Reverse genetics generation of chimeric infectious Junin/Lassa virus is dependent on interaction of homologous glycoprotein stable signal peptide and G2 cytoplasmic domains

The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.     

Emerging role of roots in plant responses to aboveground insect herbivory

Plants have evolved complex biochemical mechanisms to counter threats from insect herbivory. Recent research has revealed an important role of roots in plant responses to above ground herbivory (AGH). The involvement of roots is integral to plant resistance and tolerance mechanisms. Roots not only play an active role in plant defenses by acting as sites for biosynthesis of various toxins and but also contribute to tolerance by storing photoassimilates to enable future regrowth. The interaction of roots with beneficial soil‐borne microorganisms also influences the outcome of the interaction between plant and insect herbivores. Shoot‐to‐root communication signals are critical for plant response to AGH. A better understanding of the role of roots in plant response to AGH is essential in order to develop a comprehensive picture of plant‐insect interactions. Here, we summarize the current status of research on the role of roots in plant response to AGH and also discuss possible signals involved in shoot‐to‐root communication.     

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.     

Susceptibility of twolined spittlebug (Hemiptera: Cercopidae) life stages to entomophagous arthropods in turfgrass

Prosapia bicincta (Say) (Hemiptera: Cercopidae), the twolined spittlebug, is an economic pest of turfgrass in the southeastern United States. No data concerning natural enemies of P. bicincta in turfgrass have been reported previously. We compared predation of spittlebug eggs, nymphs, and adults in the laboratory by potential generalist predators commonly found in turfgrass: bigeyed bugs Geocoris uliginosus Say and Geocoris punctipes Say; red imported fire ant, Solenopsis invicta Buren; wolf spiders (Lycosa sp. Walckenaer); carabid beetles Harpalaus pensylvanicus DeGeer and Calosoma sayi Dejean; and tiger beetles Megacephala carolina carolina L. Eggs were readily consumed by generalist predators. S. invicta consumed 100% of the eggs offered. H. pensylvanicus and C. sayi were also significant predators of P. bicincta eggs. Nymphs live in spittlemasses that protect them from attack by predators, but exposed nymphs were susceptible to attack when mechanically removed from their spittlemasses. S. invicta and M. carolina carolina caused significant mortality of exposed nymphs. P. bicincta adults are aposematic and have the ability to reflex bleed; however, reflex bleeding did not prevent attack by predators. S. invicta and M. carolina carolina killed 100% of the adult spittlebugs offered in laboratory bioassays. Lycosa sp. are less voracious predators of adults. Sound background knowledge about P. bicincta and its potential natural enemy complex is important for the development and implementation of a detailed, site-specific, biologically based pest management program in turfgrass.     

Response of a complex foraging phenotype to artificial selection on its component traits

In nature, where predators must often track dynamic and dispersed prey populations, predator consumption rate, conversion efficiency, dispersal, and prey finding are likely to be important links between foraging and predator–prey population dynamics. Small differences in predator foraging caused by variation in any of the abovementioned traits might lead to significant differences in predator success as well as population dynamics. We used artificial selection to create lines of the predatory mite, Phytoseiulus persimilis in order to determine the potential for or constraints on the evolution of predator foraging behaviors. All four foraging traits demonstrated considerable phenotypic variation. They also exhibited significant realized heritabilities after artificial selection, except that prey finding did not respond to downward selection. Lines that responded to selection did so rapidly, and high-consumption, high-conversion efficiency, and high- and low-dispersal were stable for at least four generations after artificial selection was relaxed. There were some indirect responses to selection among the foraging traits. For example, there was positive correlation between consumption and dispersal. However, none of the correlated responses were of the magnitude of the direct responses we measured on the same trait. We also observed some correlations between foraging traits and life-history traits such as low-consumption and development time (negative), high-consumption and fecundity (positive), and high-conversion efficiency and fecundity (positive), but these were more likely to represent non-genetic constraints. Intrinsic rates of increase in low-consumption and low-conversion efficiency lines were lower than in their respective high lines and the unselected control, whereas rates of increase in dispersal and olfactory response lines did not differ from the unselected control. Thus, traits that make up foraging share partially overlapping genetic architectures with highly heritable phenotypic components, suggesting that each foraging trait will be able to respond rapidly to changes in the density and distribution of resources.     

