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Genotype I (GI) Japanese encephalitis virus (JEV) that replaced GIII virus has become the dominant circulating virus in Asia. Currently, all registered live and inactivated JEV vaccines are derived from genotype III viruses. In Taiwan, the compulsory JEV vaccination policy recommends that children receives four doses of formalin-inactivated Nakayama (GIII) JEV vaccine.
Methodology/Principal Findings
To evaluate the influence of genotype replacement on the post-vaccination viral neutralizing ability by GIII and GI viruses, the small panel of vaccinated-children serum specimens was assembled, and the reciprocal 50% plaque-reduction neutralizing antibody titers (PRNT50) were measured against Nakayama vaccine strain, CJN GIII human brain isolate and TC2009-1 GI mosquito isolate. The seropositivity rate (PRNT50≥1∶10) and geometric mean titers (GMT) against the TC2009-1 virus were the lowest among the three viruses. The protective threshold against the CJN and TC2009-1 viruses could only be achieved when the GMT against Nakayama virus was ≥1∶20 or ≥1∶80, respectively. Using undiluted vaccinees' sera, the enhancement of JEV infection in K562 cells was observed in some low or non-neutralizing serum specimens.
Our preliminary study has shown that neutralizing antibodies, elicited by the mouse brain-derived and formalin-inactivated JEV Nakayama vaccine among a limited number of vaccinees, have reduced neutralizing capacity against circulating GI virus, but more detailed studies are needed to address the potential impact on the future vaccine policy.
Author Summary
Genotype I (GI) Japanese encephalitis virus (JEV) that replaced GIII virus has become the dominant circulating virus in Asia; however, all available JEV vaccines are derived from genotype III viruses, and no study has been conducted on the cross-neutralization and protection elicited by GIII JEV vaccines against GI viruses using vaccinated children's serum specimens collected from the general population. Genotype I virus was first detected in Taiwan in 2008, and became the dominant circulating JEV, and was island-wide within a year. In the present study, the small panel of GIII virus vaccinated-children serum specimens were not only showed lower strain-specific neutralization against GI virus as compared to the GIII vaccine and human isolates but also observed the enhancement of GI virus infection in K562 cells in some low or non-neutralizing serum specimens. These preliminary results indicated the reduced neutralization potency due to genotype replacement should be closely monitored in the JE epidemic/endemic regions in the future.
Citation: Fan Y-C, Chen J-M, Chiu H-C, Chen Y-Y, Lin J-W, Shih C-C, et al. (2012) Partially Neutralizing Potency against Emerging Genotype I Virus among Children Received Formalin-Inactivated Japanese Encephalitis Virus Vaccine. PLoS Negl Trop Dis 6(9): e1834. https://doi.org/10.1371/journal.pntd.0001834
Editor: Sunit Kumar Singh, Centre for Cellular and Molecular Biology, India
Received: May 25, 2012; Accepted: August 13, 2012; Published: September 27, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was supported by the grant NSC 98-2313-B-005-014-MY3 from the National Science Council, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
South and Southeast Asia are Japanese encephalitis (JE) endemic areas in which approximately 10% of the susceptible populations are infected with JE virus (JEV) each year, based on the ratio of asymptomatic to symptomatic infections of 200 to 1 [1], [2], [3]. The most cost-effective control strategy for JE is vaccination, and there are several licensed vaccines, including live-attenuated, chimeric live-attenuated and inactivated SA14-14-2; inactivated Nakayama; P3 and Beijing-1 vaccines [3], [4], [5], [6]. In Taiwan, compulsory vaccination has been implemented since 1968 using the mouse-brain derived and formalin-inactivated Nakayama vaccine, and since then clinical JE cases have decreased dramatically to 20–30 cases each year [7]. It has been estimated that vaccine effectiveness is in the range of 85% to 90% after immunization with two doses of inactivated Nakayama vaccine [7], [8].
We have witnessed dramatic changes in the molecular epidemiology of circulating JEV in the past two decades. Historically, genotype III (GIII) viruses were the most widely distributed JEV in South and Southeast Asia [9]. However, genotype I (GI) JEV, having emerged in the 1970s in Thailand/Cambodia, has replaced GIII as the dominant circulating virus in JE endemic/epidemic regions since the 1990s [10]. Genotype I viruses first appeared in Japan, and by the 1990s the majority of Japanese JEV isolates belonged to GI [11]. Subsequently, the phenomena of genotype replacement were observed in many countries, including Korea, Vietnam, Thailand, and China [12], [13], [14]. Genotype I JEV was first detected in Taiwan in 2008, and became the dominant circulating genotype island-wide within a year [15], [16].
The nucleotide and amino acid variation between the envelope (E) glycoproteins of GIII and GI JEV is in approximately 12% and 3%, respectively [9]. All licensed JEV vaccine strains, including SA14-14-2, Nakayama, P3, and Beijing-1, belong to GIII. The reduced capacity of neutralizing antibody against field-isolated GIII viruses had been reported among vaccinated human serum samples [17], [18]. Thus, strain-specific neutralizing antibodies elicited by GIII JEV vaccines in vaccine recipients need to be assessed against GI virus. The protective efficacy of inactivated JE-VAX (suckling mouse brain-derived Nakayama vaccine) and P3, and live-attenuated SA14-14-2 vaccines has been evaluated in a mouse model. Beasley et al. have shown that mice that received JE-VAX vaccine or were passively transferred JE-VAX-vaccinated mouse sera had lower neutralizing antibody titers and were less protected against GI virus as compared to GIII virus, but the strain-dependent protection could not be excluded [19]. However, Liu et al. showed that the live-attenuated SA14-14-2 and inactivated P3 vaccines protected vaccinated mice equally against GIII and GI viruses [20]. In a series of ex vivo studies, Van Gessel et al. eloquently demonstrated that mice receiving passively transferred immune sera collected from adult human volunteers vaccinated with JE-VAX or SA14-14-2-derived IC51 (tissue culture-derived inactivated) vaccine were protected against GIII and GI viruses, and firmly established for the first time that the strain-specific correlate of protection (PRNT50≥10) could be accurately estimated by measuring the reciprocal neutralizing antibody titer at the time of viral challenge [21].
The cross-neutralization and protection elicited by GIII JEV vaccines against GI viruses are not consistent in mouse models. More importantly, no study has been conducted using vaccinated children's serum specimens collected from the general population. In the present study, the panel of specimens collected from children immunized with mouse brain-derived and formalin-inactivated Nakayama vaccine in Taiwan were assembled and used to assess the potency of neutralizing antibodies against the vaccine strain, a GIII local human isolate, and a newly introduced GI mosquito isolate.
