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Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.
West Nile virus and St Louis encephalitis virus (WNV and SLEV, respectively; family Flaviviridae) are closely related members of the Japanese encephalitis serocomplex and are both transmitted between Culex mosquitoes and avian hosts. Both viruses have the capacity for eliciting encephalitis in humans and have been associated with large epidemics. Despite utilizing the same avian and mosquito species for their enzootic maintenance, marked epidemiological differences exist between the viruses. For instance, since its introduction into North America in 1999, WNV has been associated with large epiornitics, whereas SLEV has never been associated with widespread mortality in avian hosts. Interestingly, SLEV also has been documented to be poorly cytopathogenic in cell culture, requiring up to 7 days to generate plaques on Vero cells. Additionally, SLEV has demonstrated greater oral infectivity for Culex mosquitoes than WNV (Reisen et al., 2008a). These differences suggest that the two viruses have adopted dissimilar evolutionary strategies for enzootic maintenance, with WNV relying on high avian replication rates to assure transmission and SLEV utilizing high mosquito oral susceptibility for its propagation. As SLEV is a Western hemisphere-endemic virus, this could reflect long-term co-adaptation of SLEV with North American Culex mosquitoes. This would be in direct contrast to WNV, a recently introduced virus that has not adapted to North American Culex vectors, but is capable of generalized high-level replication in a broad range of avian hosts, some of which, such as the house sparrow (Passer domesticus), have been introduced from the Old World (Robbins, 1973). The emergence and proliferation of a new North American genotype with increased dissemination capacity suggests a potential subsequent evolutionary adaptation of WNV in North American mosquitoes (Moudy et al., 2007).
Culex tarsalis mosquitoes are a major vector of WNV and SLEV in western North America, based on field isolations and laboratory vector-competence studies (Goddard et al., 2002; Reisen et al., 2006). This species has previously been demonstrated to be infected at lower oral titres with SLEV than WNV (Reisen et al., 2005). House sparrows and house finches (Carpodacus mexicanus) are considered to be major amplification hosts of WNV and SLEV in North America due to their abundance, susceptibility to infection, development of high-magnitude viraemias in laboratory studies and rapid population-replacement rates (Komar et al., 2003; Langevin et al., 2005; Mahmood et al., 2004; Reisen et al., 2004).
WNV and SLEV both possess positive-sense, ssRNA genomes of approximately 11 kb in length, expressed as a single ORF that is cleaved co- and post-translationally to produce three structural [capsid (C), pre-membrane/membrane (prM/M) and envelope (E)] and seven non-structural (NS) (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins. Previous reports have indicated that prME structural proteins are important for modulation of mosquito infectivity (McElroy et al., 2006b), presumably as a direct result of altered efficiency of virus–cell receptor binding. Alternatively, WNV NS genetic elements have been associated with altered growth in avian hosts (Brault et al., 2007) that could be dictated by enhanced intracellular replication in key target cells or altered replicative capacity in febrile avian hosts (Kinney et al., 2006). Therefore, structural genetic elements (prME) of SLEV could dictate increased oral infectivity of Culex vectors, whereas the NS proteins could modulate growth within avian hosts and produce the higher cytopathogenicity observed following WNV infection. To test this association, chimeric viruses were generated in which the structural components of WNV and SLEV were interchanged. The resulting mutant viruses generated from these chimeric constructs, WNV-prME/SLEV.IC and SLEV-prME/WNV.IC, were assayed in avian, mammalian and mosquito in vitro systems to determine virus-growth and cytopathogenic phenotypes.
Recombinant WNV from an infectious cDNA based on the NY99 genotype (strain 382-99) was generated and used as a surrogate for the parental strain, based on previous studies demonstrating the viruses to be phenotypically identical (Kinney et al., 2006). Infectious virus was generated successfully from the parental SLEV infectious clone-derived virus (SLEV.IC) following transfection of C6/36 cells, representing the first generation of a purely recombinant SLEV. Sequence analysis performed on the rescued SLEV.IC virus demonstrated complete identity with the genomic sequence of the original IMP115 SLEV isolate, GenBank accession no. JF460774. Chimeric viruses, SLEV-prME/WNV.IC and WNV-prME/SLEV.IC, were also rescued successfully following transfection in C6/36 cells. Sequences for all predicted amino acid substitutions were maintained following transfection in C6/36 cells for both chimeric viruses. Each chimeric virus shared complete sequence identity within the prME-encoding region for the heterologous flavivirus, containing a total of 191 amino acid substitutions (58 prM and 133 E substitutions; Table 1). WNV and SLEV shared predicted N-linked motifs for glycosylation at prM-15/prM-17, respectively, as well as at E-154.
To ensure the retention of antigenic specificity of the structural elements after chimerization, IFA was performed using WNV-specific (3.67) and SLEV-specific (4A4C-4) mAbs that target the envelope protein. mAb 3.67 reacted positively with Vero cells inoculated with WNV and WNV-prME/SLEV.IC; however, this antibody failed to detect cells inoculated with SLEV.IC or SLEV-prME/WNV.IC (Fig. 1a, c, e, g). Conversely, mAb 4A4C-4 was reactive with cells inoculated with SLEV.IC or SLEV-prME/WNV.IC, but not with WNV or WNV-prME/SLEV.IC viruses (Fig. 1b, d, f, h), thereby indicating the conservation of antigenic specificity of the two chimeric viruses.
Plaques of the WNV-NY99 isolate (strain 382-99) and the WNV infectious clone-derived virus (WNV.IC) averaged approximately 2 mm in diameter at 72 h post-infection (p.i.), similar to previous reports (Kinney et al., 2006). Plaques of the IMP115 SLEV strain and the SLEV.IC averaged 6 mm in diameter at 168 h p.i. (Fig. 2) and were statistically indistinguishable (P>0.05). Comparatively, WNV.IC plaques were clearly visible (2 mm) at 72 h p.i., whereas SLEV.IC plaques were <1 mm at this time point (Figs 2 and and3).3). The SLEV-prME/WNV.IC chimeric virus exhibited plaques of 2 mm at 72 h p.i. and ≥20 mm by 168 h p.i., consistent with the plaque morphology of WNV.IC (Figs 2 and and3).3). In contrast, WNV-prME/SLEV.IC plaques were statistically indistinguishable in size from those of SLEV.IC (P>0.05), measuring <1 mm at 72 h p.i. and <6 mm at 168 h p.i.
Changes in Vero cell-culture viability based on electrical impedance (Fang et al., 2011) supported the plaque-morphology results. Overall, WNV/NY99, WNV.IC and SLEV-prME/WNV.IC viruses demonstrated statistically similar (P>0.05) cell-viability patterns, initiating at a mean cell index of 9.7±0.2 and decreasing to 5.4±0.4 by 72 h p.i. and to 0.2±0.1 by 120 h p.i. (Fig. 4). However, the SLEV/IMP115, SLEV.IC and WNV-prME/SLEV.IC viruses exhibited a different cell-viability pattern, starting with a cell index value of 9.4±0.1 and reaching a mean cell index of 5.7±0.8 by 120 h p.i., similar to the pattern of the uninfected-control cells until 96 h p.i., at which point there was a more pronounced drop in cell index values for SLEV-infected cells (Fig. 4). The mean cell index did not differ significantly (P>0.05) among SLEV/IMP115, SLEV.IC and WNV-prME/SLEV.IC viruses, but did differ significantly (P<0.05) from WNV-NY99, WNV.IC and SLEV-prME/WNV.IC viruses (Fig. 4).
Previous work has demonstrated high growth rates of WNV in avian hosts and a rapid induction of cytopathic effect (CPE) in vertebrate cells. The validity of using vertebrate cells for assessing the genetic determinants associated with altered vertebrate replication and CPE was evaluated in vitro using growth profiles of the parental strains and chimeric viruses in avian [duck embryonic fibroblast; DEF (ATCC CCL-141)] and mammalian (Vero) cell lines. Preliminarily, the SLEV clone-derived virus was compared with the IMP115 strain in DEF cells and the growth patterns were indistinguishable (Fig. 5); SLEV.IC was therefore used as a surrogate for IMP115 for all subsequent in vitro growth comparisons.
The WNV.IC virus grew to an approximately 100-fold higher peak titre than the SLEV.IC virus in DEF cells. The WNV.IC virus titre peaked at 144 h p.i. at 8.5±0.2 log10(p.f.u. ml−1), significantly greater (P<0.05) than the peak SLEV.IC titre of 7.3±0.5 p.f.u. ml−1 occurring at 72 h p.i. (Fig. 6a). The mean peak titre for WNV.IC virus was not statistically different (P>0.05) from the mean peak titre for SLEV-prME/WNV.IC. Similarly, the peak titre of 7.2±0.6 log10(p.f.u. ml−1) for the WNV-prME/SLEV.IC chimeric virus at 72 h p.i. was indistinguishable (P>0.05) from that of the SLEV.IC virus. Growth patterns for both viruses containing the SLEV NS proteins plateaued at 72 h p.i., whereas the viruses that expressed the WNV NS proteins exhibited elevated growth until 96–144 h p.i. (Fig. 6a). A repeated-measures ANOVA for viruses over all time points followed by a Tukey–Kramer multiple comparison test demonstrated significant differences (P<0.05) in overall mean titres between the SLEV.IC and SLEV-prME/WNV.IC viruses, but failed to identify differences between the SLEV.IC and the chimeric virus with the homologous NS gene region, WNV-prME/SLEV.IC.
