protects ferrets against homologous and heterologous challenge.
BACKGROUND: Multiple cases of transmission of avian H5N1 influenza viruses to humans illustrate the urgent need for an efficacious, cross-protective vaccine. METHODS: Ferrets were immunized with inactivated whole-virus vaccine produced by reverse genetics with the hemagglutinin (HA) and neuraminidase genes of A/HK/213/03 virus. Ferrets received a single dose of vaccine (7 or 15 microg of HA) with aluminum hydroxide adjuvant or 2 doses (7 microg of HA each) without adjuvant and were challenged with 10(6) 50% egg infectious doses of A/HK/213/03, A/HK/156/97, or A/Vietnam/1203/04 virus. RESULTS: One or 2 doses of vaccine induced a protective antibody response to the vaccine strain. All immunization regimens completely protected ferrets from challenge with homologous wild-type A/HK/213/03 virus: no clinical signs of infection were observed, virus replication was significantly reduced (P<.05) and was restricted to the upper respiratory tract, and spread of virus to the brain was prevented. Importantly, all vaccinated ferrets were protected against lethal challenge with the highly pathogenic strain A/Vietnam/1203/04. The 2-dose schedule induced higher levels of antibodies that were cross-reactive to antigenically distinct H5N1 viruses. CONCLUSIONS: H5N1 vaccines may stimulate an immune response that is more cross-protective than what might be predicted by in vitro assays and, thus, hold potential for being stockpiled as "initial" pandemic vaccines.
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The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04.
H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like alpha-(2,3)-linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.
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Combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza A variants.
Rapid development of resistant influenza variants after amantadine treatment is one of the main drawbacks of M2 blockers. On the other hand, the emergence of variants with low susceptibility to the neuraminidase (NA) inhibitors is limited. In the present study we examined whether combination therapy with two classes of anti-influenza drugs can affect the emergence of resistant variants in vitro. We observed that virus yields of human A/Nanchang/1/99 (H1N1), A/Panama/2007/99 (H3N2), and A/Hong Kong/156/97 (H5N1) viruses in MDCK cells were significantly reduced (P<0.005) when the cells were treated with the combination of amantadine and low doses of oseltamivir carboxylate (< or =1microM). After five sequential passages in MDCK cells, the M2 protein of viruses cultivated with amantadine alone mutated at positions V27A and S31N/I. Viruses cultivated with oseltamivir carboxylate (> or =0.001microM) possessed mutations in the hemagglutinin (HA) protein. These variants showed reduced efficiency of binding to sialic acid receptors and decreased sensitivity to NA inhibitor in plaque reduction assay. Importantly, no mutations in the HA, NA, and M2 proteins were detected when the drugs were used in combination. Our results suggest that combination chemotherapy with M2 blocker and NA inhibitor reduced the emergence of drug-resistant influenza variants in vitro. This strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored.
Acetamides↗
Importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of influenza viruses.
Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.
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Detection of amantadine-resistant variants among avian influenza viruses isolated in North America and Asia.
Here, we report the diversity of amantadine-resistant mutants among avian influenza A viruses with pandemic potential (H5, H6, H7, and H9 hemagglutinin subtypes). Drug-resistant variants were not detected among 1979--83 isolates, whereas 31.1% of H5 and 10.6% of H9 strains from Southeast Asia isolated in 2000-04 carried mutations in M2 protein. In North America, resistant variants occurred among H7 viruses only (16.4% of those tested). H6 viruses were amantadine-sensitive. These findings prompt concern regarding the control of pandemic influenza, the possibility that the next pandemic virus will be amantadine-resistant and the need to monitor the use of the drug in poultry.
Amantadine↗
Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines.
If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.
Amino Acid Sequence↗
Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice.
BACKGROUND: Control of highly pathogenic avian H5N1 influenza viruses is a major public-health concern. Antiviral drugs could be the only option early in the pandemic.METHODS. BALB/c mice were given oseltamivir (0.1, 1, or 10 mg/kg/day) twice daily by oral gavage; the first dose was given 4 h before inoculation with H5N1 A/Vietnam/1203/04 (VN1203/04) virus. Five- and 8-day regimens were evaluated.RESULTS. Oseltamivir produced a dose-dependent antiviral effect against VN1203/04 in vivo (P<.01). The 5-day regimen at 10 mg/kg/day protected 50% of mice; deaths in this treatment group were delayed and indicated the replication of residual virus after the completion of treatment. Eight-day regimens improved oseltamivir efficacy, and dosages of 1 and 10 mg/kg/day significantly reduced virus titers in organs and provided 60% and 80% survival rates, respectively (P<.05). Overall, the efficacy of the 5- and 8-day regimens differed significantly (death hazard ratio, 2.658; P<.01). The new H5N1 antigenic variant VN1203/04 was more pathogenic in mice than was A/HK/156/97 virus, and a prolonged and higher-dose oseltamivir regimen may be required for the most beneficial antiviral effect.CONCLUSIONS. Oseltamivir prophylaxis is efficacious against lethal challenge with VN1203/04 virus in mice. Viral virulence may affect the antiviral treatment schedule.
Acetamides↗
Efficacy of H5 influenza vaccines produced by reverse genetics in a lethal mouse model.
We studied the efficacy, in mice, of 2 H5 influenza vaccine viruses produced by reverse genetics. Mice were immunized with inactivated viruses and then inoculated with a human H5N1 1997 or 2003 virus or an avian H5N1 2001 virus. Vaccine viruses that we tested raised high levels of hemagglutination-inhibiting (1:160-1:1280) and virus-neutralizing (1:900-1:1900) antibodies on day 21 after a single dose of vaccine and decreased or prevented virus replication in mouse lungs; 54.5%-100% of immunized mice survived, whereas all control mice died. Protection was achieved despite antigenic differences and incomplete matching of the vaccine strain and the challenge virus. Therefore, high levels of cross-protection are predicted in the mouse model.