Materials and Methods
Amino Acid Analysis
The nucleotide and amino acid sequences of JEV GI, GIII, and Nakayama viruses were retrieved from GeneBank and analyzed using BioEdit version 7.1.3 [16], [22]. To localize the amino acid substitutions, the protein structure of JEV E glycoprotein was downloaded according to recent report [23] and analyzed by Swiss-Pdb Viewer 3.7 structure analysis software. The antibody-accessible amino acids should have at least a 35% surface accessibility potential based on the result of structure analysis [24]. In order to assess the impact of amino acid substitutions on the impact of E protein, the stability calculation was performed for all amino acid substitutions by using the Prediction of Proteins Mutations Stability Changes server (http://babylone.ulb.ac.be/popmusic/index.html).
Study Subject
In Taiwan, a compulsory vaccination program was implemented in 1968 which utilizes mouse-brain derived and formalin-inactivated Nakayama vaccine. Children receive an initial dose of vaccine at one-and-one-half-years of age, and subsequent doses two weeks later, one year later, and in the first grade of elementary school. We also collected serum samples from children who received varying numbers of doses of vaccine and at different time post final booster vaccination.
Ethics Statement and Human Sera
All the serum samples used in this study were collected from an already-existing collection in two hospitals, the Mennonite Christian Hospital in Hulian and the Tungs' Taichung Metroharbor Hospital in Taichung, in 2010 and were anonymized. For serum sample collection, the clinical protocols were reviewed and approved by the institutional review boards of these two hospitals (10-03-007-ER and 99006). Serum was obtained from whole blood after clotting at room temperature and then centrifuged at 3,000 rpm for 10 min. The aliquot of serum was stored at −20°C until use.
Serological Assay
Five JEV strains were used in the serological assay, including: the GIII, cluster III Nakayama vaccine strain; the GIII, cluster I CJN strain isolated from human brain in 1998; the GIII, cluster II T1P1 strain isolated from a mosquito pool in 1997 [15]; and the GI, cluster I TC2009-1 strain and GI, cluster II YL2009-4 strain isolated from mosquito pools in 2009 [16]. Viruses were amplified in C6/36 mosquito cells and stored in aliquots at −80°C until use.
Plaque reduction neutralization tests (PRNT) are the most suitable method for assessing neutralizing antibodies against JEV. The PRNT protocol used is similar to that in our previous report with some modification [17]. The BHK-21 cells were dispensed into each well of 6-well polystyrene plates (Costar, Cambridge, MA, USA). The plates were incubated at 37°C for 36 h to form a monolayer. Serum samples were inactivated at 56°C for 30 min before a two-fold serial dilution was carried out. A target dose of about 100 plaque forming units (PFUs) of JEV were then incubated with the previously diluted serum samples. The mixture of diluted test serum and control virus was added onto the BHK-21 monolayers. After adsorption for 1 h at 37°C, the infected cells were overlaid with 4 ml/well of 1.1% methyl cellulose (Sigma) in MEM containing 2% FBS and 1% antibiotics. After an additional incubation for 3.5–4 days, the cells were fixed with 10% formalin and stained with 1% crystal violet. The PRNT titer was obtained from the reciprocal of the dilution of serum that reduced the plaque number by at least 50% relative to the virus-only control.
To determine the potential for antibody-dependent enhancement (ADE) of different genotypes of JEV, K562 cells were used to measure viral yield and infection rate [25]. The undiluted serum samples were used in the assay. Briefly, 100 µl serum was mixed with 2×105 K562 cells and 2×104 PFUs virus (MOI = 0.1), and incubated with gentle rotation at 37°C for 2 h. After being washed twice, the cells were incubated with RPMI-FBS medium at 37°C for one day. The culture supernatants were collected and the viral yield was determined by plaque count using BHK-21 cell monolayers; also, infected K562 cells were collected, stained with mouse anti-JEV HIAF, and the cell infection rate was estimated by flow cytometry. The monoclonal antibody 4G2 (obtained from Dr. Chang GJ of US CDC, Fort Collins, CO) was diluted 1∶100 and was used as the positive ADE control.
A cut-off value to determine seropositivity of neutralizing antibody titer was defined as PRNT50≥10. Seropositive subjects were defined as those having a reciprocal antibody titer above or equal to the cut-off value; seronegative subjects, those falling below the cut-off value. Antibody titers below the cut-off value of 1∶10 were given an arbitrary value of 5 for geometric mean titer (GMT) calculation.
The cut-off value for virus yield and cell infection rate for differentiating neutralization and enhancement was calculated from the average of five repeat measurements plus two times the standard deviations (SD) of the viral yield and cell infection rate obtained with the negative controls. In the negative controls, RPMI-FBS was substituted for the serum specimen in the virus–serum–cell preparation. Virus yield and cell infection rate of at least 2SDs above the negative control was determined to represent a statistically significant event.
Student's t rest was used for all analyses, and statistical significance was defined as a P value <.05.
Amino Acid Analysis
The GIII Nakayama strain has been used exclusively for the manufacture of formalin-inactivated suckling mouse brain-derived human vaccine in Taiwan since 1968. An extensive field investigation and molecular epidemiological study outlined in our previous report indicates that the replacement of JEV GIII by GI occurred in 2009 in Taiwan [16]. JEV envelope (E) protein is the primary antigen eliciting protectively neutralizing antibodies. The amino acid differences in the E protein region between Nakayama vaccine strain and GI and GIII sequences of virus isolated from Taiwan are shown as Table S1. Amino acid differences in the E protein between the vaccine strain and strains used in this study are highlighted in Figure 1. There are eight amino acid differences between Nakayama and the human brain isolate GIII CJN virus, including E protein amino acid positions 33, 51, 83, 176, 227, 242, 276, and 290 (Figure 1). Among these residues, position 33, 176, and 290 are located in E domain I (EDI); 51, 83, 227, 242, and 276 are located in EDII; and none are in EDIII. There are 13 amino acid variations between Nakayama and mosquito isolate GI TC2009-1 virus, including E protein amino acid positions 33, 51, 83, 123, 129, 176, 222, 227, 242, 276, 290, 327, and 366 (Figure 1). Among these residues, positions 33, 176, and 290 are located in EDI; 51, 83, 123, 129, 222, 227, 242, and 276 are located in EDII; and 327 and 366 are located in EDIII. The unique differences distinguish the CJN and GI strains from the vaccine Nakayama strain were at E protein amino acid positions 33, 51, 83, 176, 227, 242, 276, and 290.
Amino acid variations between the Nakayama vaccine strain and the GIII brain-isolated CJN strain are indicated in blue, and variations between the Nakayama vaccine strain and the GI field-isolated TC2009-1 strain are indicated in both blue and red.
Strain-Specific Neutralizing Antibody
A pilot experiment was conducted to determine the difference of neutralizing antibody titers against five JEVs, GIII Nakayama (cluster III), GIII T1P1 (cluster II), GIII CJN (cluster I), GI TC2009-1 (cluster I), and GI YL2009-4 (cluster II), were evaluated using the small panel of serum samples (Figure S1) [15], [16]. The correlation of PRNT50 between T1P1 and CJN or TC2009-1 and YL2009-4 was 0.75 and 0.92 (Figure S1), respectively. Due to insufficient amount of some serum samples, the GIII Nakayama, GIII CJN, and GI TC2009-1 viruses were selected as viral strains for further analysis.
