Liposomal lidocaine to improve procedural success rates and reduce procedural pain among children: a randomized controlled trial

BackgroundHistorically, children have been undertreated for their pain, and they continue to undergo painful cutaneous procedures without analgesics. A new topical anesthetic, liposomal lidocaine 4% cream (Maxilene, RGR Pharma, Windsor, Ont.), has become available. It has pharmacologic properties that are superior to other topical anesthetics, including an onset of action of only 30 minutes. We sought to determine the success rate of cannulation, analgesic effectiveness, procedure duration and rate of adverse skin reactions when liposomal lidocaine is used before intravenous cannulation of children.MethodsIn this double-blind randomized controlled trial, children aged 1 month to 17 years received liposomal lidocaine or placebo before cannulation. Success on first cannulation attempt was recorded, and, among children 5 years and older, pain was evaluated before and after the attempt by the child, parents and research assistant using a validated measure (Faces Pain Scale-Revised). For children younger than 5 years, pain was evaluated by the parents and research assistant only. The total duration of the procedure and adverse skin reactions were also recorded.ResultsBaseline characteristics did not differ (p > 0.05) between children who received liposomal lidocaine (n = 69) and those who received placebo (n = 73). Cannulation on the first attempt was achieved in 74% of children who received liposomal lidocaine compared with 55% of those who received placebo (p = 0.03). Among children 5 years of age and older (n = 67), lower mean pain scores during cannulation were reported by those receiving liposomal lidocaine (p = 0.01). Similarly, lower mean pain scores during cannulation were reported by the parents and research assistant for all children who received liposomal lidocaine than for all those who received placebo (p < 0.001). The mean total procedure duration was shorter with liposomal lidocaine (6.7 v. 8.5 minutes; p = 0.04). The incidence of transient dermal changes was 23% in both groups (p = 1.0).ConclusionsUse of liposomal lidocaine was associated with a higher intravenous cannulation success rate, less pain, shorter total procedure time and minor dermal changes among children undergoing cannulation. Its routine use for painful cutaneous procedures should be considered whenever feasible.Painful medical procedures are routinely performed on children for diagnostic and therapeutic reasons. The provision of analgesia for these procedures, however, remains uncommon.¹ Untreated pain has both short-term and long-term consequences. In the short term, there is pain during the actual procedure. This contributes to a lack of cooperation by the child, unsuccessful procedure attempts, repeated attempts, additional pain and a prolonged total procedure time. In the long term, repeated painful procedures can lead to conditioned anxiety responses and increased pain perception.^2,3 Inadequate analgesia during an initial procedure may diminish analgesic effectiveness at subsequent procedures.⁴ Moreover, there is a relation between painful procedures in childhood and blood-injection-injury phobia,⁵ a condition that affects up to 10% of adults and may cause people to avoid medical care.⁶ In light of the cumulative evidence of the negative consequences of untreated pain in childhood, interventions are needed to diminish pain among children undergoing medical procedures and to facilitate successful completion of procedures.Intravenous cannulation is a common, painful medical procedure. Although local anesthetics reduce the pain of cannulation,^7,8,9,10,11 most preparations are not feasible for routine use. The “gold standard” for skin anesthesia, lidocaine–prilocaine 5% cream, requires a 60-minute application time. In addition, it causes vasoconstriction,¹² which potentially obscures landmarks and makes cannulation more difficult.¹³ Another commercially available preparation, amethocaine 4% gel, requires a 30-minute application time. However, it frequently causes vasodilatation and may induce hypersensitivity with repeated use.¹⁴ An alternative option, subcutaneous injection of lidocaine, requires only a few minutes to administer, but it is associated with an extra and painful puncture and is therefore not routinely used.¹⁵Liposomal lidocaine 4% cream¹⁶ (Maxilene, RGR Pharma, Windsor, Ont.) was launched in Canada in 2003. The liposome-encapsulated formulation protects the anesthetic from being metabolized too quickly.¹⁷ Liposomal lidocaine has the advantages of “needle-free” administration, a short onset of action and minimal vasoactive properties that minimize any potential interference with cannulation success. It is not associated with methemoglobinemia, a systemic side effect of lidocaine–prilocaine.¹⁸Among children, liposomal lidocaine is as effective as lidocaine–prilocaine for decreasing pain from venipuncture¹⁹ and intravenous cannulation,^20,21 and as effective as buffered lidocaine injection for decreasing intravenous cannulation pain.¹⁵ Previous studies have not compared liposomal lidocaine with placebo. We conducted such a comparison to determine whether liposomal lidocaine improves cannulation success rates. We also sought to determine whether it reduces pain and procedure duration and is associated with a low frequency of dermal reactions.     

