A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus (family Paramyxoviridae) that can cause severe respiratory illness and/or encephalitis in a wide variety of mammals, including horses, pigs, and humans (7, 23). HeV was identified as the causative agent of an acute respiratory disease in horses in 1994 in Queensland, Australia (23), and to date there have been 14 outbreaks in Australia since, with at least one occurrence per year since 2006, most recently in May 2010 (ProMed-mail no. 20100522.1699 [International Society for Infectious Diseases, http://www.promedmail.org]). Every outbreak of HeV has involved horses as the initial infected host, and there have been a total of seven human cases arising from exposure to infected horses. Four human fatalities have occurred (22), with the most recent occurring in August of 2009 (ProMed-mail no. 20090826.2998 and 20090903.3098). All patients initially presented with influenza-like illnesses (ILIs) after an incubation period of 7 to 16 days. While two individuals recovered from ILI, one patient developed pneumonitis and died from multiorgan failure. Three of the lethal cases developed encephalitic manifestations (mild confusion and ataxia), with two patients experiencing seizures (22, 23, 27).Data on the histopathology of fatal human HeV cases are limited, but the pathology includes small necrotic plaques in the cerebrum and cerebellum, in addition to mild parenchymal inflammation (21, 27). Severe parenchymal inflammation and necrosis were observed in the lungs. More extensive histopathologic data are available from 32 autopsies of fatal human NiV cases (28). Similarly to the HeV cases, pathology was characterized by systemic vasculitis and parenchymal necrosis in the central nervous system (CNS), while in the lung, pathological findings mainly included vasculitis, fibrinoid necrosis, alveolar hemorrhage, pulmonary edema, and aspiration pneumonia. Other organs that were affected included heart, kidney, and spleen and showed generally mild or focal inflammation. The development of syncytial multinucleated endothelial cells is characteristic of both HeV and NiV (27, 28). At present, the details of the pathogenesis and histopathological changes mediated by either HeV or NiV infection in humans are naturally derived from only the late phases of the disease course, and therefore a relevant animal model is needed that mimics the disease progression seen in humans.Pteropid fruit bats, commonly known as flying foxes in the family Pteropodidae, are the principle natural reservoirs for both NiV and HeV (reviewed in reference 3). However, these henipaviruses display a broad species tropism, and in addition to bats, horses and humans, natural and/or experimental infection of HeV has been demonstrated in guinea pigs, hamsters, pigs, cats, and ferrets (25). Experimental infections of Syrian hamsters with HeV is lethal, and animals show disease similar to that of human cases, including respiratory and neurological symptoms, depending on the dose (11; unpublished data). In this model, viral RNA can be detected in various organs of infected hamsters, including brain, lung, kidney, heart, liver, and spleen. The main histopathological findings included parenchymal infection in various organs, including the brain, with vasculitis and syncytial multinucleated endothelial cells in many blood vessels (11). While this model is useful in studying pathogenesis, it is limited in the availability of reagents to do so.There are currently no vaccines or treatments licensed for human use. Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1, 2, 29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6). Of the seven recorded human HeV cases, three patients were treated with ribavirin, one of whom survived (22). In the most recent outbreak of HeV in Australia, three additional people received ribavirin treatment in combination with chloroquine after suspected exposure to HeV-contaminated secretions from infected horses. While all three individuals survived, infection was not confirmed, and therefore it remains unknown whether the treatment had any beneficiary effect (ProMed-mail no. 20090826.2998). In addition, two animal studies in hamsters showed that ribavirin treatment delays but does not prevent death from NiV or HeV infection (8, 10). Therefore, an animal model with greater relevance to humans and that recapitulates the disease processes seen in human cases of HeV is needed to get a better answer to whether ribavirin might be effective against henipavirus infections. In addition, the U.S. FDA implemented the “Animal Efficacy Rule,” which specifically applies to the development of therapeutic products when human efficacy studies are not possible or ethical, such as is often the case with highly virulent pathogens like HeV (24). Essentially, this rule allows for the evaluation of vaccines or therapeutics using data derived from studies carried out in at least two animal models. The licensure of any therapeutic modalities for HeV will require a thorough evaluation of HeV pathogenesis in nonhuman primates (NHPs).In the present study, we report the development and characterization of a new nonhuman primate (NHP) model of lethal HeV infection in the African green monkey (AGM). The pathogenesis and disease progression in the AGM upon HeV infection essentially mirrored the lethal disease episodes seen among human cases of HeV. Using this new model, the efficacy of ribavirin treatment against lethal challenge with HeV was examined. Here we have shown that ribavirin treatment can significantly delay but not prevent death of AGMs from lethal HeV infection. In addition to severe respiratory symptoms in all animals, prolonged disease progression in ribavirin-treated animals was also marked by the appearance of neurological symptoms.     

