A molecular mechanism for the low-pH stability of sialidase activity of influenza A virus N2 neuraminidases

Four human pandemic influenza A virus strains isolated in 1957 and 1968, but not most of the epidemic strains isolated after 1968, possess sialidase activity under low-pH conditions. Here, we used cell-expressed neuraminidases (NAs) to determine the region of the N2 NA that is associated with low-pH stability of sialidase activity. We found that consensus amino acid regions responsible for low-pH stability did not exist in pandemic NAs but that two amino acid substitutions in the low-pH-stable A/Hong Kong/1/68 (H3N2) NA and a single substitution in the low-pH-unstable A/Texas/68 (H2N2) NA resulted in significant change in low-pH stability.     

Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-alpha-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.     

Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

Duck and human pandemic influenza A viruses retain sialidase activity under low pH conditions

The majority of influenza A viruses isolated from wild birds, but not humans, can replicate in the duck intestinal tract. Here we demonstrate that all duck isolates tested universally retain sialidase activities under low pH conditions independent of their neuraminidase (NA) subtypes. In contrast, the sialidase activities of most isolates from humans and pigs practically disappear below pH 4.5, with the exception of four human pandemic viruses isolated in 1957 and 1968. Sequence comparisons among duck, human, and swine N2 NA subtypes indicate that amino acids at positions 153, 253, 307, 329, 344, 347, 356, 368, 390, and 431 may be associated with the low pH stability of duck and human pandemic N2 NAs. This finding suggests that the low pH stability of duck influenza A virus NA may be a critical factor for replication in the intestinal tract through the digestive tract of ducks, and that the properties of NAs are important for understanding the epidemiology of the influenza virus.     

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.     

Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus.

The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30% of the world's population and resulted in the death of 40 million people. The complete genomic sequence of the 1918 influenza virus will be deduced using fixed and frozen tissues of 1918 influenza victims. Sequence and phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase (NA) genes show them to be the most avian-like of mammalian sequences and support the hypothesis that the pandemic virus contained surface protein-encoding genes derived from an avian influenza strain and that the 1918 virus is very similar to the common ancestor of human and classical swine H1N1 influenza strains. Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33 or fowl plague viruses. The complete sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced and tested for the hypothesis that the enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. The results from these experiments were inconclusive. Sequence analysis of the 1918 pandemic influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help to elucidate how pandemic influenza strains emerge and what genetic features contribute to their virulence.     

A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence

The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N-infected mice compared with wild-type 1918 virus-infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.     

Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics

The current pandemic influenza A (H1N1) virus has revealed a complicated reassortment of various influenza A viruses. The biological study of these viruses, especially of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), is urgently needed for the control and prevention of H1N1 viruses. We have generated H1N1-2009 and H1N1-1918 pseudotyped particles (pp) with high infectivity. Combinations of HA1918 + NA2009 and HA2009 + NA1918 also formed infectious H1N1pps, among which the HA2009 + NA1918 combination resulted in the most highly infectious pp. Our study demonstrated that some reassortments of H1N1 viruses may hold the potential to produce higher infectivity than do their ancestors.     

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.     

Amino Acids Responsible for the Absolute Sialidase Activity of the Influenza A Virus Neuraminidase: Relationship to Growth in the Duck Intestine

The 1957 human pandemic strain of influenza A virus contained an avian virus hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for the human receptor, N-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an alpha2-6 bond (NeuAcalpha2-6Gal). Although the NA retained considerable specificity for NeuAcalpha2-3Gal, its original substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift in substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient viral growth in the intestines of orally inoculated ducks. The growth-supporting capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, we localized the determinants of high specific NA activity to a region containing six amino acid substitutions in A/England/12/62: Ser331-->Arg, Asp339-->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and Pro431-->Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs for their cellular receptor.     

Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host.     

Structural characterization of the 1918 influenza virus H1N1 neuraminidase

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 "Spanish flu" (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-A and 1.45-A resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.     

Elicitation of anti-1918 influenza virus immunity early in life prevents morbidity and lower levels of lung infection by 2009 pandemic H1N1 influenza virus in aged mice

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.     

Plasminogen-binding activity of neuraminidase determines the pathogenicity of influenza A virus

When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.     

Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding

Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.     

Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein

The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.     

Eurasian-origin gene segments contribute to the transmissibility, aerosol release, and morphology of the 2009 pandemic H1N1 influenza virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus.     