Dynamics of Influenza Virus Infection and Pathology

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon (“cytokine storms”), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.Influenza A virus causes an acute respiratory disease in humans and other mammals; in humans, it is particularly important because of the rapidity with which epidemics develop, its widespread morbidity, and the seriousness of complications. Every year, an estimated 500,000 deaths worldwide, primarily of young children and the elderly, are attributed to seasonal influenza virus infections (49). Influenza pandemics may occur when an influenza virus with new surface proteins emerges, against which the majority of the population has no preexisting immunity. Both the emergence of H5N1 virus (34) and the current H1N1 virus pandemic (43) underline the importance of understanding the dynamics of infection and disease. A key question is, what regulates virus abundance in an individual host, causing the characteristic rapid decline in virus shedding following its initial peak? The main contenders in primary influenza virus infection are depletion of susceptible target cells and the impact of the host''s innate immune response (2, 20).On infection, the influenza virus elicits an immune response, including a rapid innate response that is correlated with the observed decline in the virus load after the first 2 days of infection (1). The slower adaptive response, including both humoral and cell-mediated components, takes several days to consolidate but is important for complete virus clearance and establishment of protective immunity. During infection of an immunologically naïve host, the innate immune response is particularly important as the first line of defense against infection. The innate immune response is regulated by chemokines and cytokines, chemical messengers produced by virus-infected epithelial cells and leukocytes (23), and natural interferon-producing cells, such as plasmacytoid dendritic cells (13). Among the key cytokines induced by epithelial cells infected with influenza A virus are type I interferons (IFNs) (IFN-α/β) (23), which directly contribute to the antiviral effect on infected and neighboring cells (38).Like other viruses, influenza A viruses have evolved strategies to limit the induction of innate immune responses (38). The NS1 protein plays a dominant role, and without it, the virus is unable to grow well or to cause pathology in an immunocompetent host (14). NS1 is multifunctional and counteracts both the induction of IFN expression and the function of IFN-activated antiviral effectors via multiple mechanisms (12, 17). Individual strains of influenza A virus possess these activities to various degrees (15, 21, 22, 26), and accordingly, NS1 has been implicated as a virulence factor (3, 17). A striking effect of the failure to control the innate response to virus infection is seen as a “cytokine storm,” which causes severe pathology (8).While there is an extensive literature on modeling influenza virus spread at the population level, the individual-host scale has received much less attention (2, 4, 5, 18, 19, 20, 27, 28). In a recent important paper, Baccam et al. modeled the kinetics of influenza A virus (2). The innate dynamics were included in the form of an IFN response that delayed and reduced virus production but did not prevent it; thus, the infection was resolved primarily through near-total depletion of epithelial cells. Their model was fitted to virus titers from human volunteers exposed to H1N1 influenza virus, but no data were available on the innate immune response or epithelial cell pathology. This has been a general difficulty in developing and validating more refined within-host models; there is a lack of detailed biological data from natural host systems, in particular, measures of immune kinetics and patterns of cellular depletion.The model presented here explicitly includes the ability of IFN to induce a fully antiviral state in order to explore the relative regulatory role of innate immunity and target cell depletion. Data from experimental infections of immunologically naïve horses with an equine influenza virus (36) allowed us to calibrate our model, not only to viral kinetics, but also to IFN dynamics and cell depletion in the context of infection of a naïve natural mammalian host. With our fitted model, we then investigate modulation of the immune response.     

Interleukin-15 Is Critical in the Pathogenesis of Influenza A Virus-Induced Acute Lung Injury

Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44⁺ CD8⁺ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8⁺ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β₂-microglobulin by gene targeting and those depleted of CD8⁺ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.Highly pathogenic influenza A viruses cause acute severe pneumonia that results in high morbidity and significant mortality (11, 12, 24, 26). Elevated levels of serum cytokines and chemokines accompany these clinical manifestations, and the possibility that this “cytokine storm” contributes to increased severity of the disease caused by avian H5N1 virus and by other strains of influenza A virus has been proposed (10, 21, 33). In fact, CCR2-deficient mice [CCR2 is chemokine (C-C motif) receptor 2] were protected from early pathological manifestations despite higher pulmonary titers of the influenza virus A/PR/8/34 (H1N1) strain (7). Tumor necrosis factor receptor 1 (TNFR-1)-deficient mice exhibited significantly reduced morbidity following challenge with H5N1 virus (31). Other cytokines or chemokines have also been investigated (8, 28, 34, 35, 38). Thus, at least some of the elevated proinflammatory cytokines may contribute to the pathogenesis of influenza A virus.Interleukin-15 (IL-15) is a pleiotropic cytokine involved in both innate and adaptive immune responses (20, 36). IL-15 utilizes the β-chain of the IL-2 receptor (IL-2R) (CD122) and the common cytokine receptor γ-chain (CD132) for signal transduction in lymphocytes and therefore shares many biological properties with IL-2 (3). Memory CD8⁺ T cells, natural killer (NK) cells, NKT cells, and intraepithelial lymphocyte (IEL) T cells (15, 23, 42) decrease in mice with defective IL-15 signaling, indicating the importance of IL-15 in their development and/or maintenance. IL-15 regulates not only the number of memory CD8⁺ T cells but also activation of their functions, including gamma interferon (IFN-γ) production and cytotoxic activity (40), which are important to target the virus (9). Therefore, it is possible that we may be able to use IL-15 as an immune-enhancing molecular adjuvant in vaccines for protection against various pathogens, including influenza A virus (37).In the present study, we demonstrate that IL-15 knockout (KO) mice exhibited high resistance against infection with mouse-adapted influenza virus A/FM/1/47 (H1N1) strain. We show for the first time that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus. In addition, our observations are important in the light of recent research into the use of IL-15 as an adjuvant for vaccination.     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