WNV.IC virus grew to a peak titre of 9.4±0.1 log10(p.f.u. ml−1) by 48 h p.i., which was significantly greater (P<0.05) than the SLEV-prME/WNV.IC virus titre of 8.8±0.1 log10(p.f.u. ml−1) that also peaked at 48 h p.i. (Fig. 6b). In contrast, the SLEV.IC and WNV-prME/SLEV.IC viruses demonstrated a lag in growth, both peaking at 96 h p.i. with statistically similar (P>0.05) titres of 8.2±0.1 log10(p.f.u. ml−1) and 8.7±0.1log10(p.f.u. ml−1), respectively (Fig. 6b). A repeated-measures ANOVA for viruses over all time points followed by a Tukey–Kramer multiple comparison test confirmed that there were statistically significant differences in mean titres among all viruses (P<0.05) and that the temporal pattern of virus growth varied among viruses because the virus×time interaction term was highly significant (P<0.0001). Nevertheless, the trend for the overall mean viraemia as a function of time indicated that the overall mean titres for viruses with the NS genetic elements of SLEV were significantly lower than those for viruses containing the NS genes of WNV.
Low oral-infection thresholds have been described previously for SLEV in Culex mosquitoes (Hardy & Reeves, 1990). To assess whether vector-competence differences between SLEV and WNV could be recapitulated in vitro and used to gauge potential genetic determinants of mosquito infection, growth phenotypes of the parental and chimeric viruses were characterized in C6/36 (Aedes albopictus) and more biologically relevant CT (Chao & Ball, 1976; Main et al., 1977) cell lines.
Overall, the mean peak titre for the WNV.IC virus was significantly higher (P<0.05) than that of the SLEV.IC virus (Fig. 7a) in C6/36 cells. The chimeric WNV-prME/SLEV.IC virus, peaking at 8.0±0.2 log10(p.f.u. ml−1) at 144 h p.i., grew to statistically indistinguishable peak titres (P>0.05) from both SLEV.IC and WNV.IC. The SLEV.IC virus peaked at 6.9±0.1 log10(p.f.u. ml−1) at 144 h p.i., statistically lower (P<0.05) than SLEV-prME/WNV.IC, which produced a peak titre of 9.0±0.1 log10(p.f.u. ml−1) at the same time point (Fig. 7a). In the repeated-measures ANOVA, overall mean titre for the SLEV.IC virus was significantly lower (P<0.05) than those for the other three viruses, and no differences were observed between overall means among WNV.IC, WNV-prME/SLEV.IC and SLEV-prME/WNV.IC viruses.
No statistically significant differences were observed for the mean peak titres among the four viruses. The WNV.IC virus peaked at 8.2±0.2 log10(p.f.u. ml−1) by 168 h p.i. (Fig. 7b), whilst the WNV-prME/SLEV.IC chimeric virus peaked at 8.1±0.2 log10(p.f.u. ml−1) at 168 h p.i. The SLEV-prME/WNV.IC and SLEV.IC viruses exhibited peak titres of 8.2±0.2 log10(p.f.u. ml−1) and 8.0±0.2 log10(p.f.u. ml−1) at 120 h p.i., respectively (Fig. 7b). Using a repeated-measures ANOVA followed by a Tukey–Kramer multiple comparison test, overall mean titres of WNV.IC and WNV-prME/SLEV.IC viruses were statistically indistinguishable from one another and were significantly greater (P<0.05) than those for SLEV.IC and SLEV-prME/WNV.IC; these means varied differently over time, as indicated by the significant (P<0.001) virus by time interaction term.
WNV and SLEV serve as an evolutionary model for two viruses that have developed contrasting adaptive strategies for maintenance in similar enzootic transmission cycles. WNV, eliciting high viraemia in large numbers of avian hosts (Komar et al., 2003), relies on elevated viraemia in the imbibed bloodmeal for infection of mosquito midgut epithelium. Ingestion of a 1 µl bloodmeal containing 108 p.f.u. (ml blood)−1 would expose each epithelial cell to approximately 10 virus particles, based on an estimated 104 mesenteric cells in an average mosquito midgut (Houk et al., 1990), potentially reducing selective pressure for maintenance of high oral susceptibility and allowing infection of a wide variety of marginally competent vectors. Interestingly, WNV has demonstrated adaptation to North American mosquitoes, with increased dissemination capacity being observed with the emergent WN02 genotype (Moudy et al., 2007), demonstrating that, despite high avian viraemogenesis, evidence exists for the presence of selective forces for increased mosquito transmissibility. In contrast, SLEV appears to depend upon a specific association between the virus and a select number of highly susceptible Culex vectors, to which the virus has adapted to maintain infectivity at low oral doses; the long history of SLEV with North American vectors would support the potential for such co-adaptation (Auguste et al., 2009; Baillie et al., 2008). High oral infectivity is probably a by-product of the considerably lower avian viraemias observed following infection with different SLEV strains (Reisen et al., 2004, 2005). Although SLEV viraemias of nestlings have been observed to approach titres commonly observed in adult birds infected with WNV (Mahmood et al., 2004), the evolutionary significance of these temporally and host-restricted viraemias as a selective factor remains unresolved. Fitness constraints imposed by the two-host transmission cycles of these viruses have probably resulted in compromised fitness in both vector and host (Ciota et al., 2009; Coffey et al., 2008). This study was designed to identify specific genetic elements that dictate differential avian- and mosquito-infection phenotypes observed between these viruses; however, future studies are warranted to assess fitness trade-offs and their relative effect on driving the contrasting evolutionary strategies of WNV and SLEV. For instance, assessing the effects of high oral infectivity and avian viraemogenesis of WNV could shed light on vector-infection restrictions that have resulted in adaptations for higher dissemination rates in North American Culex mosquitoes (Moudy et al., 2007) as opposed to higher midgut infectivity, as has been observed for SLEV (Reisen et al., 2008a).
Specific genetic differences between WNV and SLEV have not been implicated in differential cytopathogenicity or the aforementioned dissimilar vector-infectivity and avian-replication/virulence phenotypes. Chimerization of flaviviruses has previously been used for the identification of genetic determinants of virulence and for functional vaccine development (Chambers et al., 1999; Charlier et al., 2010; Huang et al., 2000; Komar et al., 2009; Pletnev & Men, 1998; Pletnev et al., 1992; Pripuzova et al., 2009). Incorporation of the prME genes from tick-borne encephalitis virus into a dengue virus 4 (DENV-4) backbone restricted entry and subsequent RNA replication of the resultant chimera in C6/36 cells (Pletnev et al., 1992), supporting the notion that structural genetic elements play a role in modulating mosquito infectivity. However, chimeras replacing prME genes from DENV-4 with Langat virus prME were viable in C6/36 cells, whereas the parental Langat virus was unable to grow in mosquito cells (Pletnev & Men, 1998). Additionally, a mutant virus generated with the prME genes from WNV inserted into the same DENV backbone failed to infect Cx. tarsalis mosquitoes, indicating that NS genetic determinants also play a role determining vector infectivity (Hanley et al., 2005). Inclusion of NS2A/NS4B genes of the 17D yellow fever vaccine strain into the wild-type Asibi virus has previously demonstrated significant dissemination impairment in A. aegypti (McElroy et al., 2006a). ChimeriVax-WNV (Johnson et al., 2003) and DENV-1–4 tetravalent (Johnson et al., 2004) viruses demonstrated that NS genes mediated in vitro mosquito replication, and specific NS genetic determinants have been identified that restrict in vivo replication of a PDK-53 DENV-2 vaccine candidate (Brault et al., 2011). Replacement of the prME genes of YFV-17D/DENV-2 with the prME genes of Modoc virus, a virus incapable of infecting mosquito cells, failed to reduce growth of the chimera significantly in comparison with YFV-17D and DENV-2 in C6/36 cells, also indicating that genetic elements exclusive of the prME genes dictate replication in mosquito cells (Charlier et al., 2010). Inclusion of the variable region of the 3′-UTR from tick-borne Langat virus in a DENV-4 genetic backbone demonstrated restricted in vitro vertebrate cell growth, but had no distinguishable effect on growth in mosquito cells, indicating the differential effect of specific mutations on host-specificity phenotypes (Tumban et al., 2011). Studies performed with yellow fever virus structural and NS chimeras indicate a potential cumulative role of both gene regions to restrict in vivo mosquito infectivity and dissemination, respectively (McElroy et al., 2006a, b). A similar multigenic scenario could encode enhanced infectivity of SLEV in Culex mosquito vectors, with structural genes governing receptor-mediated virus entry and NS genes modulating intracellular replication through mosquito cell-specific RNA interference (RNAi) antagonism (Keene et al., 2004).