Decreased neuraminidase activity is important for the adaptation of H5N1 influenza virus to human airway epithelium

Highly pathogenic avian H5N1 influenza viruses remain a pandemic threat. Antiviral drugs such as neuraminidase (NA) inhibitors will be crucial for disease control in the event of a pandemic. Should drug-resistant H5N1 viruses develop, all defense strategies will be compromised. To determine the likelihood and mechanisms of emergence of NA inhibitor-resistant H5N1 variants in humans, we serially passaged two H5N1 viruses, A/Hong Kong/213/03 and A/Turkey/65-1242/06, in normal human bronchial epithelial (NHBE) cells in the presence of oseltamivir, zanamivir, or peramivir. To monitor the emergence of changes associated with the adaptation of H5N1 viruses to humans, we passaged the strains in the absence of drugs. Under pressure of each NA inhibitor, A/Turkey/65-1242/06 developed mutations in the hemagglutinin (HA) (H28R and P194L/T215I) and NA (E119A) proteins that reduced virus binding to α2,3-sialyl receptor and NA activity. Oseltamivir pressure selected a variant of A/Hong Kong/213/03 virus with HA P194S mutation that decreased viral binding to α2,6 receptor. Under peramivir pressure, A/Hong Kong/213/03 virus developed a novel NA mutation, R156K, that reduced binding to all three drugs, caused about 90% loss of NA activity, and compromised replication in NHBE cells. Both strains were eliminated in NHBE cells when they were cultivated in the absence of drugs. Here, we show for the first time that decreased NA activity mediated through NA inhibitors is essential for the adaptation of pandemic H5N1 influenza virus to humans. This ability of decreased NA activity to promote H5N1 infection underlines the necessity to optimize management strategies for a plausible H5N1 pandemic.     

Effect of 1918 PB1-F2 expression on influenza A virus infection kinetics

Relatively little is known about the viral factors contributing to the lethality of the 1918 pandemic, although its unparalleled virulence was likely due in part to the newly discovered PB1-F2 protein. This protein, while unnecessary for replication, increases apoptosis in monocytes, alters viral polymerase activity in vitro, enhances inflammation and increases secondary pneumonia in vivo. However, the effects the PB1-F2 protein have in vivo remain unclear. To address the mechanisms involved, we intranasally infected groups of mice with either influenza A virus PR8 or a genetically engineered virus that expresses the 1918 PB1-F2 protein on a PR8 background, PR8-PB1-F2(1918). Mice inoculated with PR8 had viral concentrations peaking at 72 hours, while those infected with PR8-PB1-F2(1918) reached peak concentrations earlier, 48 hours. Mice given PR8-PB1-F2(1918) also showed a faster decline in viral loads. We fit a mathematical model to these data to estimate parameter values. The model supports a higher viral production rate per cell and a higher infected cell death rate with the PR8-PB1-F2(1918) virus. We discuss the implications these mechanisms have during an infection with a virus expressing a virulent PB1-F2 on the possibility of a pandemic and on the importance of antiviral treatments.     

In silico genome analysis and drug efficacy test of influenza A virus (H1N1) 2009

The H1N1 2009 virus is pandemic in many countries. The genome of this virus contains eight segments. Among the eight segments maximum numbers of mutation occur at the segment 1 and segment 4 which codes for PB2 subunit and hemagglutinin (HA) and less number of mutations occur in segment 6 which codes for neuraminidase (NA) protein. Neuraminidase (NA) inhibitors (Oseltamivir and Zanamivir) are presently used as an anti-flu drugs. In the present study, the in silico efficacy of different drugs was tested against the swine flu virus. 3D structures of neuraminidase (NA) proteins of H1N1 2009 were generated using Geno3D. The 3D structure of H1N1 1918 was downloaded from PDB. Interaction study was done using Arguslab 4 and PyMol view. Oseltamivir and Zanamivir have good number of interactions with H1N1 2009 virus and the scoring function also support to this result. When compared with the 1918 H1N1 viral protein, 2009 H1N1 NA protein shows more number of interaction and good scoring function. The RMSD value of before and after docking are found to be same at 0.04A° for both the drugs. The force field energy of NA protein 2009 was found to be −15603.529 KJ/mol before docking. The force field energy was found to be decreased after docking at −17620.740 KJ/mol with Tamiflu and −17652.242 KJ/mol with Zanamivir. The number of interaction and scoring function shows that Oseltamivir and Zanamivir will be able to effectively control the present pandemic H1N1 virus 2009.