Impact of Temperature on Ladderane Lipid Distribution in Anammox Bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria have the unique ability to synthesize fatty acids containing linearly concatenated cyclobutane rings, termed “ladderane lipids.” In this study we investigated the effect of temperature on the ladderane lipid composition and distribution in anammox enrichment cultures, marine particulate organic matter, and surface sediments. Under controlled laboratory conditions we observed an increase in the amount of C₂₀ [5]-ladderane fatty acids compared with the amount of C₁₈ [5]-ladderane fatty acids with increasing temperature and also an increase in the amount of C₁₈ [5]-ladderane fatty acids compared with the amount of C₂₀ [5]-ladderane fatty acids with decreasing temperature. Combining these data with results from the natural environment showed a significant (R² = 0.85, P = <0.0001, n = 121) positive sigmoidal relationship between the amounts of C₁₈ and C₂₀ [5]-ladderane fatty acids and the in situ temperature; i.e., there is an increase in the relative abundance of C₁₈ [5]-ladderane fatty acids at lower temperatures and vice versa, particularly at temperatures between 12°C and 20°C. Novel shorter (C₁₆) and longer (C₂₂ to C₂₄) ladderane fatty acids were also identified, but their relative amounts were small and did not change with temperature. The adaptation of ladderane fatty acid chain length to temperature changes is similar to the regulation of common fatty acid composition in other bacteria and may be the result of maintaining constant membrane fluidity under different temperature regimens (homeoviscous adaptation). Our results can potentially be used to discriminate between the origins of ladderane lipids in marine sediments, i.e., to determine if ladderanes are produced in situ in relatively cold surface sediments or if they are fossil remnants originating from the warmer upper water column.Anaerobic ammonium-oxidizing (anammox) bacteria possess the unique ability to oxidize NH₄⁺ with NO₂⁻ to N₂ under anoxic conditions (42). Since the discovery of the anammox process in a wastewater treatment plant in the Netherlands (21), studies have indicated that anammox bacteria are omnipresent in low-oxygen environments around the world. Anammox therefore forms an important link in both the oceanic (4, 7, 17, 18, 31) and freshwater (14, 33) nitrogen cycles. Unlike other Planctomycetes, anammox bacteria contain a unique “organelle” called the anammoxosome (19, 37, 44-46). The membrane of this compartment contains unusual “ladderane” lipids (37). The core ladderane lipids consist of C₁₈ and C₂₀ fatty acids containing either 3 or 5 linearly concatenated cyclobutane rings, which are ester bound to a glycerol backbone or ether bound as alkyl chains (35). In addition, the intact polar lipids containing the core lipid structures may have different types of polar head groups, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylglycerol (PG) (1, 22). In silico density simulation modeling experiments with a ladderane lipid-containing membrane (glycerol-bound mixed ether-ester containing both ladderane moieties) have indicated that ladderane lipids could provide a denser cell membrane than conventional membrane lipids (37). Since the anammoxosome appears to be impenetrable to fluorophores, the ladderane membrane could function in cell energy conservation (37, 44).Experimental evidence has shown that anammox bacteria isolated from wastewater treatment reactors grow over a wide range of temperatures (20 to 43°C) and have an optimum temperature of about 35°C (39). In the natural environment the anammox process has been reported to occur at temperatures as low as −2.5°C in sea ice (5, 26) and as high as 70°C in hot springs and hydrothermal vent areas (3, 12). Furthermore, “Candidatus Scalindua spp.” has been successfully enriched from marine sediment (Gullmarsfjord, Sweden) in sequencing batch reactors at temperatures of 15 and 20°C (43). In other bacteria containing common fatty acids temperature adaptation can be achieved by (among other things) modifying the composition of the membrane bilayers to deal with alterations in membrane viscosity due to changes in temperature. This process has been well documented and is termed “homeoviscous adaptation”; i.e., the fatty acid composition is changed to maintain membrane fluidity (23, 27, 34, 40). Currently, it is not known how anammox bacteria, with their highly unusual ladderane lipids, react to temperature. To investigate this, we analyzed the ladderane lipid composition of anammox bacteria grown at different temperatures in sequencing batch reactors and in samples from different natural environments covering a wide range of temperatures.     

Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge,Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.Enhanced biological phosphorus removal (EBPR) has been applied in many wastewater treatment plants to reduce the phosphorus that causes eutrophication in surface waters. EBPR employs polyphosphate-accumulating organisms (PAOs), which are enriched through alternating aerobic-anaerobic cycles (34). Since PAOs are essential for an understanding of EBPR, many candidates have been proposed as potential PAOs, such as Acinetobacter spp. (11), Tetrasphaera spp. (31), Microlunatus phosphovorus (36), Lampropedia spp. (40), and Gram-positive Actinobacteria (24). However, those organisms do not exhibit all of the characteristics of the EBPR biochemistry model. Recently developed culture-independent approaches such as PCR-clone libraries, fluorescence in situ hybridization (FISH), and microautoradiography (MAR) have highlighted an uncultured Rhodocyclus-related bacterium, “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), as one of the most important PAO candidates (2, 5, 16, 22, 23, 27, 28, 47).Numerous studies have sought to investigate uncultured “Ca. Accumulibacter” and have shown the presence of genetically and physiologically different members with a global geographic distribution (3, 9, 22, 27, 39). For example, Kong et al. (22) identified two morphologically different “Ca. Accumulibacter” cells of small cocci and large coccobacilli labeled with PAOmix (PAO462, PAO651, and PAO846) in laboratory-scale EBPR reactors. Additional results showing phenotypic and morphological diversities of “Ca. Accumulibacter” cells also existed with respect to the different roles of denitrifying PAO (DPAO) in the EBPR process (3, 9, 23). Carvalho et al. (3) detected two different morphotypes of “Ca. Accumulibacter” with different nitrate reduction capabilities. The presence of other “Ca. Accumulibacter” strains with 15% genome sequence divergence from the dominant strains in metagenomic analyses is likely to explain these morphological and phenotypic differences (12). McMahon et al. (33) suggested the use of the polyphosphate kinase (ppk) gene, which is involved in the production of polyphosphate, for a finer elucidation of “Ca. Accumulibacter” diversity. He et al. (15) grouped “Ca. Accumulibacter” strains into five distinct clades, designated clades I, IIA, IIB, IIC, and IID, using ppk gene sequence information. Flowers and colleagues (9) previously reported that “Ca. Accumulibacter” cells of clade IA had nitrate reduction activity with phosphorus uptake but that “Ca. Accumulibacter” cells of clade IIA did not.FISH-fluorescence activated cell sorting (FACS) techniques have been used for the separation of specific microbial cells from complex microbial consortia and their metabolic gene analysis (14, 46). For example, Miyauchi et al. (35) sorted PAOmix probe-labeled “Ca. Accumulibacter” cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity. In the current study, we found that four different “Ca. Accumulibacter” clades (Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4) were present in the EBPR sludge of laboratory-scale reactors supplied with acetate as the sole carbon source. We analyzed their morphological characteristics and ppk gene sequence information using a suite of FISH and FACS approaches and linked fine-scale phylogenetic diversities of “Ca. Accumulibacter” strains with their morphological characteristics and metabolic genes. This study will be useful for further genetic and physiological studies of different “Ca. Accumulibacter” clades.     

The Ultramicrobacterium “Elusimicrobium minutum” gen. nov., sp. nov., the First Cultivated Representative of the Termite Group 1 Phylum

Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191^T is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 μm long and 0.17 to 0.3 μm wide). The isolate grows heterotrophically on sugars and ferments d-galactose, d-glucose, d-fructose, d-glucosamine, and N-acetyl-d-glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191^T belongs to the “intestinal cluster,” a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called “endomicrobia” lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name “Elusimicrobium minutum” gen. nov., sp. nov. (type strain, Pei191^T = ATCC BAA-1559^T = JCM 14958^T) for the first isolate of this deep-branching lineage and the name “Elusimicrobia” phyl. nov. for the former TG1 phylum.Insect intestinal tracts harbor an enormous diversity of as-yet-uncultivated bacteria that are characterized only by their 16S rRNA gene sequences and whose biology is typically completely obscure (9, 17, 49). As in other environments (45), many of these sequences form deep-branching phylogenetic lineages that do not contain a single isolate (18, 28). One of these lineages is the termite group 1 (TG1), which was originally discovered by Ohkuma and Kudo (37) and recognized as a phylum-level group (candidate division) by Hugenholtz et al. (20). TG1 bacteria form a major proportion of the microbial community in the hindgut of lower termites (17, 69), where they inhabit the cytoplasm of the intestinal flagellates (38, 53). These so-called “endomicrobia” are specific for the respective flagellate species (21) and, at least in the case of “Candidatus Endomicrobium trichonymphae,” are cospeciating with their flagellate host (22).However, the TG1 phylum also comprises several other deep-rooting lineages (>15% 16S rRNA gene sequence divergence). They are present in a variety of environments, including soils, sediments, and intestinal tracts (14). One of these lineages, the “intestinal cluster,” comprises sequences originating exclusively from intestinal habitats, including the termite gut, but is only distantly related to the lineage comprising the “endomicrobia” (14). Here, we report the isolation of a member of the intestinal cluster from the hindgut of a humivorous scarab beetle larva and its physiological and ultrastructural characterization. We propose a new species, “Elusimicrobium minutum” gen. nov. sp. nov., and define the phylogenetic framework for the first cultivated representative of the TG1 phylum.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.     

Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.Two classes of antiviral drugs are currently available for the treatment and prophylaxis of influenza, the adamantanes and the neuraminidase (NA) inhibitors (NAIs). The adamantanes were the first agents to be recognized to have anti-influenza virus activities as early as 1964 (2) although the rapid emergence of drug-resistant influenza virus strains has limited their clinical effectiveness (12). The NAIs, zanamivir (Relenza) and oseltamivir (Tamiflu), were the first drugs to be specifically designed as anti-influenza virus agents and have been available on the market in many countries since 1999. During oseltamivir clinical trials, 1 to 4% of treated adults (6) and 5 to 6% of treated children were found to shed resistant viruses (30) although more recent studies have reported resistance in 16 to 18% of viruses from oseltamivir-treated children (20, 29). In contrast to the frequency of resistance seen following oseltamivir treatment, only one occurrence of significant zanamivir resistance has been observed following zanamivir treatment. The zanamivir-resistant strain, an influenza B virus with an R152K NA mutation, was isolated from an immunocompromised patient undergoing prolonged zanamivir treatment (7).In addition to the analysis of influenza viruses isolated from patients undergoing either oseltamivir or zanamivir treatment, surveillance studies that analyze the NAI susceptibility of circulating viruses, predominantly from patients not undergoing NAI treatment, have also been conducted. Studies that have tested viruses isolated prior to the release of the NAIs (1996 to 1999) (23) and after the initiation of clinical use of these drugs (2000 to 2006) (16, 24) have found either no resistance or a very low frequency of resistance. In contrast, analysis of circulating seasonal influenza viruses from Europe during the 2007 to 2008 season revealed that 14% (59/437) of influenza A (H1N1) viruses had significantly decreased susceptibility to oseltamivir (21). Since this initial report, oseltamivir-resistant influenza A (H1N1) strains have spread throughout Europe (11) and have been detected at high frequencies in other countries including the United States (4), Japan (28), South Africa (1) and Oceania and Southeast Asia (17). These influenza A (H1N1) viruses have a mutation of histidine to tyrosine at residue 274 of the NA (N2 NA numbering; residue 275 by N1 NA numbering), which confers a high level of resistance to oseltamivir (10) but has no effect on susceptibility to zanamivir or to the adamantanes.Prior to May 2008, when the oseltamivir-resistant variants became the dominant influenza A (H1N1) strain in Oceania and Southeast Asia (17), NAI sensitivity monitoring conducted at the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, identified a number of influenza A (H1N1) viruses with reduced zanamivir susceptibility. These viruses contained a previously undescribed mutation at residue 136 of the NA. Here, we report on the detection of these mutant viruses from geographically distinct locations, the in vitro and in vivo fitness of the strains, and the finding that the mutant viruses appear to have been preferentially propagated during viral culture in Madin-Darby canine kidney (MDCK) cells.     

A Single Immunization with CoVaccine HT-Adjuvanted H5N1 Influenza Virus Vaccine Induces Protective Cellular and Humoral Immune Responses in Ferrets