Higher peak growth titres were observed for the parental WNV than for SLEV in C6/36 cells, but no statistical differences in peak titres were observed in CT cells. Although the overall mean titre analyses implicated the prME structural proteins with defining CT-cell growth phenotypes, both structural and NS gene regions were associated with modulation of C6/36 growth. Although differences were observed between WNV and SLEV growth patterns in both mosquito cell types, WNV consistently grew to higher titre in both mosquito cell lines. This finding somewhat contradicts the notion that SLEV would be capable of replicating more efficiently in mosquitoes than WNV, as would be expected based on higher observed SLEV in vivo infection rates (Reisen et al., 2005). The finding that C6/36 cells lack a functional RNAi pathway could dictate such a discordance between mosquito in vitro and in vivo infection models (Brackney et al., 2010), as could differences in virus internalization predicated by altered receptor expression in non-differentiated cell-culture progenitor cultured cell types. For instance, SLEV could have evolved specific structural protein motifs to bind specifically to Culex midgut epithelial-cell receptors that are not present in cultured mosquito cells derived from eggs. Delayed uptake of viral RNA and consequent lag in viral RNA replication in C6/36 cells could have resulted in lower peak titres due to SLEV structural proteins limiting entry into these cells. Once the cells were infected, enhanced intracellular replication could have been afforded by the presence of WNV NS genes in the absence of a functional RNAi response. This could explain the observed enhanced growth of the SLEV-prME/WNV.IC chimeric virus compared with SLEV.IC virus in C6/36 cells. It is intriguing that, in this study, overall growth patterns were determined to be dictated by prME gene regions for CT cells, a cell line derived from a relevant enzootic SLEV vector, although viruses expressing the SLEV prME exhibited lower growth levels than the WNV prME-expressing viruses. Virus entry and replicative capacity in cultured cells may not be reflective of the physiological environment that modulates a virus's ability to traverse mosquito-midgut and salivary-gland barriers (Ciota et al., 2007). Further studies in vivo will be needed to determine the influence of flavivirus genetic components on oral infection thresholds and tissue tropisms in mosquitoes.
Plaque-morphology and impedance data indicated that NS genes influenced cytopathogenicity profiles for WNV and SLEV; however, because the chimeric viruses maintained homologous capsid proteins, the role that this protein serves in eliciting differential cytopathogenicity cannot be excluded. WNV exhibited CPE more rapidly than SLEV on Vero cells, as measured by the timing of plaque formation and the decreased cellular impedance. Several flaviviral replicon systems have identified mutations in the NS1, NS2A, NS3, NS4B and NS5 genes associated with diminished capacities for eliciting CPE, suggesting that a variety of NS genetic differences between WNV and SLEV could be responsible for the alternative cytopathogenicity phenotypes observed (Liu et al., 2004; Rossi et al., 2007).
WNV grows to high titres in competent avian hosts and causes variable mortality, dependent upon the relative susceptibility of the avian host species (Brault et al., 2004; Langevin et al., 2005); SLEV, in contrast, generates low virus titres in birds and has not been associated with avian mortality (Reisen et al., 2003, 2004, 2005, 2008b). In DEF cells, the WNV-prME/SLEV.IC chimeric virus exhibited a similar growth profile to that of SLEV.IC, whereas SLEV-prME/WNV.IC demonstrated a comparable profile to WNV.IC. These data, in contrast to our finding that structural elements dictate invertebrate-cell growth differences, indicated that genetic elements exclusive of the prME gene region of WNV mediate increased virus replication in DEF cells. Previous studies using infectious cDNA clones of WNV strains with different avian-pathogenicity phenotypes (Brault et al., 2004) implicated a single NS viral helicase mutation in altered viraemogenic potential and mortality in American crows (Brault et al., 2007). These reports, coupled with the findings herein, demonstrate that genetic elements exclusive of the prME genes govern replicative fitness of WNV/SLEV chimeras in an avian cell line. Once again, the role of the capsid protein cannot be excluded, due to the chimerization of only the prME gene regions. The finding that the capsid protein of WNV is capable of serving as an apoptosis agonist (Yang et al., 2008) as well as a cellular serine/threonine phosphatase inhibitor (Hunt et al., 2007) could indicate the importance of the capsid for increased vertebrate pathogenesis of WNV; however, avian-inoculation experiments performed with identical WNV genetic backbones differing by single NS amino acid substitutions have demonstrated dramatic differences in avian virulence, indicating that such virulence variation is not associated with capsid polymorphisms between WNV strains (Brault et al., 2007).
Single point mutations in the NS4B and NS5 regions of WNV have demonstrated altered plaque-morphology, murine-neuroinvasiveness and temperature-sensitivity phenotypes (Wicker et al., 2006). The NS1 protein may also modulate the interferon (IFN) response by inhibiting signal transduction of the TLR3 receptor in addition to inhibiting complement activation by binding to regulatory factors H, C4 and C5 (Avirutnan et al., 2010; Chung et al., 2006; Wilson et al., 2008). Incorporation of an A30P mutation in the NS2A protein of Kunjin virus was shown to disable IFN-α/β induction (Liu et al., 2006), and the NS5 protein of WNV has been determined experimentally to effectively suppress IFN-mediated gene expression and inhibit IFN-α/β induction of the JAK/STAT pathway (Laurent-Rolle et al., 2010).
This study has ascertained that genetic regions exclusive of the prME genes are influential in flavivirus in vitro replication in mammalian and avian cell types, whereas flaviviral structural genetic elements modulate virus growth in mosquito (CT) cells. These data could potentially explain the disparate virological and epidemiological host and vector profiles exhibited by WNV and SLEV. However, the potential that in vitro models do not reflect in vivo physiological factors that might dictate variability in vector infectivity of differentiated midgut epithelial cells or viraemogenesis in an avian host will necessitate further in vivo characterizations of these parental and chimeric viruses in biologically relevant mosquito and avian species. The close genetic identity between WNV and SLEV (71.5% amino acid identity for the prME genes) makes this an intriguing model for the elucidation of specific genetic determinants associated with the structural or NS genes for modulation of vector infectivity, as well as extra-prME determinants for modulation of vertebrate viraemogenesis. Future studies that utilize these viruses and further refined chimeras to assess specific genetic motifs for altered vertebrate pathogenesis and midgut epithelial infection of reservoir avian species and mosquito vectors will be necessary to assess more completely the hypothesis that WNV and SLEV have evolved differential strategies for enzootic maintenance. Identification of flaviviral genetic determinants of vertebrate host tropism and mosquito infectivity will provide a wealth of information on the evolutionary history of these viruses, allow for potential risk assessments of the emergence of viruses with altered epidemiological patterns and provide insight into novel control methodologies.
SLEV strain IMP115, isolated from a pool of Cx. tarsalis mosquitoes from Imperial Valley, CA, USA, in 2003 with one Vero cell passage, was sequenced in its entirety and used for generation of an SLEV infectious cDNA. At least 2 µg total nucleic acids was used for RT-PCR. The genome of the IMP115 SLEV strain was amplified in six overlapping fragments spanning appropriate unique restriction sites. Viral cDNAs were synthesized from RNA by using reverse transcriptase according to the manufacturer's recommendations (SuperScript II; Invitrogen). Amplicons (primer sequences available as Supplementary Table S1 in JGV Online) were generated by using the high-fidelity Pfu Ultra Hotstart polymerase (Stratagene) and products were cloned into the pCR-XL-TOPO vector (Invitrogen) and sequenced directly. In order to clone viral cDNAs into the final constructions, a specific low-copy-number, kanamycin-resistant vector pMA3, with convenient unique restriction sites, was created (sequence available upon request). Due to bacterial toxicity/stability issues encountered, a two-plasmid cloning strategy was utilized similar to that reported previously (Kinney et al., 2006). The first amplicon (nt 1–2012 of the 5′ end of the SLEV genome) was cloned into a separate 5′ (68M) plasmid, whereas the remaining five amplicons were engineered sequentially into a 3′ (79M) plasmid.