Fluxomics with Ratiometric Metabolite Dyes

Today''s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g., regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.^1–3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals.⁴ This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants.^5,6 As it turns out, the cell registers many metabolites, and flux through the pathways is regulated using complex signaling networks that involve calcium as well as hormones.Key Words: flux, fluxome, glucose, glutamate, phosphate, sucrose, fluorescence resonance energy transfer, biosensorOne of the reasons for the fable for hormones lies in the simple fact that it is easier to observe macroscopic changes, such as changes in the architecture of a plant than to determine metabolite levels, but also here new tools are urgently needed that allow quantification of these small molecules. Visualization of starch levels provided a significant advance, and in combination with mutant screens allowed to identify fundamental components of starch metabolism.^7–9 The biggest advance for the signaling field was the development of advanced chemical and genetically encoded calcium dyes.^10–12 No such dyes are available for hormones or metabolites, as soon as we try to determine levels of metabolites (or signaling molecules), we run into the issues of compartmentation and cellular differences in tissues. Today, the same enzymatic assays used decades ago are still widely used to determine metabolite levels. Although significant advances in chromatography and mass spectrometry based metabolite analysis have moved the study of metabolism to ‘omics’ era, compartmentalization of metabolism still presents a major challenge. Especially the large vacuoles of plant cells are a major obstacle, since even fractionation studies suffer from contamination. Moreover, with the current set of tools it is not possible to determine the dynamic changes in metabolite levels in different subcellular compartments in real time in vivo. Radiotracers have helped a lot to identify and quantify intermediates and to assemble pathways, originally using pulse labeling followed by paper chromatography. Today ¹³C-labeling is used together with mass spectrometry to obtain insights into metabolic flux control.¹³ This tool set for the first time enabled the comparison of mutants and study regulatory networks involved in sugar signaling. While significant, advances in radiotracer experiments do not provide cellular or subcellular information and only limited temporal resolution, they do provide efficient means for studying metabolite fluxes through complex and/or not well-defined pathways. Thus there is a clear need for metabolite specific dyes that can be targeted to subcellular compartments and that would enable flux measurements in response to environmental cues helping to push metabolic research back into the focus of signaling-related biology.In 2002, we developed the first prototype “metabolic dye” FRET sensor for maltose.^14,15 A similar glucose sensor was recently employed for measuring tracer-independent transport of glucose across the ER membrane of liver cells.¹⁶ After resolving some issues such as low signal-to-noise and gene silencing in plants, we are now able to compare glucose levels between cells in an intact root in real time.¹⁷ The parallel development of sucrose and phosphate sensors complements the set of tools, in future experiments providing a comparison of sucrose, phosphate and glucose fluxes in intact tissues with both temporal (below seconds) and spatial resolution (cellular and subcellular).^18,19The first experiments already led to a big surprise: glucose supplied to the root is rapidly taken up and is rapidly metabolized.¹⁷ Roots expressing the highest affinity sensor FLIPglu170n responded to glucose perfusion suggesting that the steady state glucose level in the root is less than 100 nM, the estimated detection limit for this sensor in these first experiments. The first experiments were limited by the mixing kinetics in the bath used for perfusion, while improvement of the chamber now allow for faster for glucose exchange. We estimate that glucose levels fall from a steady state level of approximately 5 mM in the cytosol when perfused with 5 mM glucose to below 100 nM in about three minutes. For the sensor with an affinity of 600 µM the rate of glucose accumulation, which is composed of the various rates that affect the steady state in the cytosol such as metabolism, compartmentation and transport across the plasma membrane, is in the range of 527 ± 77 µM glucose/min and that for glucose removal is 317 ± 37 (Fig. 1; Chaudhuri B, Frommer WB, unpublished). Questions that arise are: Which transport systems drive uptake? How much does the vacuole contribute to the observed flux and steady state levels? Is the capacity of hexokinase at levels below its K_m still sufficient to phosphorylate glucose efficient enough to pull glucose below 100 nM or does hexokinase have different properties in vivo compared to what we know from the purified enzyme? Are there different transporters and enzymes contributing to flux in the low (1–10 mM) and the ultrahigh affinity (low µM) phases? Are there spatial differences in the root? Why do roots take up glucose so efficiently in the first place? The combination of the sensors with information from the expression-LEDs from Birnbaum and Benfey²⁰ and specific knock-out mutants should help answering some of these questions.Open in a separate windowFigure 1Quantitative analysis of glucose flux from an Arabidopsis root expressing FLIPglu-600µΔ13, a FRET sensor for glucose with an affinity of 600 µM. The root of a 10 day-old seedling was placed into a perfusion chamber and perfused with hydroponic medium with or without 5 mM glucose. eCFP was excited and emission was recorded for eCFP and eYFP every 10 seconds (essentially as decsribed in ref. 17). The emission intensities for a region-of-interest were averaged and the emission ratio was determined at the two wavelengths for each image of a time series and plotted on the Y-axis against time on the X-axis. Addition of glucose is indicated.Another big surprise is the dramatic gradient of glucose across the plasma membrane, which has important implications for our understanding of transport processes across the plasma membrane as well as the intracellular membranes.¹⁷ Information about the gradients is relevant in the context of apo- and symplasmic unloading routes in roots²¹ and the contribution of proton-coupled transporters in cellular export.²² It will thus be interesting to follow the extracellular levels using surface-anchored sensors. Now that besides high sensitivity glucose FLIPs¹⁷ we also generated nanosensors for sucrose¹⁹ and phosphate,¹⁸ complementing the similar tool sets for calcium²³ and pH,²⁴ it is possible to compare multiple parameters and to follow flux at different levels and to calibrate against other influences.The improvements of the signal-to-noise ratio of the FRET-based metabolite sensors²⁵ makes the FLIPs a standard tool for every lab interested in measuring ion-, sugar- or amino acid flux in living cells. Since the nanosensors are genetically encoded, they can be used to characterize intracellular fluxes^16,26 in any organism for which transformation protocols have been established. The existing sets of sensors are simple to use, constructs are available through Addgene and Arabidopsis lines from the Arabidopsis Stock Center. Detailed instructions for imaging can be found at: http://carnegiedpb.stanford.edu/research/frommer/research_frommer_protocols.php. These tools will hopefully become a standard system not only for physiological analyses, but in addition provide a new way for high throughput fluxomics studies.     