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 μg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.Since the first human case of infection with a highly pathogenic avian influenza A virus of the H5N1 subtype in 1997 (9, 10, 37), hundreds of zoonotic transmissions have been reported, with a high case-fatality rate (10, 44). Since these viruses continue to circulate among domestic birds and human cases are regularly reported, it is feared that they will adapt to their new host or exchange gene segments with other influenza A viruses, become transmissible from human to human, and cause a new pandemic. Recently, a novel influenza A virus of the H1N1 subtype emerged. This virus, which originated from pigs, was transmitted between humans efficiently, resulting in the first influenza pandemic of the 21st century (8, 45). Although millions of people have been inoculated with the (H1N1)2009 virus, the case-fatality rate was relatively low compared to that for infections with the H5N1 viruses (11, 31). However, the unexpected pandemic caused by influenza A/H1N1(2009) viruses has further highlighted the importance of rapid availability of safe and effective pandemic influenza virus vaccines. Other key issues for the development of pandemic influenza A virus vaccines include optimal use of the existing (limited) capacity for production of viral antigen and effectiveness against viruses that are antigenically distinct. Ideally, a single administration of a low dose of antigen would be sufficient to induce protective immunity against the homologous strain and heterologous antigenic variant strains. However, since the population at large will be immunologically naïve to a newly introduced virus, high doses of antigen are required to induce protective immunity in unprimed subjects (23, 36). The use of safe and effective adjuvants in pandemic influenza virus vaccines is considered a dose-sparing strategy. Clinical trials evaluating candidate inactivated influenza A/H5N1 virus vaccines showed that the use of adjuvants can increase their immunogenicity and broaden the specificity of the induced antibody responses (2, 7, 19, 23, 27, 36, 41). These research efforts have resulted in the licensing of adjuvanted vaccines against seasonal and pandemic influenza viruses (17). The protective efficacy of immune responses induced with candidate influenza A/H5N1 virus vaccines was demonstrated in ferrets after two immunizations (1, 22, 24, 25) or after a single immunization. The latter was achieved with a low dose of antigen in combination with the adjuvant Iscomatrix (26).Recently, a novel adjuvant that consists of a sucrose fatty acid sulfate ester (SFASE) immobilized on the oil droplets of a submicrometer emulsion of squalane in water has been developed (4). It has been demonstrated that the addition of this novel adjuvant, called CoVaccine HT, to multiple antigens increased the immune response to these antigens in pigs and horses and was well tolerated in both species (4, 16, 40). Furthermore, it was shown that the use of CoVaccine HT increased the virus-specific antibody responses in mice and ferrets after vaccination with a cell culture-derived whole inactivated influenza A/H5N1 virus vaccine (5, 13). One of the mode of actions of CoVaccine HT is the activation of antigen-presenting cells such as dendritic cells, most likely through Toll-like receptor 4 (TLR4) signaling (5).In the present study, we evaluated the protective potential of CoVaccine HT-adjuvanted cell culture-derived whole inactivated influenza A/H5N1 virus (WIV) vaccine in the ferret model, which is considered the most suitable animal model for the evaluation of candidate influenza virus vaccines (6, 14, 15). To this end, ferrets were vaccinated once or twice with various antigen doses with or without the adjuvant to test whether dose sparing could be achieved. The use of CoVaccine HT increased virus-specific antibody responses and T cell responses. A single administration of 3.8 μg hemagglutinin (HA) of WIV NIBRG-14 vaccine preparation in combination with CoVaccine HT conferred protection against challenge infection with the homologous highly pathogenic A/H5N1 virus strain A/VN/1194/04 and partial protection against infection with a heterologous, antigenically distinct strain, A/IND/5/05. Therefore, it was concluded that the use of CoVaccine HT in inactivated influenza virus vaccines induced protective virus-specific humoral and cell-mediated immune responses and that it could be suitable as adjuvant in (pre)pandemic A/H5N1 virus vaccines. Further clinical testing of these candidate vaccines seems to be warranted.     

Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of ∼60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur.The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds α(2,6)-linked sialic acids while that of avian viruses preferentially bind α(2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment.Human respiratory tract epithelial cells can possess surface glycans with α(2,3)- and α(2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface α(2,3)- and α(2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that α(2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with α(2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accordance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses.Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations.     

Oseltamivir-Resistant Variants of the 2009 Pandemic H1N1 Influenza A Virus Are Not Attenuated in the Guinea Pig and Ferret Transmission Models