Internal BamHI and DraIII restriction sites at genomic nucleotide positions 466 and 2472 within the prME coding region of the 68M plasmid described above were ablated by mutagenesis. The mutated 68M plasmid was used as a template for generation of an SLEV prME region with 5′ and 3′ flanking restriction sites for incorporation into the 5′ WNV plasmid. Primers utilized for the generation of this amplicon were designed to contain BamHI and DraIII restriction sites at the 5′ and 3′ ends of the SLEV prME fragment. Amplicons were generated by using the high-fidelity Pfu Ultra Hotstart polymerase (Stratagene), and the subsequent BamHI–SLEV prME–DraIII fragments were inserted in a pCR-XL-TOPO vector (Invitrogen). A BamHI restriction site was created upstream of the prM gene of the 5′ WNV receptor plasmid [the first plasmid of the bipartite system for strain NY99 (Kinney et al., 2006)]. The (BamHI–prME–DraIII) fragment was cloned directly into the 5′ WNV plasmid with the newly created BamHI and existing DraIII sites in the 5′ WNV plasmid. The BamHI site was removed from the final clone by site-directed mutagenesis to restore the native amino acid identity upstream of the prM gene in the chimeric plasmid. This modified chimeric plasmid containing the SLEV prME genes was subsequently used for in vitro ligation with the WNV 3′ plasmid, for generation of SLEV prME/WNV.IC virus described below.
The prME gene region of WNV was amplified from the 5′ WNV plasmid with flanking primers containing the BglII and BssHII restriction sequences and the product was subcloned into a pCR-XL-TOPO vector. The 68M SLEV 5′ plasmid was modified to contain an additional BglII site at nt 462 for direct incorporation of the WNV prME BglII/BssHII amplicon. The presence of the AgeI in vitro ligation site used for the generation of the recombinant parental SLEV at the C terminus of the E protein necessitated the incorporation of 126 nt of SLEV sequence from the 5′ portion of the SLEV plasmid 79M at the 3′ end of plasmid 68M for inclusion of the complete prME gene sequences. To allow for inclusion of this fragment into the chimeric WNV prME 5′ plasmid, a NotI site was incorporated by mutagenesis after the last viral nucleotide. Direct PCR amplification of the 126 nt SLEV fragment from plasmid 79M with corresponding flanking NotI and BssHII sites at nt 10940 and 2152 and subsequent ligation into the modified 68M plasmid were performed. The BglII and NotI sites were removed from the modified 68M plasmid and the corresponding 126 nt sequence was removed from the 79M plasmid by mutagenesis. This cloning process ablated the AgeI restriction site used for in vitro ligation of the SLEV 5′ and 3′ plasmids and, as such, necessitated the use of the introduced BssHII site for in vitro ligation.
For generation of virus from the SLEV infectious cDNA, plasmids 68M and 79M were digested with SphI and NotI, respectively, treated with calf intestinal alkaline phosphatase (New England Biolabs), purified over spin columns (QIAquick PCR Purification kit; Qiagen) and digested with AgeI. Ligation was performed at the AgeI site for generation of in vitro transcription template. The AgeI restriction site was also used for in vitro ligation of the chimeric SLEV prME 5′ plasmid with the WNV 3′ plasmid. Ligation of the WNV prME 5′ plasmid with the SLEV 3′ plasmid was performed with an engineered BssHII site.
Following in vitro ligation, cDNAs were treated with proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1; Amresco) and chloroform, and ethanol-precipitated. Viral genomic RNA was transcribed for 2 h at 37 °C (AmpliScribe T7 Transcription kit; Epicentre). Reactions included 6 mM m7G-(5′)ppp-(5′)A cap analogue (New England Biolabs), 20% of the manufacturer-recommended concentration of ATP in order to facilitate incorporation of the cap analogue, and in vitro-ligated template cDNA. Transcribed RNA was treated with DNase I (Epicentre), extracted with phenol:chloroform:isoamyl alcohol and chloroform, and precipitated with ethanol. RNA pellets were resuspended in 20 µl RNase-free dH2O and 2 µl aliquots were analysed by non-denaturing agarose gel electrophoresis to visualize genome-length RNA transcripts. Transcribed RNA was transfected by electroporation into C6/36 cells (Kinney et al., 1997) in six-well plates using a Petri pulser (ECM 630; BTX). Supernatants were harvested 7 days post-transfection and RT-PCR and plaque assay on Vero cells were performed to confirm the presence of viral RNA and infectious virus, respectively.
Similarly, chimeric SLEV-prME/WNV.IC and WNV-prME/SLEV.IC viruses were recovered from in vitro ligation of the modified 5′ plasmids containing the reciprocal prME genes with either the 3′ plasmids of WNV or SLEV. All rescued viruses were sequenced fully to ensure complete sequence identity with parental strains and to ensure the exclusion of any spurious mutations that might have occurred during the cloning, assembly or rescuing processes.
IFA was performed with the parental and clone-derived chimeric viruses. Briefly, confluent chamber-well slides of Vero cells were inoculated individually with parental or chimeric viruses at an m.o.i. of 10. Medium was removed 24 h p.i. and cells were scraped and added to each well of a 12-well spot slide. The slides were dried for 30 min and fixed with an acetone:DPBS (3:1) mixture. Slides were washed and incubated with a 1:400 dilution of either WNV-specific (3.67) or SLEV-specific (4A4C-4) mAbs (CDC). The slides were incubated in a humid chamber at 37 °C for 1 h, washed and incubated similarly for 1 h with a goat anti-mouse FITC-conjugated antibody (Fisher Scientific) at a dilution of 1:20 with (2%) Evans' blue used as a counterstain. Following DPBS washes, 1,4-diazabicyclo-octane (DABCO, anti-fade solution) was added to each well and coverslips were added to each slide and scored by fluorescence microscopy.
C6/36, CT (Chao & Ball, 1976), DEF (ATCC CCL-141) and Vero cells were utilized for in vitro growth-profile assays. C6/36 and Vero cells were maintained at 28 and 37 °C, respectively, in Dulbecco's modified Eagle's medium (Gibco-BRL) containing 5% FBS (Hyclone), penicillin (100 U ml–1) and streptomycin (100 µg ml–1). CT and DEF cells were maintained in Schneider's Drosophila media (Lonza BioWhittaker) and Eagle's MEM containing 5% FBS and penicillin (100 U ml–1) and streptomycin (100 µg ml−1) at 28 and 37 °C, respectively. All cells were grown to 90% confluence in triplicate 25 cm2 flasks prior to inoculation with virus at an m.o.i. of 0.1. Virus was absorbed for 1 h at 37 °C, after which cells were washed twice with DPBS and fresh medium was added. Aliquots (50 µl) of supernatant were collected daily for 7 days p.i., added to 450 µl MEM containing 20% FBS and stored at −80 °C until titrated by Vero-cell plaque assay for quantification and plaque-morphology (3 and 7 days p.i.) assessment. Each growth-profile experiment for all cell types was replicated to assure the reproducibility of the reported findings.
A real-time cell-analysis system using electrical impedance (formerly RT-CES System; ACEA Biosciences) was utilized for quantification of CPE elicited by the respective viruses as described previously (Fang et al., 2011). Impedance was expressed as a dimensionless parameter, the cell index, representing cell status based on the change of impedance of cells relative to cell-negative control wells. Initial cell concentration was measured, and decreasing impedance related to cell death was plotted over time. Vero cells were trypsinized, washed twice with DPBS, added to wells and infected at an m.o.i. of 0.1 (six wells per virus). Real-time impedance values were measured every 15 min until day 5 p.i.
A repeated-measures ANOVA statistically compared plaque sizes, impedance values at selected time points, and growth-profile data over time among viruses, with viral means grouped by an a posteriori Tukey–Kramer multiple comparison test with α=0.05 (ncss statistical software). Peak titres for specific time points were compared in a mixed-model ANOVA. Both viruses and time points were treated as fixed effects throughout.
Funding for these studies was provided by the Pacific Southwest Regional Center for Excellence (AI065359) and by grants from NIH (AI061822, AI55607), CDC (CI000235) and the University of California Mosquito Research Program. We would like to thank Jason Velez for providing cell-culture support for these studies.
A supplementary table is available with the online version of this paper.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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C. 1990 Vintage 6-9mm Pink Coral Bead Necklace With 14kt Yellow Gold. 16"
Item#: 869949
C. 1990. Light and feminine, this necklace from our Estate collection boasts 6-9mm powder-pink coral beads. Fastens with a 14kt yellow gold lobster clasp. Pink coral bead necklace. Exclusive, one-of-a-kind Estate Jewelry.
Product Details (for C. 1990 Vintage 6-9mm Pink Coral Bead Necklace With 14kt Yellow Gold. 16")
1/4-3/8 inches
14kt close 14KT: Karat (abbreviated kt or K) is a unit that indicates the purity of gold in the metal. 14kt is 58.33% gold. In the United States, 14K yellow gold is most favored and used in jewelry. Yellow Gold close YELLOW GOLD: Gold is a precious metal with a natural yellow color. It is quite soft and often alloyed with other metals to increase its strength. The purity of the metal is indicated by a unit called a "karat," with 24kt having the highest percentage of natural gold.
Polished close POLISHED: A buffed-out finish used to produce a mirror-like shine.
Chain Size:
6-9 mm
Lobster close LOBSTER CLASP: named after the pinching style of the hook, this clasp is often shaped like a lobster's claw. Use your finger to work the lever to open the clasp.