A total of 157 serum samples from vaccinated children were collected and grouped based on age (0–15 years) and dosage of vaccination (0–4 doses) at the time of sampling (Table 1). The seroprotection or seropositivity rate, defined by the PRNT50≥10 using BHK-21 cells against homologous Nakayama strain, was 66.7% after primary vaccination, peaking after the receipt of the 4th dose of vaccine (80.0% for 8–9-years-old), but decreased gradually to 60.0% in children aged 14–15 years (Figure 2). The trend in strain-specific seroprotection rate against the GIII CJN strain was similar to that of Nakayama virus, but the seroprotection rate themselves were generally lower (ranged from 0 to 21.5%) as compared to those for the Nakayama strain. The seroprotection rate against GI TC2009-1 virus were much lower as compared to other two viruses, especially in the 14–15-years-of-age group (P<.05, Figure 2).
Seropositivity is defined by a reciprocal titer of ≥10 in a 50% plaque-reduction neutralization test using BHK-21 cells. Significantly different (P<.05) seropositivity rates against CJN or TC2009-1 as compared to that for Nakayama virus are indicated by asterisks.
The geometric mean titers (GMT) of strain-specific PRNT50 against the GIII vaccine and CJN viruses and the TC2009-1 GI virus are shown in Table 1. The GMT of neutralizing antibodies against homologous Nakayama strain was 25.2 after primary vaccination and reached a peak (38.0) following the third dose of vaccine, but decreased gradually after the final booster and dipped below the protective threshold of 10 in children aged 14–15 years. The strain-specific GMT of neutralizing antibodies against the GIII CJN strain trended similarly to those against the Nakayama virus, but the titers were lower. Interestingly, the GMT of antibodies neutralizing field-isolated GI TC2009-1 virus were significantly lower than those for the other two viruses (P<.05) and the titers were below the presumptive protective threshold of 10 with the exception of children aged 2.5–4 years who had received a third dose of vaccine.
Protective Threshold for GI Virus
Neutralizing antibodies elicited by the mouse brain-derived, formalin-inactivated Nakayama vaccine could be protective against circulating GIII and GI virus in Taiwan (Figure 2 and Table 1). Serum specimens capable of neutralizing Nakayama virus were selected and stratified into groups with PRNT50 titers of 10, 20, 40, 80, 160, and ≧320. The strain-specific GMT from each group was calculated for the human brain GIII CJN and mosquito GI TC2009-1 viruses (Table 2). The grouping results suggest that the GMT reach the presumptive protective threshold (PRNT50 = 10) against CJN and TC2009-1 viruses when the neutralizing titer against Nakayama virus is 1∶20 or 1∶80, respectively.
Potential of Antibody-Dependent Enhancement of Infection
Antibodies elicited by the inactivated Nakayama vaccine are less potent in neutralizing GI virus, and the majority of vaccinees' PRNT50 titers are below the protective threshold (Table 2). The enhancement of virus infection resulting from vaccination has been suggested [26]. Thus, 26 serum samples from vaccinated children were selected, and the undiluted serum samples were used to estimate the potential for ADE of GI JEV infection in K562 cells by measurement of viral yield (Figure 3). The three serum samples most highly neutralizing against Nakayama (PRNT50≧80) also strongly inhibited the infectivity of GI JEV. Of the 16 weakly neutralizing serum samples (PRNT50 = 10 to 40 against Nakayama), only three exhibited some increase in virus yields as compared to the control serum. However, most (6/7) of the non-neutralizing serum samples (PRNT50<10 against Nakayama) enhanced GI virus infection.
The magnitude of enhancement of viral yield is calculated as follows: Log10 (serum-treated viral titer/untreated viral titer). The K562 cells infected with labeled samples C86, TC36, B19, D7, D15, 113, I5 and I6, were subjected to infection rate analysis by flow cytometry (Figure 4).
The risk of ADE of GI JEV infection in K562 cells was also analyzed by flow cytometry to determine the infection rate of cells infected with serum-treated virus. The results of selected samples, not all samples were included due to insufficient amount individual serum, are shown in Figure 4. At an MOI = 0.1, the untreated K562 infection rate by TC2009-1 was 6.9%. Thus, an infection rate of less than 6.9% can be interpreted as neutralization, and greater than 6.9% as enhancement. The flavivirus group-cross reactive murine monoclonal antibody 4G2, used as an ADE control at a 1∶100 dilution, resulted in an infection rate of 20.5%, significantly higher than that of the virus control. The T36 (PRNT50 = 20) and C86 (PRNT50 = 160) samples neutralized GI virus, and the cell infection rate were reduced to 1.4–1.5%, significantly lower than that of the virus control (P<0.05). The B19 serum neutralized Nakayama and CJN viruses, but not TC2009-1 virus. Treatment with B19 serum resulted in some degree of enhancement of TC2009-1 infection (10.5% infection) (P>.05). Sera I6, 113, D7, D15, and I5, non-neutralizing against all three viruses, showed significant enhancement of GI JEV infection (P<.05).
The virus control is GI TC2009-1 virus-infected K562 cells in the absence of serum samples. The ADE of TC2009-1 virus in K562 cells by using 1∶100 diluted 4G2 monoclonal antibody as a positive ADE control and the serum samples collected from vaccinated children including C86, TC36, B19, I6, 113, D7, D15, and I5. The K562 cells were collected, stained with mouse anti-JEV HIAF, and analyzed for infection rate by flow cytometry.
In addition, we conducted an ex vivo ADE experiment with vaccinees serum samples, and the result was shown in Supplemental Figure 2. Due to the availability of vaccinee's serum, the undiluted serum from TC36, C86, D7, and I5 only were preincubated with 1000 PFU of TC2009-1 virus and injected intraperitoneally into suckling mouse (four mice per serum sample). The brain of infected suckling mouse was collected at four days post-inoculation; and the virus titer was determined by the plaque forming assay in BHK21 cells. The TC36 and C86 serum samples almost completely neutralized TC2009-1 virus replication in suckling mouse. However, compared to virus control, the mouse brain virus titers were significantly increased to 5.53- and 5.79-fold when the TC2009-1 virus was pre-incubated with D7 or I5 serum samples, respectively. The result of this ex vivo ADE experiment was consisted with the result of flow analysis (Figure 4).