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Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility.
Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.
Acetamides↗
Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004.
The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.
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Influenza H5 virus escape mutants: immune protection and antibody production in mice.
Avian H5N1 influenza A viruses are considered to be of high pandemic potential as they are able to cross the avian-human species barrier and cause disease in humans. In the present study we assessed the impact of amino acid substitutions in the hemagglutinin (HA) of antigenic escape mutants of influenza A/Mallard/Pennsylvania/10218/84 (H5N2) (Mld/PA/84-MA) virus on the level of neutralizing antibodies and the ability to protect mice against challenge with the wild type H5 influenza virus. beta-Propiolactone-inactivated vaccines prepared from eight different H5 escape mutants could be separated into two groups based on levels of protection. One group of escape mutants [m46(7), m46(7)-24B9, m46(7)-55, and m46(7)-55-24B9] was characterized by providing high levels of protection (90.0-95.4% survival) to mice against subsequent challenge with 5 LD(50) of wild type Mld/PA/84-MA virus. The other group of escape mutants [m176/26, m55(2), m55(2)-24B9, and m24B9-176/26] provided moderate level of protection (57.1-66.6% survival) in mice. Analysis of the amino acid substitutions in the HA revealed that two amino acid changes in antigenic site B of the HA molecule (D(126)-->N and K(152)-->N) were associated for decreases in the levels of antibody and the immune protection afforded by vaccination with these H5 virus escape mutants. The phenotypic effects of mutations in HA gene of H5 virus may be of importance to appraise the extent and direction of H5 influenza viruses antigenic evolution.
Amino Acid Substitution↗
Neuraminidase inhibitor-rimantadine combinations exert additive and synergistic anti-influenza virus effects in MDCK cells.
There is insufficient information about combination therapy with approved anti-influenza agents. We tested combinations that paired a neuraminidase (NA) inhibitor (zanamivir, oseltamivir carboxylate, or peramivir) with rimantadine against infection of MDCK cells with H1N1 and H3N2 subtypes of influenza A virus and characterized their mode of interaction. When reduction of extracellular virus was analyzed by individual regression models and three-dimensional representations of the data, all three combinations showed additive and synergistic effects with no cytotoxicity. Maximum synergy against A/New Caledonia/20/99 (H1N1) virus infection was observed with <2.5 microM rimantadine paired with low concentrations of NA inhibitors. All combinations reduced the extracellular yield of A/Panama/2007/99 (H3N2) influenza virus synergistically. However, our findings were different for the cell-associated virus yield. At some drug concentrations, the yield of cell-associated virus was inhibited antagonistically. Therefore, the method of analysis can be a crucial factor in evaluating the interactions of drugs with different mechanisms. We hypothesize that assays based on cell-associated virus yield may underestimate the efficacies of drug combinations that include an NA inhibitor. Taken together, our results suggest that regimens that combine NA inhibitors and rimantadine exert synergistic anti-influenza effects in vitro. These findings provide baseline information for therapeutic testing of the drug combinations in vivo.
Acetamides↗
Influenza: emergence and control.
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Generation of high-yielding influenza A viruses in African green monkey kidney (Vero) cells by reverse genetics.
Influenza A viruses are the cause of annual epidemics of human disease with occasional outbreaks of pandemic proportions. The zoonotic nature of the disease and the vast viral reservoirs in the aquatic birds of the world mean that influenza will not easily be eradicated and that vaccines will continue to be needed. Recent technological advances in reverse genetics methods and limitations of the conventional production of vaccines by using eggs have led to a push to develop cell-based strategies to produce influenza vaccine. Although cell-based systems are being developed, barriers remain that need to be overcome if the potential of these systems is to be fully realized. These barriers include, but are not limited to, potentially poor reproducibility of viral rescue with reverse genetics systems and poor growth kinetics and yields. In this study we present a modified A/Puerto Rico/8/34 (PR8) influenza virus master strain that has improved viral rescue and growth properties in the African green monkey kidney cell line, Vero. The improved properties were mediated by the substitution of the PR8 NS gene for that of a Vero-adapted reassortant virus. The Vero growth kinetics of viruses with H1N1, H3N2, H6N1, and H9N2 hemagglutinin and neuraminidase combinations rescued on the new master strain were significantly enhanced in comparison to those of viruses with the same combinations rescued on the standard PR8 master strain. These improvements pave the way for the reproducible generation of high-yielding human and animal influenza vaccines by reverse genetics methods. Such a means of production has particular relevance to epidemic and pandemic use.
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Structure of antigenic sites on the haemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants.
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.
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Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China.
The H5N1 influenza virus, which killed humans and poultry in 1997, was a reassortant that possibly arose in one type of domestic poultry present in the live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are still circulating in poultry in southern China, the reassortment event that generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1; Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1 virus, we investigated the continued circulation, host range, and transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused weight loss and death in some mice inoculated with high virus doses. Transmission of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in infection of all birds but limited signs of overt disease. In contrast, oral inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to 50% of infected geese. Transmission from infected geese to chickens occurred only by fecal contact, whereas transmission to quail occurred by either aerosol or fecal spread. This difference is probably explained by the higher susceptibility of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N1/97 virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds, especially quail, supports the wisdom of separating aquatic and land-based poultry in the markets in Hong Kong and the need for continued surveillance in the field and live-bird markets in which different types of poultry are in contact with one another.
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