Gemstone 1:
Coral close CORAL: This prized sea stone conjures up images of exotic oceans and faraway places. With shades ranging from soft muted pinks to vibrant bold reds, coral jewelry complements every skin complexion with a warm glow. (Color: Pink, Quantity: 58, Shape & Cutting Style: Bead, Size: 6-9 mm)
All weights and measures are approximate.
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|Reviews for Never understood
| nervousdreamwriter19 chapter 36 . 7/3
This was super entertaining and I am looking forward to future updates!
| Demonic girl111 chapter 3 . 5/14
You repeated this. Ch2 and Ch3 are the same
| Sakusha chapter 6 . 4/30
That's so cool it's like blood bending sorta
| Haha chapter 16 . 4/29
So sakura basically called suigetsu soup thingy guy. hahahahhahahahha!
| Sakuras cherry tree chapter 8 . 4/19
The 1,2,3,9 are red those tailed beasts are red
| Guest chapter 4 . 1/26
I'd say blood because cherry blossoms are fairly useless, all things considered. But blood is OP and rather cliche to top it off.
| Guest chapter 19 . 12/29/2015
In the last sentence it says 'Saku-chan' instead of what I believe to be supposed to be 'Sasu-chan'.
| Unidentified Gem chapter 36 . 12/19/2015
| Line464d chapter 36 . 12/5/2015
Pleeeeeeaaaaseeeee update this SOON! It's absolutely, amazingly, wonderfully, awesomely gorgeous! I absolutely love Gaara and Sakura's relationship and their character development! Please.. Just... Just update soon, okay? Darque, Raven? Now, maybe?
| Line464d chapter 3 . 12/4/2015
Uhm... Now we're at it, chapter 2 and 3 are the same...
| Line464d chapter 1 . 12/4/2015
Just started here, and I can already feel it will be awesome~! You're using my favorite character - Gaara - after all!
Anyway, planned on first reviewing at the last posted chapter, but... Nah
Well, what I really wanted to say was that the ages are a little mixed up, the numbers Sakura and Gaara gives Tsunade differ from the ones she has on her information-scrolls, so... Yeah, might wanna take a look at that sometime ;)
| Wolfe chapter 35 . 7/24/2015
Hey, that's my birthday too!
| iPonimeOtaku chapter 33 . 7/20/2015
How is Ami on Team Kakashi? As far as I remember, she didn't even pass the Academy Genin Test
| MidnightWhisper15 chapter 36 . 7/16/2015
ThiS is awesome! Can't wait for update xD
| KumorikoKumoriko chapter 36 . 6/21/2015
Do hope you plan to update yeah.


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GRB Detection Date: 10/03/17 04:43:54.37 UT
GRB Notice Date: Wed 17 Mar 10 04:45:13 UT
Center of Error Pattern RA: 175.059d {+11h 40m 14s} (J2000), Dec: -46.314d {-46d 18' 50"} (J2000),
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Distance to Moon: 141.03
Type of Notice (see web page for details): Swift-BAT GRB Position
Comments from GCN: SWIFT-BAT GRB Coordinates. This is an image trigger. (The RATE_SIGNIF & BKG_{INTEN, TIME, DUR} are undefined.) A point_source was found. This does not match any source in the on-board catalog. This does not match any source in the ground catalog. This is a GRB. This trigger occurred at longitude,latitude = 224.43,-5.37 [deg].
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LOL Of The Day - "Hillary for Prison" banner plane spotted over Philadelphia
Congress approves Monsanto Dark Act 2.0, destroying consumers' chances of having on-package labels
Don't let the globalists start a race war!
If you grow food in cinder blocks, you may be poisoning yourself with heavy metals
Doctor at psychiatric hospital accused of experimenting on children with 'truth serum' drug used on soldiers during WWII
Ohio moves towards mandatory vaccines, proposing to eliminate philosophical exemptions entirely
Scientists 'REVERSE' menopause...
Obama Administration Technocrats On Fire: Obama's Brain Mapping Initiative Is Racing Forward
French government orders destruction of Nice attack CCTV footage officially to prevent leaks
White House Moves To Reauthorize Military Equipment For Police
Verizon Nears Deal to Acquire Yahoo
Leaked DNC Emails Confirm Democrats Rigged Primary, Reveal Extensive Media Collusion
VIDEO: The Militarization Of Police In Cleveland
DoD Creating Virus-Fighting Viruses For Outbreak Emergency
New York Lawsuit Claims 2016 Election Was Rigged
Edward Snowden Develops Phone Case to Alert Users if Their Data is Being Compromised
In Light of Recent Attacks on Police, White House Considers Re-Militarization
Human DNA sent into Space to 'back-up' species so we're Never extinct
Twitter Executives Refuse to Answer Whether They Believe in Freedom of Speech
Kentucky woman files suit after state denies license to marry animal
Exploding Meteor Shakes Buildings in Argentina
Justice Department Lynch, sues Pennsylvania town for rejecting mosque
Verizon Set to Buy Yahoo for $5 Billion
Philly Police Union mad police victims have voice at DNC, not cops' widows
CNN posted 100 (one-hundred) videos about SANDY HOOK! WHY did CNN suddenly post 100 Sandy Hook videos in 1 HOUR??
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Results will be available for students at 8.30am on August 18th in the assembly hall.
Please see the revised school bus timetable which will take effect from Monday 5 September 2016. Revised Bus Timetable - September 2016
The buses will be leaving promptly for Blackpool after the last school bus drops students off in the morning and not 8 am as stated in the letter. Students should come directly to the Dining Hall to be registered. Buses will drop students off at Lismore Place and then Edgehill Road (Sports Centre) on the return journey.
The France trip is taking place. We are monitoring the situation and taking guidance from the Foreign & Commonwealth Office and our insurance company, both of which have no qualms about the trip going ahead. The area around Nice is the area of focus, and this is 1000km from our destination. Please contact Mrs Black or Miss Lowrey if you have any questions.
End of Term Arrangements July 2016
The last day of term is Thursday 21 July 2016. Students will leave school at 12.15 pm after an early lunch. School buses will leave school at 12.15 pm.
End of term arrangements July 2016Proposed Berlin Trip - July 2017
The school is proposing the above visit next summer which will be open to students in all year groups. Please see details on the attached letter. Berlin Visit Letter - July 2017University Application Process
Year 12 University Application Process information is attached from Head of Sixth Form - Year 12 University Application Process
We are delighted to announce that our popular Sports Awards Dinner is being held on Thursday 14 July 2016 at The Creighton Rugby Club. Students have been invited to thank them for their commitment to school sports during the past year. The evening will commence at 6.00 pm with pre-dinner refreshments followed by a buffet and awards ceremony at 6.30 pm. The evening will come to a close at 9.30 pm. There is a formal dress code.
Sports Awards Dinner - Thursday 14 July 2016
The Rewards Trip to Blackpool Pleasure Beach is on Wednesday 20 July 2016 - to qualify for this special day out your child's good work, positive attitude and behaviour throughout the year have been recognised. Please ensure the permission slip, medical form and payment is returned to Student Services by Friday 8 July 2016. The bus will drop off at Lismore Place and then Edgehill Road
Rewards Trip Letter - Blackpool - Wednesday 20 July 2016
Newman School Prom - 2016
Prom photos can be accessed by clicking on the Keltic Rose logo below.
Please find attached a letter to parents/carers outlining the school's progress to date School Progress Letter - April 2016Changes to bus information
Number 5 Route - Wigton Road - Thomson Coaches will no longer be supplying the bus for this route. Stagecoach will now run this bus service. Same times apply.
School Transport Arrangements - Update
Good news - there will be a slight change to the bus arrangements. There will no longer be a bus at 4.00 pm from school for students who are attending after school clubs. Instead students who attend will be given a mega rider which will take them directly to the nearest bus stop to their home, including transfers to another route. Tickets should be collected after the club at reception from Mrs Fisher.
School Bus Routes - Jan 2016Exam InformationSummer exam timetable has now been published and can be viewed here
Dear All
Newman is a genuinely comprehensive school. We believe that each student brings a special talent to our community. Strong pastoral care is vital to our success and creates a happy, caring atmosphere.
As a smaller school, we get to know our students well; their wellbeing is our foundation. This strong pastoral care enables us to support our students, not only with their academic work, but also in the varied extra-curricular activities which are so important as they develop. We endeavour to meet their needs as individuals, and help them to use their talents, discover new skills and to face the future with optimism.
We have high expectations of our students; we expect them to work hard, to play hard and to take a full part in the life of the school. In return, we strive to provide the best teaching and guidance in a caring and supportive environment, where students are challenged and give of their best.
Newman is a Catholic school with Christian values. Our school is open to all students, regardless of faith. We simply ask that you share our aims for high standards of discipline, trust and respect, commitment to achievement and success for all students at every level of ability.