Long-term evolution, geographical barriers and host-parasite interactions have resulted in many flaviviruses evolving into multiple genotypes, including dengue virus serotypes 1 through 4, West Nile virus, and JEV [27], [28], [29], [30], [31], [32]. Genotype replacement in dengue virus serotypes 3 and 4 in Sri Lanka and Puerto Rico, respectively, has resulted in increased transmissibility and epidemic potential of the new viruses [33], [34]. Vaccination is the most effective strategy to control flavivirus epidemics [35]. Genotype I JEV has replaced GIII as the dominant genotype throughout Asian countries, and human clinical cases due to GI virus infection have been observed in China [10], [36]. The GIII mouse brain-derived, inactivated Nakayama vaccine and live-attenuated SA14-14-2 vaccine are the most widely distributed vaccines in Asian countries, including Japan, Korea, Taiwan, Vietnam, Thailand, Malaysia, China, India, and Nepal [3], [4]. The replacement of GIII by GI virus provides the opportunity to evaluate the contribution of genotype replacement to the strain-specific vaccine effectiveness of JEV. The reported protective efficacies of GIII JEV vaccines against GI virus are not consistent due to differences in evaluation models used, including virus strain, passive or active immunization, and challenge dosage (17, 18, 19).
The Nakayama JEV was isolated in 1935, and the vaccine derived from it was developed in 1956 [37]. Differences between vaccine strain and circulating GIII viruses are expected, but the vaccine is proven to be highly protective [38]. The efficacy of formalin-inactivated Nakayama JEV vaccine in vaccinees who received one, two, and three doses of immunization is 85.59%, 91.07% and 98.51%, respectively [8]. The trial of mouse brain-derived inactivated JEV vaccine, including Nakayama or Nakayama plus Beijing-1 strains, was conducted in Thailand in 1984–1985, and the estimated efficacy was 91%. But, several JE-confirmed cases were diagnosed among vaccinated group indicating the potential of primary vaccine failure. The JEV genotype I was first isolated in the Southeast Asian countries; it has been suggested that the switch from genotype III to I in the 1980s might contribute to the primary vaccine failure without confirmed virological evidence [13], [39]. Our study also showed that the vaccine, used in Taiwan, offered strain-specific neutralization against field-isolated GIII CJN virus. This result suggests and supports that the amino acid variations, located in EDI and EDII, between these two viruses, are not critical in eliciting neutralizing antibody as compared to residues located in domain III [40].
Our previous report demonstrated GI JEV replaced GIII in Taiwan in 2009, and here we conducted the first study using serum samples from vaccinated children to systematically evaluate strain-specific neutralizing antibodies elicited by the GIII Nakayama vaccine [16]. Previous report indicated that the GMT titers against GIII isolates, including T1P1, CC27, CJN, and CH1392, were 2-fold lower than that against Nakayama strain; and Shyu et al., reported that only 37.9% of vaccinees sera in the 15–19 year-old group can actually neutralize JE5 Taiwanese isolate [17], [18]. In presented study, the neutralizing titer against GI virus was 8-fold lower (Table 2) than against Nakayama strain, and the seroprotection threshold against GI virus was 10% of vaccinees in the vaccinated 14–15 year-old group. Thus, the lower titers of antibody against GI in general in Taiwan might relate directly to overall antigenic variability between GIII and GI, but strain-dependent neutralization could not be totally excluded. The seroprotection rate should be appropriately estimated against currently circulating JE strains rather than against vaccine strain.
There were only five informative amino acids variations between GIII and GI JEV in the E protein: residues 123, 129, 222, 327, and 366. However, the antibodies elicited by the GIII JEV vaccines were only weakly neutralizing against circulating GI virus as compared to the human GIII isolate. Three of the five amino acid differences occur in domain II, which is involved in weakly or non-neutralizing antibody binding [41], and residues 129 and 222 are not accessible for antibody binding. Thus, the remaining two amino acid variations, residues 327 and 366, might play an important role in lower strain-specific neutralization against GI virus as compared to the GIII vaccine and human isolates. These two residues are accessible for antibody binding and are located in domain III of the E protein, which has been shown to be the most important region eliciting neutralizing antibody. Antibodies targeting domain III of E protein make up a relatively small proportion of the polyclonal human antibody response against flaviviruses, thus more detail studies are need to clarity the role of residues 327 and 366 [42].
GI virus-specific residues at position 327 and 366 located in the BC and DE loops on domain III of E protein, respectively, may play an important role in eliciting genotype-specific neutralizing antibodies. Genotype- and strain-specific neutralizing MAbs have been characterized against dengue virus serotypes 1, 2 and 3 [36]–[38]. Monoclonal antibody E104, derived from mice immunized with GII of DENV-1 virus, is a genotype-specific MAb, which recognizes residues 328, 330, 361 and 362 in the BC and DE loops of EDIII. Residues 328 and 329, located in the BC loop, are recognized by another DENV-3-derived genotype-specific MAb. Thus, the amino acids located in the BC and DE loops on domain III of E protein may involve in the induction of genotype-specific neutralizing antibodies.
Non-neutralizing antibodies are a possible risk factor for ADE in dengue pathogenesis during infection with heterologous or homologous dengue viruses, but the role of ADE contributes to JE disease is unclear [43]. Vaccine-induced enhancements of virus infection have been documented for members of different virus families as well [26]. Our preliminary in vitro and ex vivo studies suggest that the potential of ADE with vaccinees serum specimens may be increased due to GIII to GI replacement. Prior to the genotype replacement in Taiwan, the average mortality rate of confirmed cases of JEV infection was 7.8% (2000–2008). However, during 2009–2010, after GI JEV became the dominant circulating virus, the average mortality rate increased to 14.2% (Official statistics of the Department of Health, Taiwan). The neurovirulence of GI and GIII viruses was similar and might not directly associate with an increasing case fatality rate of JE cases [19], [20]. The potential for ADE, measured by in vitro assays and supplemented by the limited number of ex vivo assays, increases dramatically when the neutralization titer of vaccinee serum decreases to below the protective threshold of 1∶10 against the vaccine strain. The statistical differences we have seen in older children, reflecting waning of neutralizing antibody to GI virus to less than seroprotective threshold (Figure 2). The presence of memory B cells, CD4+ and CD8+ T cells, and anamnestic response has been indicated after received JEV inactivated vaccine [44], but rapidly decline of seroprotection rate against field-isolated JEVs also has been suggested [45]. Thus, the duration of immunity among vaccinated adults should be evaluated comprehensively ten years after the final booster vaccination.
The conclusion of current study is limited by the small sample size and the volume of vaccinee's serum. With the Institution Approved Protocol in the further, we plan to increase the sample size and collect larger serum volume to increase more selection of GIII and GI strains in the analysis. The most cost-effective control strategy for JE is vaccination, but genotype replacement in JEV endemic/epidemic regions may reduce the efficacy of traditional GIII virus-based vaccines. The efficacy of GIII JEV vaccines should be closely monitored at the national or regional level. The potential impact due to genotype replacement could be overcome in the future by 1) increasing the effective immunogenic dose or incorporating a novel adjuvant in the vaccine formulation to improve the immunogenicity of the current vaccine, or 2) replacing the GIII vaccine strain with a dominant GI isolate and conducting a non-inferiority study of GI strain-specific immune responses.
