Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza A virus

The antiviral neuraminidase inhibitor oseltamivir (OSV) is widely used to suppress viral replication in the treatment of influenza. Here, we report that OSV administration significantly suppressed respiratory mucosal secretory IgA responses with respect to antigen (Ag)-specific antibody (Ab) production and also the induction of Ag-specific IgA Ab-forming cells, but not systemic IgG responses, in weanling mice as a model of pediatric influenza. Neutralizing activities of the airway fluids in oral OSV-treated mice were significantly less than those of sham-treated mice. Our findings suggest the risk of re-infection in patients showing a low mucosal response following OSV treatment.     

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.     

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC₅₀ for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.     

Molecular dynamics simulation of oseltamivir resistance in neuraminidase of avian influenza H5N1 virus

The outbreak of avian influenza virus H5N1 has raised a global concern because of its high virulence and mutation rate. Although two classes of antiviral drugs, M2 ion channel protein inhibitors and neuraminidase inhibitors, are expected to be important in controlling the early stages of a potential pandemic. Different strains of influenza viruses have differing degrees of resistance against the antivirals. In order to analyze the detailed information on the viral resistance, molecular dynamics simulations were carried out for the neuraminidase (NA) complex with oseltamivir. The carboxylate of Glu276 of H252Y NA faces toward the O-ethyl-propyl group of oesltamivir, Glu276 of wild-type NA adopts a conformation pointing away from the oesltamivir. τ2 and τ3 torsional angles fluctuation of the oesltamivir are relatively high for the H252Y mutant NA complex. In addition, there are fewer hydrogen bonds between the oesltamivir and H252Y mutation NA. The results show that H252Y mutation NA has high resistance against the drug.     

Analysis of oseltamivir resistance substitutions in influenza virus glycoprotein neuraminidase using a lentivirus-based surrogate assay system

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.     

Mx is dispensable for interferon-mediated resistance of chicken cells against influenza A virus

The type I interferon (IFN) system plays an important role in antiviral defense against influenza A viruses (FLUAV), which are natural chicken pathogens. Studies of mice identified the Mx1 protein as a key effector molecule of the IFN-induced antiviral state against FLUAV. Chicken Mx genes are highly polymorphic, and recent studies suggested that an Asn/Ser polymorphism at amino acid position 631 determines the antiviral activity of the chicken Mx protein. By employing chicken embryo fibroblasts with defined Mx-631 polymorphisms and retroviral vectors for the expression of Mx isoforms in chicken cells and embryonated eggs, we show here that neither the 631Asn nor the 631Ser variant of chicken Mx was able to confer antiviral protection against several lowly and highly pathogenic FLUAV strains. Using a short interfering RNA (siRNA)-mediated knockdown approach, we noted that the antiviral effect of type I IFN in chicken cells was not dependent on Mx, suggesting that some other IFN-induced factors must contribute to the inhibition of FLUAV in chicken cells. Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.     

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity.

The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.     

Gamma interferon is not required for mucosal cytotoxic T-lymphocyte responses or heterosubtypic immunity to influenza A virus infection in mice

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.     

The NB protein of influenza B virus is not necessary for virus replication in vitro

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.     

Homologous recombination is very rare or absent in human influenza A virus

To determine the extent of homologous recombination in human influenza A virus, we assembled a data set of 13,852 sequences representing all eight segments and both major circulating subtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametric test for mosaic structure, we identified 315 sequences (approximately 2%) in five different RNA segments that, after a multiple-comparison correction, had statistically significant mosaic signals compatible with homologous recombination. Of these, only two contained recombinant regions of sufficient length (>100 nucleotides [nt]) that the occurrence of homologous recombination could be verified using phylogenetic methods, with the rest involving very short sequence regions (15 to 30 nt). Although this secondary analysis revealed patterns of phylogenetic incongruence compatible with the action of recombination, neither candidate recombinant was strongly supported. Given our inability to exclude the occurrence of mixed infection and template switching during amplification, laboratory artifacts provide an alternative and likely explanation for the occurrence of phylogenetic incongruence in these two cases. We therefore conclude that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human influenza A virus.     