Partnership with families is very important and we seek at all times to work effectively with parents and carers to make our students' days at Newman rewarding and successful. Students are more likely to flourish when they know their parents and the school are working together for their benefit. We invite you to choose Newman for your child's secondary education and play a vital role in this partnership.
Have a look at our after school clubs. There's a club for everyone
Here you will find some very good reasons why you should choose Newman Catholic School.
Additional Optional Clothing:
Black tracksuit bottoms, additional hoodie/waterproof jacket to go over the top of the school rugby shirt.
High Performance Badminton Club is back up and running at the Sands Centre on Wednesdays after school. Badminton club is also running at school on Thursdays and Fridays at school 3.05 - 3.55 pm.
Compulsory PE Kit:
School Rugby Shirt (All boys and girls for all lessons), Black Shorts, Red socks, Trainers for the 3G astro- turf.
Ofsted 2012
The proportion of students attaining 5 or more GCSEs at grade A* to C has risen strongly and is above the national average.
Ofsted 2012
Behaviour is good and the school is very safe.
Pastoral care is a strength of the school.


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Wolfenstein's Assassin trailer
Sneaky little blighters, assassins. Always creeping about, sticking stuff into to people.
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Pokémon Go guide, best tips and tricks Best ways to catch and level up Pokémon out in the real world.
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"In a complex global supply chain, the U.S. apparel and footwear industry will find the updated AAFA Restricted Substances List to be a helpful tool as part of an overall robust chemical management program," said AAFA Environmental Committee Chairman John Eapen, Vice President of Environmental, Health & Safety at America & Efird, Inc. "We extend our gratitude to the AAFA Environmental Committee, the members of the RSL Task Force, and our RSL peer review group for their time and significant contributions to update this important list."
"As the U.S. apparel and footwear industry continues to confront challenges posed by state, federal, and international chemical management and product safety requirements, the AAFA Restricted Substances List is just one of several tools AAFA offers to provide greater clarity," said AAFA President and CEO Kevin M. Burke. "The AAFA RSL can be a helpful resource for the industry as we pursue collaboration and harmonization of complicated rules and restrictions. We are delighted to offer this tool to the industry for free."
In an effort to foster greater harmonization among industry stakeholders, the AAFA RSL contains several new components, including the addition of Washington Children's Safe Products Act requirements in the reporting annex, a link to the updated AAFA-member only AAFA RSL Covering California Proposition 65, and a link to the AAFA List of Non-Regulated Substances Contained in Apparel Footwear International RSL Management (AFIRM) RSL.
The AAFA RSL, first released in the summer of 2007, is the first of its kind. The list covers chemicals and other substances whose presence in a product is restricted through a government regulation or law. The list identifies the most restrictive iteration of that regulation worldwide. Every six months, the publication is reviewed, updated, and the latest version is made available for free on the AAFA website.
This eleventh release has been updated to reflect changes spurred by the Washington Children's Safe Products Act, as well as other state and international regulations.
The AAFA RSL is available in four languages: English, Chinese, Vietnamese, and Spanish.
In addition to the global RSL, AAFA continues to offer training sessions and workshops on regulating restricted substances in apparel, footwear, travel goods, home textiles, and other fashion accessories, as well as on broader product safety, sustainability, and environmental compliance issues.
American Apparel & Footwear Association (AAFA)
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Chinese Film Classics Collection: An Orphan on the Streets / The Watch
Longji Wang &
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sku: QR48
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DVD Features:
- Rated: Unrated
- Run Time: 2 hours, 58 minutes
- Video: Black & White
- Encoding: Region 1 (USA & Canada)
- Released: March 4, 2008
- Originally Released: 1949
- Label: Cinema Epoch
- Packaging: Keep Case
- Aspect Ratio: Full Frame - 1.33
- Audio:
- (unspecified) - Chinese/Mandarin
- Subtitles - English
Performers, Cast and Crew:
|Longji Wang & Zhao Qiansun
|Han Mingde
|Directed by
|Zhao Ming, Yan Gong & Zuo Lin
Product Description:
AN ORPHAN ON THE STREETS: An orphan resorts to unusual tactics to find someone to look after him--by attaching a "for sale" sign to his body--in this feature film from 1949.
THE WATCH: A poor young boy named Calf steals a watch and has to face some dire consequences for his actions in THE WATCH.
THE WATCH: A poor young boy named Calf steals a watch and has to face some dire consequences for his actions in THE WATCH.
Plot Keywords:
Romance | Romances
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text:
Having been raised in a Greek culture in wilmington I was happily surprised to find a restaurant during my charleston visit that had the taste of the old country. We had a wonderful mazes appetizer, mousaka and Greek chicken. Good sized portions. We had to get a carryout box.
Charleston Greek Restaurants
1. Olympik Restaurant & BakeryMenu
This is my father's restaurant. I've eaten here more than most anyone else, and can say even with my unbiased opinion that the food is always wonder…
2. Eat A Pita
The advertising chicken wings and pulled pork....but there was none...the rest of the food was decent some rice would have gone nicely with some of …
3. Athens Greek Restaurant
Having been raised in a Greek culture in wilmington I was happily surprised to find a restaurant during my charleston visit that had the taste of th…
4. Manny's Neighborhood GrillMenu
1680 Old Towne RdCharleston, SC 29407
5. Odyssey Greek Restaurant
915 Folly RdCharleston, SC 29412
From Business: on urbanspoon.com
6. Athens ExpressMenu
Athens Greek Restaurant on James Island is the best Greek Restaurant in Charleston Period! The food is outstanding, and the wine & drink list is…
7. TabbuliMenu
2026 Savannah HwyCharleston, SC 29407
8. Olde Towne Grill & SeafoodMenu
229 King StCharleston, SC 29401
9. Athens Pizza ExpressMenu
1798 Ashley River RdCharleston, SC 29407
10. PaPa ZuZu'sMenu
Great food horrible service is my experience, If you have lots of time and like to be ignored this is the restaurant is for you. I did like the food…
11. Manny's Mediterranean CafeMenu
The tzatziki is awesome! I could make a meal of just that and pita. Plus, they deliver!
12. Doe's Pita PlusMenu
5134 N Rhett AveNorth Charleston, SC 29405
13. Nick's Gyros & Phillys
Sure you wait in line! The food is worth the wait! Where can you go these days and not wait? Wish they would put one in Goose Creek....
14. Continental Corner GreekMenu
123 W Richardson AveSummerville, SC 29483
From Business: Open for Lunch and Dinner. Sunday Brunch 11:30 AM to 2:30 PM. Visit our site for menus at www.continentalcorner.net
15. Opa CafeMenu
This establishment has a very friendly and pleasant atmosphere. The portions are almost too large! The food is fresh and well prepared and deliver…
16. Dimitrios' House of Pizza Restaurant
656 Bells HwyWalterboro, SC 29488
Showing1-16 of 16results


text:
The 11 Hottest Runway Trends Inspired By '90s Barbies
Overalls? Quirky prints? '90s Barbies wore them first.
1. Bib Overalls
3.1 Phillip Lim
Grunge meets…hip-hop? Or something.
2. Crop Tops
Just Barbie's casual workout gear.
3. Printed Pants
And Troll Doll pants at that!
4. Irridescent Fabrics
Strikingly similar, no?
Quinceañera realness.
5. Neon Hues
Diane Von Furstenberg
Note the lovely t-shirt flounces.
Paris Hilton tried to pull this look off once. It didn't work.
6. Boyfriend Jeans
G-Star Raw
Daytime Rave Barbie.
7. Quirky Prints
Breakfast florals! But of course.
Barbie took her profession(s) seriously.
8. Statement Earrings
Dolce and Gabbana
And if that's not enough…beach themed medallions!
9. Disney Character Sweater
Marc Jacobs
All that crimped hair is making Daisy Duck giddy.
10. Coordinating Separates
Diane Von Furstenberg
And why stop at matching just one outfit when you can coordinate clothes with your friends too? Double the bleached double denim fun.
11. Animals As Acessories
Tracy Reese
This is basically the plot of Rust and Bone, so Barbz clearly inspired that too.
Possibly a confusing time for Barbie.
- Hillary Clinton made her debut with VP pick Tim Kaine, who dipped into Spanish and spoke on support for immigration reform and gun control.
- The gunman who killed at least 10 people at a Munich, Germany mall on Friday was an 18-year-old "obsessed" with mass shootings, police said.
- ISIS has claimed responsibility for a bombing that killed at least 80 people in Afghanistan Saturday.


text:
Iron Kingdoms: Five Fingers—Port of Deceit (OGL)
Pathfinder Miniatures: Merisiel, Iconic Elf Rogue—Version 2
Iron Kingdoms RPG: Kings, Nations, and Gods Hardcover
Pathfinder Flip-Mat: Slum Quarter
Hero: Immortal King—The Den of Dementia
Asmodee Editions
Venture forth to explore the Den of Dementia—or see to its defense! Lead a team of adventurers, fight your way through the monster-infested corridors and face the master of the domain… Unless you decide to take that very role to try and eliminate the intruders! Hero: Immortal King is designed for two players but can also be played solitaire, and each dungeon in the series can be played independently or combined with the others.