Andes Virus Disrupts the Endothelial Cell Barrier by Induction of Vascular Endothelial Growth Factor and Downregulation of VE-Cadherin

Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infection. High levels of virus replication occur in microvascular endothelial cells but without a virus-induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus-induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease.Hantaviruses, of the family Bunyaviridae, are rodent-borne RNA viruses. Members of the Hantavirus genus have been identified as etiologic agents of two severe human diseases: hemorrhagic fever with renal syndrome (HFRS), which is caused by the Old World hantaviruses, and hantavirus pulmonary syndrome (HPS), which is caused by the New World hantaviruses (38, 39). Sin Nombre virus (SNV) and Andes virus (ANDV) are the main causes of HPS in the Americas. The major hantavirus target in humans is the microvascular endothelium, and the basis of HPS and HFRS is attributed to microvascular leakage (9, 34, 57). Common clinical features of HPS are interstitial pneumonitis with variable amounts of mononuclear cell infiltration, congestion, and both interstitial and alveolar edema (4, 34, 57). Despite the prominent accumulation of viral antigen in the infected vascular endothelium, no evidence of cellular destruction has been observed (57). Absence of a cytopathic effect has also been reported in in vitro studies of hantavirus infection of human primary endothelial cells (35, 46). In general, it is believed that induction of an uncontrolled immune response to the hantavirus infection, rather than the viral infection per se, is the cause of the microvascular leakage and ultimately HPS and HFRS (3, 48, 57). So far, a limited number of in vitro permeability studies have reported either no significant changes in the vascular permeability upon hantavirus infection or a significant increase only when mediators of increased permeability are exogenously added to the hantavirus-infected cells (12, 22, 46).Endothelial cell permeability is a highly regulated process and is maintained by both tight and adherens junctions (47). The disruption of adherens junctions is sufficient to disturb the endothelium barrier function and cause an increase in permeability and formation of edema (25, 47). Adherens junctions are largely composed of vascular endothelial (VE) cadherin (VE-cadherin), an endothelial cell-specific member of the cadherin family of adhesion protein (51, 52). Adherens junctions and in particular VE-cadherin are targets of the signaling pathway of agents that increase vascular permeability (7, 8, 10). Vascular endothelial growth factor (VEGF), one of the most potent vascular permeability agents, exerts its effects after binding to its homologous membrane tyrosine kinase receptor, VEGF-R2, whose expression is restricted to endothelial cells. It is known that VEGF-R2 interacts with VE-cadherin, and together they maintain the endothelial cell barrier (26). When VEGF is present, it binds to VEGF-R2, and that initiates the internalization and degradation of VE-cadherin and disruption of the adherens junctions (10, 54).In general, increase of vascular permeability is an important component of severe disease progression in hemorrhagic fevers (36). A number of studies have investigated the cause of increased vascular permeability in viral hemorrhagic fevers induced by viruses such as Dengue virus or Ebola virus (41, 42, 50, 53, 56). Studies of vascular permeability during hantavirus infection in vitro have mainly been performed in the presence of various inflammatory agents and growth factors (12, 15, 19, 22, 46). A recent study demonstrated that pathogenic hantaviruses sensitize the endothelium and cause hyperpermeability in response to high levels of exogenously added VEGF (12). We show here that VE-cadherin downregulation can be observed in ANDV-infected cells in the absence of exogenous VEGF. The downregulation of VE-cadherin in the absence of exogenous VEGF led us to the discovery that endothelial cells infected with ANDV induce the production of VEGF at early times postinfection. The early increased secretion of VEGF coincides with the initiation of downregulation of the adherent junction protein VE-cadherin and an increase in permeability of endothelial cells. The involvement of VEGF-R2 in VE-cadherin downregulation was demonstrated by antibody blockage of VEGF-R2 that resulted in significant recovery of VE-cadherin levels. These data indicate that the increased vascular permeability seen in HPS could be a direct result of hantavirus infection of the endothelium and may occur through a pathway involving VEGF-induced downregulation of VE-cadherin at early times postinfection.     

Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase

Binding of tumor necrosis factor-alpha (TNFalpha) to its receptor, TNF-R1, results in the activation of inhibitor of kappaB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFalpha to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFalpha, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

Prostaglandin D₂ was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G₂ was inhibited more strongly by PGD₂ than by PGE₁. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE₁ than by PGD₂ the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD₂ has less hypotensive effects on the cardiovascular system than PGE₁ suggest the possible use of PGD₂ as an antithrombotic agent.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

In rat adrenal glands incubated in Locke's solution in vitro norepinephrine and phenylephrine inhibited the release of epinephrine. PGE₂ and PGE₁ also inhibited the release of catecholamines but PGF_α1 had no effect on the adrenal. Thus, catecholamine release from adrenal cells may be regulated by the same mechanisms as in adrenergic nerve endings.