Supporting Information
Figure S1.
Selection of JEVs for neutralizing antibody assay. Four JEVs, GIII T1P1 (cluster II), GIII CJN (cluster I), GI TC2009-1 (cluster I), and GI YL2009-4 (cluster II), were evaluated using a panel of serum samples (N = 10 and 15 for panel A and B, respectively) by a plaque-reduction neutralization assay. The correlations of PRNT50 were determined between (A) T1P1 and CJN or (B) TC2009-1 and YL2009-4. Number in black circle corresponding to the specimen number.
Figure S2.
Semi-ex vivo ADE experiment. The undiluted serum was pre-incubated with 1000 PFU of TC2009-1 virus and inoculated intraperitoneally into four suckling mice per serum. Brains of inoculated sucking mice were harvested and virus titers determined by plaque forming assay in BHK-21 cells.
Table S1.
Amino acid variations among JEV E protein between genotype and cluster.
We thank Ann Hunt for the assistance with the English writing while preparing this manuscript.
Author Contributions
Conceived and designed the experiments: YCF SSC. Performed the experiments: YCF JMC YYC JWL. Analyzed the data: YCF CCC GJJC SSC. Contributed reagents/materials/analysis tools: HCC CCS CMC. Wrote the paper: YCF SSC.
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- 19. Beasley DW, Li L, Suderman MT, Guirakhoo F, Trent DW, et al. (2004) Protection against Japanese encephalitis virus strains representing four genotypes by passive transfer of sera raised against ChimeriVax-JE experimental vaccine. Vaccine 22: 3722–3726.
- 20. Liu X, Yu Y, Li M, Liang G, Wang H, et al. (2011) Study on the protective efficacy of SA14-14-2 attenuated Japanese encephalitis against different JE virus isolates circulating in China. Vaccine 29: 2127–2130.
- 21. Van Gessel Y, Klade CS, Putnak R, Formica A, Krasaesub S, et al. (2011) Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO((R))) induced neutralizing antibody titers. Vaccine 29: 5925–5931.
- 22. Chiou SS, Chen WJ (2007) Phenotypic changes in the Japanese encephalitis virus after one passage in Neuro-2a cells: generation of attenuated strains of the virus. Vaccine 26: 15–23.
- 23. Luca VC, Abimansour J, Nelson CA, Fremont DH (2012) Crystal structure of the Japanese encephalitis virus envelope protein. J Virol 86: 2337–2346.
- 24. Crill WD, Chang GJ (2004) Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. J Virol 78: 13975–13986.
- 25. Konishi E, Tabuchi Y, Yamanaka A (2010) A simple assay system for infection-enhancing and -neutralizing antibodies to dengue type 2 virus using layers of semi-adherent K562 cells. J Virol Methods 163: 360–367.
- 26. Huisman W, Martina BE, Rimmelzwaan GF, Gruters RA, Osterhaus AD (2008) Vaccine-induced enhancement of viral infections. Vaccine
- 27. McMullen AR, May FJ, Li L, Guzman H, Bueno R Jr, et al. (2011) Evolution of new genotype of West Nile virus in North America. Emerg Infect Dis 17: 785–793.
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- 45. Ku CC, King CC, Lin CY, Hsu HC, Chen LY, et al. (1994) Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children. J Med Virol 44: 122–131.


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Algeria is an Arab and Berber country in North Africa. It has a Mediterranean Sea coastline in the north. It is surrounded by Morocco to the northwest, Tunisia to the northeast, Libya to the east, Niger to the southeast, Mali to the southwest, Mauritania and Western Sahara to the west. After Sudan, Algeria is the second-largest country in Africa.
Much of recent Algerian history has been dominated by civil wars and subsequent warlordism. That said, the country is gradually restoring order and will prove an interesting, if difficult, destination.

subset: commoncrawl


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Special equipment: 1 package (6-inch) wooden skewers
Soak wooden skewers in water for 30 to 60 minutes. Dry them off with a towel.
Prepare a grill or broiler.
Lay out skewers on a plate and brush with cinnamon oil. Skewer a few pieces of fruit onto each skewer. Grill or broil fruit skewers for 30 seconds on each side.
The skewers look great when served in a pineapple. Just lay a pineapple down on its side and stick the pointed end of the skewer into the top.


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This month Americans welcomed the three millionth refugees since 1975, helping them build new lives, homes, and communities in all 50 states. The United States is proud of its history of welcoming immigrants and refugees.
The 1980s saw primarily refugees resettling in the United States from Vietnam, Laos, and the Soviet Union. The 1990s brought large numbers of Bosnians as war engulfed the former Yugoslavia. In the 21st century, we welcomed refugees from Burma, Bhutan, Iran, Iraq and Somalia, among others, reflecting a more diverse and expansive refugee resettlement program.
Historically the United States has provided protection to such well-known individuals as former Secretaries of State Henry Kissinger and Madeline Albright, Nobel Prize winner Albert Einstein, performer Gloria Estafan, and marathoner Mebrahtom Keflezighi.
The stories of many other lesser-known refugees reflect the same determination and perseverance:
- Mr. Bol B. Aweng, a "Lost Boy" from Sudan, came to the United States in 2001. He fled at the age of six, without his parents, when helicopter gunfire and aerial bombs destroyed his village. He saw friends shot, eaten by crocodiles, and die of food poisoning. He took refuge in Ethiopia and finally in Kenya before being resettled to the U.S. As a boy in the refugee camp, Mr. Aweng first began drawing in dirt and then on cardboard. He brought his love of art with him to the U.S. He graduated from Ohio State University in 2009, where he majored in Fine Arts, specializing in digital media.
- Mr. Bertine Bahige, from the Democratic Republic of the Congo, arrived in the United States in 2003. When Mr. Bahige was fifteen years old, rebels came to his house and tried to forcibly recruit him. Traveling alone, he fled first through Zambia, then spent five years in a refugee camp in Mozambique. When he was resettled in Maryland, he worked multiple jobs and went to community college. In 2006, he was offered a scholarship to the University of Wyoming. Today, he serves as a high school and community college math teacher, soccer and cross-country coach, and liaison/interpreter for Spanish-speaking parents.
- Mr. Anh "Joseph" Quang Cao was the first Vietnamese-American elected to the U.S. Congress, serving Louisiana's second Congressional District from 2009 until 2011. Mr. Cao left his home country with his aunt and two siblings in a military transport plane three days before the fall of Saigon, while his mother and the rest of his family stayed behind to wait for his father.
- Mr. Wilmot Collins, from Liberia, arrived in the United States in 1994. Mr. Collins and his wife have been U.S. citizens since 2001. He is an administrative officer at the Veterans Hospital in Helena. He has an M.A. in Human Resources Management, and is working towards a Ph.D. in Clinical Psychology. He is also a member of the U.S. Naval Reserves.