Playing Time: about 30 minutes
Product Availability
Discontinued This product has been discontinued by the manufacturer or is no longer being carried by our distributor.
Are there errors or omissions in this product information? Got corrections? Let us know at firstname.lastname@example.com.
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Psychic bloodline sorcerer - is it still an "arcane spellcasting class"?,
so what kinda move would you like to copy from.....,
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Ghostbusters Trailer,
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The Monkey's Treefort,
Kemonomimi: Moe Races (PFRPG) PDF,
>>Ask *James Jacobs* ALL your Questions Here!<<,
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Pathfinder's Mischievous Goblins Return to Wreak Havoc on Free RPG Day in New We B4 Goblins Module,
Trapdoor Technologies Releases Officially Licensed Digital Toolset for the Pathfinder Roleplaying Game,


text:
This page is part of a frozen web archive of this mailing list.
You can still navigate around this archive, but know that no new mails
have been added to it since July of 2016.
Click here to be taken to the new web archives of this list; it includes all the mails that are in this frozen archive plus all new mails that have been sent to the list since it was migrated to the new archives.
Subject: Re: [OMPI devel] non-portable code in examples/Makefile
From: Jeffrey Squyres (jsquyres_at_[hidden])
Date: 2012-02-21 06:47:21
On Feb 21, 2012, at 6:39 AM, TERRY DONTJE wrote:
>> Heads up that in the upcoming fortran revamp, we *only* use FC. I.E., there's only mpifort wrapper compiler (mpif77 and mpif90 still exist, but only as sym links to mpifort, signifying that mpifort is the way of the future).
>> This was done because there have been no f77 compilers for decades (literally), and no f90 compilers for 10+ years. All the fortran compiler vendors have long-since moved to a single compiler executable name (e.g., ifort, gfortran), so mpifort just reflects that.
> Hmmm, well Oracle's compiler is still named f90 :-). (now to duck and cover)
Yes, multiple vendors still have <foo>f90 (and/or <foo>f77)-named compilers. But these are just multiple entry points to a common back end, usually for legacy reasons (just like we'll still have mpif90 and mpif77).
Another fun fact: MPI-1 was never compliant with Fortran 77. The most obvious/easiest point to cite is that F77 only allowed 6-character variable and subroutine names. :-)
Jeff Squyres
For corporate legal information go to: http://www.cisco.com/web/about/doing_business/legal/cri/


text:
Prolimatech MK-26 Video Card Cooler Review
Final Thoughts
There are a couple of issues I want to address up front, but oddly they aren't about the MK-26, it is with what you plan to use this cooler in that is going to be an issue. Most cases are wide enough to take on a 160mm CPU cooler, but the problem here is that once on the video card, the MK-26 stands taller than that, so be sure your case has a ton of room or you run open air as I do. The next issue that needs addressed is the combined weight of the cooler and fans. You can shave a few grams with a selective fan choice, but again if you are running in a standard case, expect a bit of droop on the right side of the video card as the PCB suspends all that weight.
Size and weight out of the way, we can now focus on the MK-26 and its performance. While I did choose some really great fans to cool the MK-26 with, the level of performance is what you would expect from a cooler this big. Once we loaded the video card, in both situations, the results were at least 20 degrees better than the stock solution. What really sells me on the whole idea of the MK-26 is that it was super simple to install. The cooler stands tall enough to allow me to use the stock cooling solution which is also a PCB support rail. So I got the best of everything, including a way to limit the droop that this cooler is bound to cause other video cards.
As I mentioned earlier in the review, the only thing that may hurt the success of the MK-26 is the actual price you will be able to get it at once it hits the US shores. Doing the basic conversion, and if you want to use the fans I chose, this setup is going to be near $110 nearest I can figure. That fact alone is going to keep the MK-26 out of the average Joe's hands and be a selection for the wealthier elitists out there. I will say this for the MK-26 from Prolimatech though; you do get exactly what you pay for with a cooler like this. It offers a very serious foundation for superior air cooling, and regardless of the fan speed recommendations on their site, I was able to easily handle the heat load of the HD 7950 with 2000 RPM fans with enough static pressure to be sure to get all the way through to the heat sinks and the PCB.
If I was in the market to air cool a single GPU video card, the MK-26 is the choice of all choices to make, but if we are looking at cooling a multi-GPU setup, this four slot cooler isn't what you are looking for.
PRICING: You can find products similar to this one for sale below.
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- Page 1 [Introduction]
- Page 2 [Specifications, Availability and Pricing]
- Page 3 [Packaging]
- Page 4 [Prolimatech MK-26 Multi-VGA Cooler]
- Page 5 [Accessories and Documentation]
- Page 6 [Installation and Finished Product]
- Page 7 [Installation and Finished Product Continued]
- Page 8 [The Test System and Thermal Results]
- Page 9 [Final Thoughts]
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- MSI announces custom GeForce GTX 1060 graphics cards
- Seagate unveils the industry's broadest 10TB portfolio


text:
Southern District of New York Cases
Cases filed
Cases 1 - 10 of 132,089
Johnson v. Warden, Attica Correctional Facility
as 1:2016cv05977
Cause Of Action: Petition for Writ of Habeas Corpus (State)
Case name Not on File
as 1:2016cv05992
Cause Of Action: Diversity Action
Rodriguez v. Delgado's Deli & Grocery et al
as 7:2016cv05976
Cause Of Action: Fair Labor Standards Act
Type: Labor › Labor: Fair Standards
3M Company v. HSBC Bank USA, N.A.
as 1:2016cv05984
Cause Of Action: Diversity Action
Eastern Savings Bank, FSB v. Thomas et al
as 7:2016cv05978
Cause Of Action: Diversity-(Citizenship)
as 1:2016cv05986
Cause Of Action: Venue
Eissenberg v. Pioneer Credit Recovery, Inc.
as 7:2016cv06010
Cause Of Action: Fair Debt Collection Act
Cardenas v. Blessed Forever, Inc. et al
as 7:2016cv05993
Cause Of Action: Fair Labor Standards Act
Type: Labor › Labor: Fair Standards
Sony Corporation et al v. Fujifilm Holdings Corporation et al
as 1:2016cv05988
Cause Of Action: Constitutionality of State Statute(s)
Plaintiff v. Defendant
as 1:2016cv05991
Cause Of Action: Diversity: Notice of Removal
Type: Civil Rights › Civil Rights: Jobs
Disclaimer: Justia Dockets & Filings provides public litigation records from the federal appellate and district courts. These filings and docket sheets should not be considered findings of fact or liability, nor do they necessarily reflect the view of Justia.


text:
|Use in Moderation / The Aftermath
|Bartle Bogle Hegarty
|Production Company
|Biscuit Filmworks UK
|Editing Company
|Own the Week-End 2
|Guardian Media Group
|The Guardian
|Date of First Broadcast/Publication
|Subscribers Only
|Business Sector
|Subscribers Only
|Subscribers Only
|Post Production
|..e M..l L...on Subscribers Only
|7...ph Subscribers Only
|Executive Creative Director
|Mr. David Kolbusz
|Creative Director
|W...ey H...s Subscribers Only
|Creative Director
|G..y Mc.....ie Subscribers Only
|Art Director
|AK Parker
|G...ge Br....ll Subscribers Only
|J..f L..e Subscribers Only
|Production Company Producer
|Mr. Kwok Yau
|Director of Photography
|.d W..d Subscribers Only
|Executive Producer
|O....do W..d Subscribers Only
|Agency Producer
|Ms. Rachel Hough
|Agency Producer
|D...d L...h Subscribers Only
|Account Manager
|F...a B....ry Subscribers Only
|Account Manager
|Mr. Jonny Price
|Post Production
|..n T...er Subscribers Only
|M..k B....tt Subscribers Only
|Mr. Sam Ashwell
|Advertising Manager
|R....rd F....ss Subscribers Only
|Advertising Manager
|Ch.....te Em....on Subscribers Only
|Advertising Manager
|Jenny Heeley
|Advertising Manager
|C...e C...ey Subscribers Only
|Chief Strategy Officer
|J...n Go.....es Subscribers Only
|Strategic Business Lead
|Ms. Zara Mirza
|Strategy Director
|A...he Gu....er Subscribers Only
|A...a K..g Subscribers Only
|Team Director
|Mr. Jon Barnes
"When the world zigs, zag"
This philosophy was born out of the first print execution we ever produced back in 1982 to launch black Levi's. It became something of a model for our own business…and how we approach Client's problems always seeking to challenge the prevailing or conventional wisdom.
The philosophy has created moments in our own history when we decided to go against the grain of our industry. We were the first agency to become financially transparent. We were the first agency to create a micro network to serve global business. We were the first agency to recognise the need for channel planning to be at the heart of all creative development so we created a discipline called Engagement planning.