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.Oseltamivir resistance was rare until 2008, when resistant seasonal H1N1 viruses were found circulating in the general Scandinavian population (15). Soon after, studies from other countries in Europe also reported the isolation of oseltamivir-resistant viruses, and eventually, oseltamivir resistance was recognized as a global phenomenon (9, 27). Prior to 2008, resistant viruses were primarily isolated from patients with nonresponsive influenza virus infections or from infected patients who received a low-dose prophylaxis regiment prior to symptom onset. At the time, these resistant isolates accounted for 1% of the circulating H1N1 virus. Drug resistance mutations were identified during oseltamivir development, including a histidine-to-tyrosine mutation at position 275 (H275Y) in N1 neuraminidase (NA). This mutation in particular was shown to attenuate virus growth and pathology in ferrets (17). Additionally, oseltamivir-resistant viruses with a nearby mutation in N2 neuraminidase transmitted less efficiently than oseltamivir-sensitive viruses in the guinea pig transmission model (4). Surprisingly, the seasonal 2008 H1N1 viral isolates that spread around the world had the same tyrosine mutation, which was previously associated with iatrogenic infections and attenuation. Furthermore, epidemiological studies concluded that this resistant virus developed independently of drug selection, suggesting that compensatory adaptations allowed an attenuating mutation to become permissible (3, 18). The ability of resistant 2008 isolates to perform on par with nonresistant 2008 isolates in growth curves, in mean plaque size, and in a transmission model was recently confirmed (2). Currently, 99% of seasonal H1N1 viruses are oseltamivir resistant; however, the prevalence of these viruses is very low due to replacement by a novel reassortant H1N1 virus (6, 8). This novel reassortant was originally identified in Mexico by doctors concerned about a jump in the number of influenza cases during the month of March in 2009 (7). Later referred to as swine-origin influenza virus, novel H1N1 virus, or 2009 pandemic H1N1 virus, this virus would continue to efficiently transmit around the world, even during the summer months of the northern hemisphere. Its robust transmission was later confirmed in aerosol transmission models, in which 86% of ferrets and 100% of guinea pigs exposed to infected animals contracted pandemic influenza (22, 28, 31). Oseltamivir was used broadly during the outbreak, treating those with complications and prophylactically treating close contacts of confirmed cases. The use of oseltamivir in this manner provided ample opportunity for oseltamivir-resistant viruses to develop. More than 225 cases of oseltamivir-resistant infections have been confirmed from the beginning of the pandemic, including four incidents of suspected aerosol transmission (21, 32, 33). Fortunately, these clinical isolates never progressed into stable transmission in the general public. This study seeks to evaluate if introducing the H275Y mutation into the pandemic 2009 H1N1 virus attenuates virus replication in vitro or in vivo using the guinea pig model and the ferret model to test aerosol transmission efficiency. Furthermore, this study evaluates if a reassortant between the circulating novel H1N1 virus and seasonal neuraminidase (NA) forms a well-adapted, resistant virus capable of efficient transmission.Currently, oseltamivir is the drug of choice for treating novel H1N1 complications and outpatient prophylaxis. Therefore, it is of great importance to study the in vitro replication and transmission phenotypes of oseltamivir-resistant novel H1N1 viruses to understand why broad oseltamivir resistance has not occurred or whether we should expect it to occur in the future.     

T-Cell Tolerance for Variability in an HLA Class I-Presented Influenza A Virus Epitope

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP_418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP_418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP_418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition.Cytotoxic T-lymphocytes (CTL) kill virus-infected cells and release antiviral cytokines upon recognition of short viral peptides displayed on the cell surface by the class I HLA molecule (36). Virus-derived peptides are processed in the cytoplasm by proteasome degradation of viral proteins (25), shuttled into the lumen of the endoplasmic reticulum (ER) by the transporter-associated protein, and loaded into the basket-like groove of the class I molecule. Class I HLA molecules await peptide loading in the ER and demonstrate specificity for viral peptides with particular anchor residues representing a good fit for the class I HLA binding groove. Once stable class I HLA-peptide complexes are formed, the class I molecule and its peptide cargo are transported via the Golgi apparatus to the cell surface, where the complex is anchored to the plasma membrane (21, 36-38). CTL then survey class I HLA-presented peptides on the cell surface. Viral peptides must therefore be processed, specifically bound by class I HLA, and presented at the plasma membrane for CTL to distinguish infected cells from uninfected tissue.A high mutation rate is one of many mechanisms utilized by viruses to escape detection by the immune system. Mutations within the genome allow viruses to accumulate and select for amino acid substitutions that (i) inhibit proteasome processing and viral peptide generation (2, 23), (ii) alter anchor residues within viral peptides to diminish class I HLA binding specificity (3, 14, 24, 32), or (iii) reduce immune recognition of the class I HLA-peptide complex by varying amino acids that come in contact with the T-cell receptor (6, 10, 27, 30, 35). While viral mutations might be advantageous for escaping immune detection, such flexibility can cost the virus in terms of replicative fitness. In order to maintain reproductive fitness and structural integrity, viruses must temper their use of genetic flexibility as a means of immune escape.Influenza viruses have the well-documented ability to escape detection by various immune epitopes (3, 10, 27). A priori, investigators often assume that variable regions of the virus represent poor immune targets because such regions will not be consistently processed, presented, or recognized (15, 20). However, we along with others continue to find that a hypervariable stretch of the influenza virus nucleoprotein consisting of residues 418 to 426 (NP_418-426) is presented to CTL by different HLA-B alleles (B*0702 and B*3501) in spite of extensive viral variability within this epitope (8, 10, 27, 34). Moreover, NP_418-426 is a dominant immune epitope (8, 10, 27, 34). The consistent processing and presentation of NP_418-426 by class I HLA can be explained by the finding that different influenza virus isolates cannot mutate the proline located at position 2 (P2) within the epitope because elimination of this proline reduces viral fitness (4, 5). Little to no variability is found at the methionine P9 anchor as well. These facts lead to the unique observation that strain-to-strain variability does not abrogate class I HLA presentation of the influenza virus NP_418-426 epitope and that CTL respond to this consistently presented viral epitope in an immunodominant fashion.In this study we took advantage of the anchor residue conservation that prompts the NP_418-426 epitope to be consistently presented to CTL by investigating the functional impact that influenza virus intraepitope variability has on CTL recognition. The amino acid alignment of human influenza A (H1N1 and H3N2) virus nucleoprotein molecules identifies 20 unique NP_418-426 peptide sequences which demonstrate amino acid diversity between the anchors. We infected HLA-transgenic mice intranasally with influenza virus and tested CTL from these animals for their ability to recognize each of the 20 NP_418-426 variants. These 20 NP_418-426 sequences represent a natural “recombinant library” of viral epitopes that the immune system has and will face. The resulting data demonstrate a gradient of viral substitutions whereby CTL recognition diminishes depending upon the number of viral substitutions and their location within the epitope. Understanding how intraepitope variability impacts CTL recognition is discussed in terms of eliciting immune responses to variants of influenza.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Persistence of Transgene Expression Influences CD8+ T-Cell Expansion and Maintenance following Immunization with Recombinant Adenovirus