- Mr. Aumer Hadi is an Iraqi refugee who resettled in Columbia, South Carolina in 2010. Following the Gulf War, Mr. Hadi and his family fled to Yemen, where they lived for eight years. With a computer science degree from Saba University in Yemen, Mr. Hadi has been able to use his education in the United States by helping his employer to develop software for the iPhone.
- Mr. Igor Kotler, originally from the Soviet Union, has lived in the United States for over 20 years. He is President and Executive Director of the Museum of Human Rights, Freedom and Tolerance. An accomplished scholar in human rights and world history, Mr. Kotler and others helped to capture 52,000 interviews of Holocaust survivors in 32 languages, representing 56 countries.
Less than one percent of refugees worldwide are ever resettled in a third country, estimates the Office of the United Nations High Commissioner for Refugees. But, in the words of one refugee, new life in the United States was "the beginning of my return to humanity."
This past year, more than 56,000 refugees from almost 70 countries, fleeing fear of persecution based upon race, religion, nationality, political opinion, or membership in a particular social group, found new homes in the United States. Until then, some lived in camps for years or even decades, such as the Burmese on the Thai border or Bhutanese in Nepal. Others, like Iraqis in Jordan, Lebanon and Syria, or Somalis and Ethiopians in Kenya, eked out meager existences in temporary and uncertain urban dwellings.
Some came with family members, most came with few belongings, but all came with hope for the future.
In the end, all Americans benefit from our nation's open doors -- the refugees, those whose lives they touch, and communities strengthened by their contributions.
The Great Seal of the United States carries the motto "E Pluribus Unum." Ninety-nine percent of all Americans trace our heritage to foreign lands. Our founding fathers would be proud that our nation continues to offer a place of refuge to the most persecuted from many ethnicities, races and religions, who now call America home.
Continue the conversation on PRM's Facebook page.Editor's Note: This entry appeared first on the Huffington Post.
Monnie in Maryland writes:
My family and I were part of the 1980's wave of immigrants to the US. We settled in the suburb outside Chicago, in Yorkville, Illinois where the whole community adopted us, where Mr. Denis Hastert was then a math teacher and my father who was a head master in our community back home in Laos settled into his humble job of being a custodian in the high school to support seven children and a wife. We are thankful, grateful and indebted to the Yorkville and Aurora communities for adopting us and showing us kindness and sharing with us the American can do spirit. Although it is still difficult for some of us after this many years (30+), I do attest that America is one of the few places remaining in the world where a family can remain hopeful and aspire to be greater. I personally thank you for the opportunity to continue to elevate! America is great! In service - Monnie :-)
Eric in New Mexico writes:
@ David Robinson,
I was watching the video of the Sec. of State and the Korean foreign minister today and noted the Secretary's comments regarding North Korean refugees in China being repatriated back to NK, and it reminded me that (since folks are still working on a solution to that issue) that I'd offered one on Dipnote quite awhile back (buried in the archives someplace), so I thought I'd offer it up for consideration again to you, in the hopes that it will offer the State Dept. a viable framwork to both China and Korea for a cooperative alternative to sending folks back into harm's way and hardship in North Korea.
A more humane way for China to deal with an influx of refugees they do not want on their soil would be to reach an understanding with Korea that should Korea be willing to accept them in the interests of reunification and/or humanitarian reasons, and be willing to take care of the travel expenses from China to the RoK, then this would solve China's basic problem, and safeguard the lives of the refugees.
It would then be up to Korea to determine on an individual case by case basis what the permanent status of these refugees would be, by their own immigration policies.
I see the State Dept's role as being the bearer of a good idea to both nations and helping to facilitate a dialoge to reach the understanding outlined above.
Should North Korea insist that these folks be sent back to North Korea, the answer to that is simply that no nation owns an idividual excercising free will to better their lives, and slavery as such has been universally outlawed within the family of nations, thus any claim upon their persons is null and void under international law.
I hope you'll let me know what you think of this idea, and I hope this helps folks resolve the issue.
Best regards,


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text:
Mar 13, 2012 | 224 Pages
Praise for The Fourth Fisherman
"Faith and hope unite the unlikely stories of Mexican fishermen lost at sea and an American television executive even more profoundly lost in his affluence. You'll be inspired by this passionate tale of intertwined lives, touched by the author's unvarnished honesty, and challenged to trust God in fresh ways. Here's an adventure story that may well launch you on your own new adventures with God."
—Lee Strobel, New York Times best-selling author
"We love this message. Joe weaves together two stories with the same central theme, being lost and adrift, and tells how one thing—faith in God—provided each of the four fishermen the courage and comfort to face the next day and its uncertainties and ultimately led them safely to shore. These are the stories that we hold tight in our memories—when days seem so uncertain and doubts scream at us that we'll never find the shore."
—Shaunti and Jeff Feldhahn, best-selling authors of For Women Only and For Men Only
"This book is about the fishermen's agony and survival, or at least that's where Joe begins. But the real book, the inner story, shows readers how God took a once high-powered salesman who was obsessed with success and image and turned him into a new man: a dedicated husband and a serious, committed Christian."
—Cecil Murphey, New York Times best-selling coauthor of 90 Minutes in Heaven
"People of faith, myself included, often put limitations on it. But, as you'll read here, real freedom comes when we finally surrender to the mystery of God and what He is doing. Thank you, Joe, for being so honest and real. I loved it!"
—Jeff Foxworthy, stand-up comedian and television personality
"You won't be able to put this book down. It is more than the true story of three courageous fishermen and one brave author. Believe it or not, hidden within their inspiring stories is your story. It is the belief that life is bigger than we think. It is the hope of wanting our lives to have purpose. It's all there. If you're looking to find that hope, a common purpose, and a reason to still believe, you've come to the right place. But be warned. The journey begins when you board this boat."
—Jeff Henderson, North Point Ministries
"Joe is an ordinary guy who has been given an extraordinary story. He answered a call that few will ever get a chance to say yes to. I wish it could have been me."
—Steve Bartkowski, NFL legend, outdoorsman, and motivational speaker


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Digestive Health
Gut health can be linked back to many other symptoms in the body, even mood and immunity! We carry a wide variety of traditional, as well as natural digestive aids and gut health products. Of course diet also plays a critical role in this area and we can help with that as well!
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November 25, 2018: If you have an AT&T (or related) email account, you will not receive board emails, and we can not contact you. Look into using an alternate ISP for email.
Daniel / Admin, web guy.
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Remember those old World War II movies? The ones where the Navy ships are trolling for enemy submarines and the only sound that cuts the tension-thick air is the "ping" of the sonar?
While the technology is considerably more advanced and the "ping" is relegated to sound effects in old movies, Kevin Smith and Norman Stovall III are, in a sense, not unlike those old battleship commanders. Instead of a sonar screen, they stare intently at spreadsheets. Instead of launching depth charges, they redline heifers. But they're using the best technology available in an incessant search for an enemy that lies hidden beneath the surface.