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10^−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10^−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.     

Overexpression of eCLCA1 in small airways of horses with recurrent airway obstruction.

The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.     

Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j

The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages. The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM). Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling. In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn420, 531Y-F, 176R-Q mutant. In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j. Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis. Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j. Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j. Our data support the possibility that Csk regulates the function of CD85j.     

Blood System Formation in the Urochordate Ciona intestinalis Requires the Variable Receptor vCRL1

Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.     

Review and advancement of the marine biotic resource use metric in seafood LCAs: a case study of Norwegian salmon feed

Purpose

Seafood life cycle assessment (LCA) studies have adopted the primary production required (PPR) indicator to account for the impact of these production systems (e.g., capture fisheries or aquaculture) on the ecosystems they harvest wild inputs from. However, there exists a large diversity in the application of methods to calculate PPR, and current practice often does not consider species- and ecosystem-specific factors. Here, we critically examine current practice and propose a refined method for applying the PPR metric in seafood LCAs.

Methods

We surveyed seafood LCAs that quantify PPR, or its derivatives, to examine the diversity of practice. We then defined and applied a refined method to a case study of the average Norwegian salmon feed in 2012. This refined method incorporates species-specific fishmeal and oil yields, source ecosystem-specific transfer efficiencies and expresses results as a percentage of total ecosystem production that PPR represents. Results were compared to those using previously applied methods based on the literature review, and the impact of uncertainty and natural variability of key input parameters was also assessed using Monte Carlo simulation.

Results and discussion

From the literature review, most studies do not incorporate species-specific fishmeal and oil yields or ecosystem-specific transfer efficiencies when calculating PPR. Our proposed method, which incorporated source species- and ecosystem-specific values for these parameters, provides far greater resolution of PPR than when employing global average values. When alternative methods to calculate PPR were applied to marine inputs to Norwegian salmon feeds, resulting PPR values were similar for some sources of fishmeal and oil. For other species, such as Atlantic herring from ecosystems with low transfer efficiencies, there was a large divergence in resulting PPR values. For combined inputs to Norwegian salmon feeds in 2012, the refined method resulted in a total PPR value that is three times higher than would result using the currently standard method signaling that previous LCA research may have substantially underestimated the marine biotic impacts of fishery products.

Conclusions

While there exists a great diversity of practice in the application of the PPR indicator in seafood LCA, the refined method should be adopted for future LCA studies to be more specific to the context of the study.

     

Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.