Source of oseltamivir resistance in avian influenza H5N1 virus with the H274Y mutation

Molecular dynamics simulations were carried out for the mutant oseltamivir-NA complex, to provide detailed information on the oseltamivir-resistance resulting from the H274Y mutation in neuraminidase (NA) of avian influenza H5N1 viruses. In contrast with a previous proposal, the H274Y mutation does not prevent E276 and R224 from forming the hydrophobic pocket for the oseltamivir bulky group. Instead, reduction of the hydrophobicity and size of pocket in the area around an ethyl moiety at this bulky group were found to be the source of the oseltamivir-resistance. These changes were primarily due to the dramatic rotation of the hydrophilic –COO⁻ group of E276 toward the ethyl moiety. In addition, hydrogen-bonding interactions with N1 residues at the -NH₃ ⁺ and -NHAc groups of oseltamivir were replaced by a water molecule. The calculated binding affinity of oseltamivir to NA was significantly reduced from −14.6 kcal mol⁻¹ in the wild-type to −9.9 kcal mol⁻¹ in the mutant-type.     

The development of larval resistance to a nucleopolyhedrovirus is not accompanied by an increased virulence in the virus

Two laboratory experiments were conducted to examine the possible coevolution of cabbage loopers (Trichoplusia ni) and their S nucleopolyhedrovirus (TnSNPV). At the conclusion of Experiments 1 and 2, T. ni had respectively evolved 4.4 × and 22 × resistance to TnSNPV. The higher level of resistance achieved in Experiment 2 could be due to marginally stronger selection, possibly greater genetic variability in larval resistance to TnSNPV, or both. However, the evolution of resistance was not accompanied by an increased virulence of TnSNPV or a change in the restriction profile of the viral DNA when digested with BamHI, EcoRI, HindIII, PstI, SalI, SstI or XhoI. Little genetic variability for virulence in the initial TnSNPV stocks, low mutation rates and possibly weak selection on the virus are some factors that may have constrained the evolution of TnSNPV. We discuss our results in light of the geographic mosaic theory of coevolution and their implications for the use of TnSNPV as a biological control agent against T. ni. This revised version was published online in November 2006 with corrections to the Cover Date.     

Parathyroid hormone-related protein is not required for normal ductal or alveolar development in the post-natal mammary gland

PTHrP is necessary for the formation of the embryonic mammary gland and, in its absence, the embryonic mammary bud fails to form the neonatal duct system. In addition, PTHrP is produced by the breast during lactation and contributes to the regulation of maternal calcium homeostasis during milk production. In this study, we examined the role of PTHrP during post-natal mammary development. Using a PTHrP-lacZ transgenic mouse, we surveyed the expression of PTHrP in the developing post-natal mouse mammary gland. We found that PTHrP expression is restricted to the basal cells of the gland during pubertal development and becomes expressed in milk secreting alveolar cells during pregnancy and lactation. Based on the previous findings that overexpression of PTHrP in cap and myoepithelial cells inhibited ductal elongation during puberty, we predicted that ablation of native PTHrP expression in the post-natal gland would result in accelerated ductal development. To address this hypothesis, we generated two conditional models of PTHrP-deficiency specifically targeted to the postnatal mammary gland. We used the MMTV-Cre transgene to ablate the floxed PTHrP gene in both luminal and myoepithelial cells and a tetracycline-regulated K14-tTA;tetO-Cre transgene to target PTHrP expression in just myoepithelial and cap cells. In both models of PTHrP ablation, we found that mammary development proceeds normally despite the absence of PTHrP. We conclude that PTHrP signaling is not required for normal ductal or alveolar development.     

Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.     

Sulfatide is required for efficient replication of influenza A virus

Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.     

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.     

Upper respiratory tract resistance to influenza infection is not prevented by the absence of either nasal-associated lymphoid tissue or cervical lymph nodes

The murine nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) are involved in the generation of local immune responses within the upper respiratory tract (URT). However, their involvement in these responses does not imply the necessity for resistance to URT infections. We surgically removed NALT or CLN to address the necessity of these lymphatic tissues for the development of a local protective immune response after a URT influenza infection. No histological evidence of the re-establishment of either tissue was detected after surgery and the subsequent infection. Removal of NALT did not elicit changes in serum or nasal mucosa-associated influenza-specific Ig levels. However, increases in PR8-specific serum IgG and nasal mucosa-associated IgA were detected after removal of CLN. Recruitment of influenza-specific CD4 T cells into the nasal mucosa was not altered by removal of NALT. The removal of NALT or CLN did not alter the recruitment of influenza-specific CD8 T cells into the URT. However, increased levels of influenza-specific CD8 T cells were observed in the tracheal-bronchial lymph nodes after CLN surgery. The rate of viral clearance from nasal mucosa and lungs was not altered by removal of NALT or CLN. These studies demonstrate that despite the participation of NALT and CLN in the generation of local immunity to influenza infections, neither tissue is essential for the development of protective immunity and viral clearance in URT.