Previous studies determined that the CD8⁺ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8⁺ T-cell maintenance following immunization with a recombinant adenovirus.Recombinant human adenovirus 5 (rHuAd5) vector vaccines have garnered considerable attention as platforms for eliciting CD8⁺ T-cell immunity due to their strong immunogenicity in numerous studies, including primate studies and preliminary human trials (30, 32, 53). While these vectors may not represent the optimal serotype for use in humans, due to the high prevalence of preexisting immunity, the robust immunogenicity of rHuAd5 in preclinical models merits further investigation, since the biological information derived from these studies will offer important insights that can be extended to other vaccine platforms.CD8⁺ T cells play an important role in host defense against tumors and viral infections. During the primary phase of the CD8⁺ T-cell response, the activated precursors undergo a rapid and dramatic expansion in cell number, followed by a period of contraction where 80 to 90% of the antigen-specific population dies off, leaving the remaining cells to constitute the memory population (44). CD8⁺ T cells mature over the course of the primary response and acquire the ability to produce gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and, to a lesser degree, interleukin 2 (IL-2). Memory T cells can be divided into central memory and effector memory T cells based on phenotype and anatomical location (44). These phenotypic differences have also been linked to functional differences; however, these relationships remain controversial (2, 16, 20, 46, 55).Various reports have revealed some unexpected qualities of the CD8⁺ T-cell response generated by intramuscular immunization with rHuAd5. The rHuAd5-induced CD8⁺ T-cell response exhibited a protracted contraction phase, and the memory population was composed primarily of effector and effector-memory cells (23, 38, 39, 41, 51). The phenotype of the rHuAd5-elicited CD8⁺ T-cell population was more consistent with the CD8⁺ T-cell population observed in persistent infections, such as polyomavirus (25), murine herpesvirus-68 (35), and murine cytomegalovirus (MCMV) (1) infections, than with that observed in acute infections, such as lymphocytic choriomeningitis virus (LCMV) (44), vaccinia virus (15), and influenza virus (24) infections. Further investigation demonstrated that, as in a persistent infection, antigen presentation persisted for a prolonged period following intramuscular immunization with rHuAd5, and transgene expression could persist at low levels for more than 1 year following infection (41, 51). These data suggest that the sustained effector phenotype may arise from prolonged, low-level transgene expression from the rHuAd5 vector, although this connection was not formally proven. It is difficult to fully appreciate the implications of these observations at this time, since chronic exposure to antigen is often associated with CD8⁺ T-cell dysfunction, yet rHuAd5 vectors have been used successfully to elicit protective immunity in many models of pathogen infection and tumor challenge (5, 54). Nevertheless, other reports have provided evidence that rHuAd5 vectors can, indeed, lead to dysfunctional CD8⁺ T-cell immunity (27, 36). Therefore, further investigation is necessary in order to properly assess the implications of the prolonged antigen expression following rHuAd5 immunization in terms of sustaining a functional memory CD8⁺ T-cell response.In the current report, we sought to determine the relationship between transgene expression and CD8⁺ T-cell maintenance and memory. To this end, we constructed an Ad vector with a doxycycline (DOX)-regulated expression cassette that would permit attenuation of gene expression at various times postinfection. Using this reagent, we addressed two key questions. (i) How does the duration of antigen expression affect the magnitude of primary CD8⁺ T-cell expansion? (ii) Is antigen expression required beyond the peak expansion to maintain the memory CD8⁺ T-cell population?