Engagement planning, not to be confused with media planning, is focused on understanding the consumer journey, bringing clarity to the roles different channels play in the marketing mix, identifying how brands can be built through interaction and participation, and most importantly using disruptive channel thinking to get to bigger ideas that deliver a step change in communication effectiveness.
At BBH, we believe in one central belief; that big, enduring ideas are the key to successful communications that create value for our Clients. It was true in 1982. It is even more true today. And in an increasingly complex and competitive world, it will be truer still tomorrow. So everything we do and all our structures support that single thought.


text:
The Scarlet Car (Silent)
Franklyn Farnum,
Edith Johnson &
Lon Chaney
Joseph De Grasse
Starring: Franklyn Farnum, Edith Johnson & Lon Chaney Director: Joseph De Grasse
Only 4 copies left
|List Price:
|You Save:
|$4.03 (21% Off)
Available: Usually ships in 1-3 business days
Format: DVD-R
sku: GRVN 0031D
Brand New
DVD Features:
- Rated: Not Rated
- Run Time: 57 minutes
- Video: Black & White
- Released: June 21, 2011
- Originally Released: 1917
- Label: Grapevine Video
- Encoding: Region 0 (Worldwide)
Performers, Cast and Crew:
|Franklyn Farnum, Edith Johnson & Lon Chaney
|Lon Chaney
|Edith Johnson
|Directed by
|Joseph De Grasse
Description by OLDIES.com:
Billy Winthrop (Franklyn Farnum) is the idle son of Samuel Winthrop (Al Filson). When Billy discovers that it's destroying his father financially to constantly get him out of trouble, he straightens up. Although Billy loves Beatrice Forbes (Edith Johnson), she is already engaged to marry Ernest Peabody (Sam deGrasse), the son of banker Cyrus Peabody (Howard Crampton). Billy and Beatrice plan to elope, but it isn't as easy for them to run off together as they thought it would be. They get tangled up in situations involving forgery and murder that need to be solved before they can get married.
Product Description:
When Chaney discovers his boss stealing from the bank coffers, he accuses him. A fight ensues, and the boss is inadvertantly killed. Fleeing, Chaney slowly goes mad while hiding in a remote cabin. Silent.
Production Notes:
- A silent film.
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text:
Behind the targeting of black shoppers
African Americans have $1 trillion in buying power
October 8, 2013
African Americans consume a lot of media compared to the average person, and they're particularly partial to media that reflects their personal experience, such as cable channels aimed at blacks or blogs with black writers. Yet advertisers are putting only a small fraction of the money spent on advertising toward black media. That's one finding of a new study from Nielsen examining African Americans' buying habits and influence. It finds that this group wields $1 trillion in buying power, and it's a number that will grow to $1.3 trillion by 2017. Meanwhile, blacks wield a lot of influence over popular culture, in the view of other groups, including whites and Hispanics. And blacks spend a huge amount of time with media, with African-American women watching more television than any other group. Yet advertisers seem to be missing opportunities to target this minority group. Just $2.24 billion of the $75 billion spent on TV, magazines, radio and digital ads last year was funneled to media focused on black audiences. Cheryl Pearson-McNeil, senior vice president of public affairs and government relations at Nielsen, talks to Media Life about how buyers can target blacks more effectively, what ad categories target this group most frequently, and why blacks influence other groups' buying power.
What did you find most surprising or most interesting about this report?
We were pleased to see the diversity in African-Americans' online digital behavior.
From the amount of time spent on education and career web sites to family and lifestyle web sites to the corporate information that blacks seek out online–it's not something that gets talked about often when a story is being told about black consumers.
What can media buyers and planners take from the report?
Some companies believe that, because there are no language barriers, a general market "one-size-fits-all" strategy is an effective way to market to African-Americans. But just the opposite is true.
It is not language that yields connectivity with blacks but rather a brand's ability to understand the cultural nuances that resonate with blacks. Blacks tend to be more receptive to messages when they feel valued, so incorporating media that focuses on culturally relevant themes among African-Americans is a great way to add value to an existing campaign.
How does African Americans' buying power compare to other minority groups, such as Hispanics?
Seventy-three percent of whites and 67 percent of Hispanics say blacks influence mainstream cultures, so the black influence extends beyond the group's $1 trillion annual buying power, because they have the ability to impact how other cultures spend as well.
How are African Americans more aggressive consumers of media than the average consumer?
Blacks watch more television (37 percent more), make more shopping trips (eight more), purchase more ethnic beauty and grooming products (nine times more), read more financial magazines (28 percent more) and spend more than twice the time at personal web sites than any other group.
What's the significance of the fact that African Americans spend more than twice the time at personal hosted websites than any other group?
It's important for marketers and advertisers to understand that this consumer segment is not monolithic. By understanding, for example, that blacks like to blog, or post, or own their own web sites points to how influential African-Americans are with other cultures and that they appreciate having a voice on matters and issues that are of importance to them.
Blacks like being able to control the story that is being told about them. So often it's not told in a positive light by others.
Are African Americans more or less brand loyal than other minority groups or whites? How receptive are they to trying new things?
Blacks spend 18 percent of their annual retail dollars on store brands, otherwise known as private-label items. While this behavior is similar to other consumer groups, there are certain categories that blacks prefer to be store brand that other consumer segments aren't as store brand loyal to–like frozen prepared foods, frozen vegetables, bottled water, juices and drinks, nuts and sugar or sugar substitutes.
Why is so little advertising focused on black audiences?
Some companies believe that they can just as easily reach blacks through general-market advertising.
But when we consider the fact that 81 percent of blacks believe that products advertised using black media are more relevant to them, we see that there's a disconnect between that belief and the fact that only 3 percent of all television, radio, magazine and internet advertising dollars were actually spent with media focused on black audiences in 2012.
Imagine how much potential growth companies leave on the table when they don't attempt to reach African Americans through black media with creative concepts that resonate with black audiences.
Which media are more likely to focus on advertising to this group?
Some companies are doing it right. The industries that are advertising the most with media that focus on black audiences are quick service restaurants, automotive, motion pictures, telephone services and insurance companies.
The top five advertisers with media focused on black audiences are Procter & Gamble, L'Oreal, McDonald's, Unilever and the U.S. government.
Tags: advertising, advertising to minorities, african american shoppers, african americans, black shoppers, blacks, minorities, nielsen, research, studies
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How Nielsen is beefing up its ad viewability options
NBC tops Fox and CBS combined on Wednesday
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- Jeff Davidoff becomes president at Walton Isaacson
- Renato Fernandez and Doug Menezes rise at TBWA
- Isaac Mizrahi becomes chair-elect at AHAA
- John Solaro and Roger Tsai join Disney|ABC Television Group
- Sarah Geismer becomes VP of comedy at 20th Century Fox TV
- Diego Rodriguez becomes chief security officer at Univision
- Chris Mancivalano becomes associate marketing director at People en Español
- Meghan Bentley becomes VP of business development at Tapad
- Damian Hurley joins the cast of E!'s 'The Royals'
- Malik Yoba joins ABC's 'Designated Survivor'
This week's broadcast ratings
This week's cable ratings
This week's top-rated movies, songs and books
This week's daypart ratings
This month's digital traffic data: May 2016
This month's new media traffic data
Needed: Print media buyer in Chicago
Digital media buyer wanted in El Segundo
Media buyer wanted in Austin
Digital media planner wanted in Salt Lake City
Direct mail media planner job in Minneapolis


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...Please review my student feedback and subject list and feel free to contact me to schedule a tutoring session if you need any assistance in the subjects that I tutor.I earned my MBA from UCLA Anderson School of Management in June 2009. The MBA covers the required materials for Business, Accounti...
Carson, CAView Tutor
I am a graduate with a Master's of Chemical Engineering degree from the University of Louisville in Kentucky. During my years there, I graded math tests in the engineering school ranging from pre-calculus to Calculus 1-3 as well as the higher courses of Differential Equations and Numerical Methods. I also provided tutoring in those classes.
Burbank, CAView Tutor
...Here's a little about myself and my style of teaching: My first tutoring job came to me unexpectedly during high school. I saw a fellow student named Raj in the library after school, cursing and pounding his fists at his chemistry textbook while trying and ultimately failing at a certain proble...
Los Angeles, CAView Tutor
...Thankfully, I had taken two years of college mathematics at Harvey Mudd College while still in high school, which enabled me to complete my double-major and still study abroad my entire junior year of college. In addition to my two years of math at Harvey Mudd, I also completed an entire year of...
Los Angeles, CAView Tutor
...I have experience tutoring in all mathematics subjects up to and including graduate-level material. I have a massive amount of experience in tutoring almost all of the subjects that I'm qualified to teach or tutor. Thus, it is very difficult to find another tutor who has as much experience in t...
Long Beach, CAView Tutor