Their enemy? Small ribeyes and scarce marbling. Their target? Cattle that consistently hit Choice and Prime on the rail. The bullseye? The higher value those cattle can and will return to the operation.
According to Smith, a commercial rancher and owner of Cimarron Commodities in Seymour, TX, his goal is to produce 50% Prime in his steers by his fifth year in the cow-calf business. Stovall, with Agri-Ventures Corp. at Graham, TX, is looking to improve the overall genetics on 1,200 commercial cows on his family-owned ranch. Both of them say ultrasound data will help them achieve their goals much more quickly.
Intense management for quality. Smith is no newcomer to the cattle business. He's fed cattle for 25 years and run 500-1,000 stockers for the past 12-14 years on both native and improved pastures and winter wheat. He had a bred-heifer operation for several years as well. Three years ago, after selling out the last of his bred heifers, he decided to add cows to the mix to enable him to use summer forage the stockers leave behind.
He figured he could handle around 100 cows. But with limited space and forage, his challenge was to make every one of those cows return the highest possible profit to the operation. That means finding the highest value signals in the market and producing calves to hit those targets.
"I think if you ask 100 people in the industry what the target is in terms of what the packer is looking for, they'd all tell you 70% Choice and mostly USDA Yield Grade (YG) 2s and 3s," Smith says. "That's not my target. My target is Prime."
He figures it this way -- based on the U.S. Premium Beef grid that he sold his steers on this year, Prime paid a $26.70/cwt. premium to Choice. "And if you're over 50% Choice, you're getting paid a Choice premium of $10.59 in addition to the Prime premium. So why not aim for Prime?"
He ultrasounds his heifers as yearlings at a cost of $10 each, using Casey Worrell with Rancher's Resource in Harper, TX to do the work. The first heifers he bought and ultrasounded are now three-year-old cows. They came from a group of 244 heifers that were top-end animals based on visual appraisal, but with no information at all on breeding and genetics. Using the ultrasound data along with another sort for conformation, he kept the best 50 head.
The second year, he bought 168 more replacements and did the same thing, keeping 61 head. He kept 22 heifers and 19 steers out of his first calf crop. The steers went to the feedyard. The heifers were ultrasounded and evaluated just as the other two groups.
The ultrasound data not only gives him solid information to use in culling, but it accelerates his timeline. "The reason I ultrasound is to save the years of feeding and testing to get information," he says. Smith's bull supplier, Rich Blair of Blair Brothers Angus at Sturgis, SD, encouraged him to use ultrasound and estimates it will speed up Smith's ability to reach his goal by 10 years.
Smith mines the ultrasound data for a couple of things. First, he concentrates on marbling, reported on the data sheet as IMF, or intramuscular fat. Since he's shooting for Prime, the higher the number, the better. An IMF of 5% equates to small 0 marbling, the bottom end of Choice. IMF of 8% is slightly abundant, the bottom end of Prime.
Then he looks at ribeye area, culling anything below 1 sq. in./cwt. And finally, he culls on visual appraisal, looking for an animal that's deep bodied with lots of capacity and bone.
While he says he's not at his 50% Prime goal yet, he's well on his way. His 19 steers came out of the feedyard averaging 1,341 lbs. and 85% Choice, with 35% qualifying for Certified Angus Beef(R). On a yield basis, 73% were YG 3 with a few 4s and no 5s. On average, they netted $107.15/head over what they would have brought on the cash market.
But that's only part of the story. "Look at the difference in value," he says as he flips through spreadsheets. "The three Select steers ranged in value from $1,068 to $1,284/carcass. The Choice steers, their value ranged from $1,260 to $1,477." From top to bottom, that's a $409/head difference. If he had any Prime carcasses, he would have netted an additional $200/head.
"What do I want with a cow that's producing a Select carcass? I have no use for her. You can run a good one in the same tracks that a bad one's in. Why not have something that will produce a calf that will pay you a premium?"
Improving the herd average. Stovall agrees. He assumed day-to-day management of the family operation two years ago and is working to improve the data on his cattle. "In order to improve my overall quality-based genetics, I'm utilizing every tool possible and I believe that ultrasounding is a very important one," he says.
Agri-Ventures Corp. runs Angus/Brangus cross cows on five different places, all bred to registered Angus bulls, and Stovall selects replacement heifers from all five herds. He also uses Worrell for ultrasounding his potential replacements. "Ultrasounding provides feedback on an average base of what each herd is producing. It is a small snapshot, but it provides data for informed decisions."
Stovall uses a combination of conformation, disposition appraisal, hip height and pelvic measurement, along with ultrasound data, to select his replacements. "I'll measure hip height at 10 months and appraise conformation and disposition as a first-round selection." At 12 months, he measures the pelvis and if the heifer passed the minimum, she is ultrasounded.
For pelvic size, his goal is to reach a 170-square centimeters (sq. cm) average. This past year, the minimum was set at 156 sq. cm and this current set of heifers averaged 164 sq. cm.
He uses ultrasound data in much the same way, aiming to improve each herd's quality average. In 2001, the first year ultrasound was used, the IMF score ranged from 2.0 to 3.5 with an average of 2.54 on 20 heifers. "Now, the range for 2007 yearling heifers was 2.9 as a minimum cutoff to the top heifer at 6.72, with an average of 3.95 on 140 head," he says. "I would like to produce an average of 5-6 IMF score as a minimum baseline on future heifers. We are working toward eliminating less than low Choice."
Considering all those traits and data gives him better options for culling decisions. Last year, he kept a heifer that had a 2.9 IMF score along with a minimum-sized ribeye area of 9.8 sq. in. (1.18 sq. in./cwt.). "She was a nice heifer that fit the hip height requirements and should reach a frame score of 6 at maturity. That size cow works well in our part of the country."
Since Stovall artificially inseminates all his replacements, he is able to select sires based on EPDs that match well with each individual heifer. For example, the heifer that had the 2.9 IMF and 9.8 ribeye area (REA) score was bred to a registered Angus sire with an IMF score of 8.95 and a 13.8 REA, thus improving two critical carcass traits in this heifer's first calf, he says.
However, both Smith and Stovall caution that it's important to make comparisons within contemporary groups, due to year-to-year differences in forage and environmental conditions. "The fly in the ointment is, if you stress these heifers on a nutritional or health basis as they are developing (4 to 8 months is the critical age), marbling during that stage can be greatly impacted," Stovall says.
This year, Stovall kept and bred 140 heifers from last year's calf crop. "I would like to increase that number to 200, accelerating the quality of cattle I'm raising and accomplishing my goal of having ultrasound data on each cow standing."
Until that goal is realized, Stovall has been able to make better-informed herd sire decisions by utilizing herd average ultrasound data, thus improving the overall quality and consistency of his calves.
Stovall hasn't fed any steers recently, but still sees a premium for the quality genetics he produces. This past spring, he sold steer and heifer lots over Superior Livestock Video Auction and buyers responded very positively to the quality genetics, backed by hard data.
"Ultrasounding is not new technology and I am surprised that more commercial producers aren't utilizing it," he says. "When there is an $8-$10/cwt. premium available for quality cattle, it makes sense to incorporate an ultrasound program into the management plan in order to know what you're producing."


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Southwestern Campus Directory
- Your best bet is to search by last name only, but you may also search by keywords like department or title.
- If you're not looking for a particular person, we have a full list of departments and services.
- Searching for only part of a name will match the full name; so searches for "Smith" will also return "Smithers" and searches for "Ian" will also return "Adriana"
- Only current faculty, staff and students are listed. Contact information for retired faculty and staff may be requested from the Human Resources Office, (512) 863-1435.
Note: This search will return only the first 20 matches to your query. Please be more specific if your search string matches too many entries.


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Hocus-Pocus: Quantum Quackery, aka Alternative Medicines for HIV/AIDS
Bob Frascino, M.D., was President and Founder of The Robert James Frascino AIDS Foundation. He had been an outspoken, popular expert in TheBody.com's "Ask the Experts" forums on safe sex and fatigue/anemia since 2000. Once a Fellow of the American Academy of Allergy, Asthma, and Immunology, and the American Academy of Pediatrics, Dr. Frascino served as Associate Clinical Professor of Medicine, Division of Immunology, Rheumatology, and Allergy, at Stanford University Medical Center from 1983 until 2001. He was a member of the American Academy of HIV Medicine and had also been a distinguished member of the executive boards of numerous state and regional associations.
We're inexpressibly saddened to share the news that Dr. Frascino passed away unexpectedly on Saturday, Sept. 17, 2011. Click here to read more and to share your thoughts.
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"Here's a hypothetical question -- and this is just hypothetical, so don't read too much into this -- what if we were to take a game like, say, Final Fantasy VI that does have the ATB system but is basically command-based. What if we were to take that game and breathe new life into it? Make it into a current-gen game? Would users be OK with it in that turn-based style?"
Very intelligent to mention FFVI in his/her question given how much of a sacred cow that game is in particular. :P
There's already X, X-2, and soon to be X-HD. Let us FFVI fans have out chance, damn it.
Quote from: zMKnightz on June 13, 2013, 01:07:58 AMThere's already X, X-2, and soon to be X-HD. Let us FFVI fans have out chance, damn it.I'm pretty sure they don't have any changes besides HDifying though, even with 10 being horribly unbalanced and desperately needing a decrease in encounter rates/grinding.


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WHEN: 7 p.m. Thursday.
WHERE: Moyse Field (Glendale High).
RECORDS: The Indians are 2-5, 2-2 in the Pacific League; the Tornadoes are 3-4, 1-3.
LAST WEEK: Burroughs lost to Muir, 21-14; Hoover defeated Pasadena, 37-8.
OUTLOOK: Burroughs Coach Keith Knoop isn't big on moral victories. But he was pleased with the way his team played last week in the loss to Muir, which many picked to run away with the league championship this season. The Indians defense stepped up and played well in the loss. Quarterback Andrew Williams also had a solid outing, throwing for a 70-yard touchdown. Hoover is likely to be sky high after its big win last week against Pasadena. In the victory, the Tornadoes snapped a 17-game league losing streak and defeated Pasadena for the first time since 1986. Hoover has a talented running back in Jesse Pina, who rushed for 138 yards in 20 carries and had a touchdown last week. The Tornadoes also have a reliable quarterback in Brandon Kerr. The game is an important one for Burroughs, which is fighting for a playoff spot with three regular-season games remaining.
SOMETHING INTERESTING: The last time the Indians failed to make the postseason was in 2005, when they finished fifth in the Foothill League.
WHEN: 7 p.m. Friday.
WHERE: Muir High.
RECORDS: Burbank is 5-2, 4-0 in the Pacific League; Muir is 4-3, 4-0.
LAST WEEK: Burbank defeated Crescenta Valley, 38-21; Muir defeated Burroughs, 21-14.
OUTLOOK: This is shaping up to be the biggest game of the season for Burbank. Both teams come into the contest as the lone remaining undefeated teams in the Pacific League. With both Burbank and Muir possessing offenses that can run up points, the contest is likely to come down to which squad plays better on the defensive end. Muir surrendered its first points of the league campaign last week after outscoring its opponents, 136-0, in the first three games. Muir has a crafty quarterback in Joshua Muema-Washington, who passed for nearly 100 yards and scored a rushing touchdown last week. Muir also likes to plug in multiple running backs into its offensive set. Burbank also has its share of talented running backs, led by James Williams (168 yards in 14 carries and two touchdowns last week) and Teddy Arlington (one receiving touchdown and one rushing touchdown last week). Sophomore quarterback Ryan Meredith has also stepped up since being given the starting job in the fourth game of the season. Both teams have something else in common — racking up penalties. Muir had 13 infractions for 125 yards last week and Burbank was penalized 18 times for 170 yards two weeks ago against Pasadena.
SOMETHING INTERESTING: The last time Burbank won a league championship was in 2009 when it shared the crown with cross-town rival Burroughs.
Jeff Tully


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Chiefs sign CB Owens, waive DL Moore
KANSAS CITY, Mo. — The Chiefs have signed cornerback Chris Owens, who will likely take over the nickel position and provide depth behind starters Sean Smith and Brandon Flowers.
Kansas City waived defensive lineman Brandon Moore on Friday to make room for Owens, who spent last season with Miami and Cleveland after four seasons with the Falcons.
The 5-foot-9 Owens doesn't fit the mold of big, physical cornerbacks preferred by Chiefs general manager John Dorsey and coach Andy Reid, but he does have the kind of speed and toughness that helps him make up for his size.
The Chiefs were desperate for depth in the defensive backfield after they let free safety Kendrick Lewis go in free agency and parted ways with veteran Dunta Robinson.


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Trickle-Down Freedom
America votes today.
Or rather, I should say, a fraction of America votes today to decide who will be our president for the next four years.
Some people -- too many -- will be turned away or frightened off by the impediments placed in their path by legislators and election officials in places like Florida (where freedom-to-vote advocates have filed a federal lawsuit for alleged attempts to disenfranchise early voters likely to cast their ballots for Democratic candidates).
Would that everyone had the passion it takes to face down the vote suppression crowd and insist on their right, as citizens of a free and democratic republic, to have their say. But a lot of people, maybe even a majority of those qualified to vote, will sit this one out, as happens every election. Some are disinterested or apathetic; some don't think their votes will matter. Some are actively disgusted; one friend advises against voting lest one be guilty, by association, of whatever atrocities the victor's administration might perpetrate.
This, too, is democracy, though in a limited way: I suppose it is a valid choice to abstain (or, if you like, go off and sulk), rather than stand for something, even if the choices at hand are not perfect.
We certainly have imperfect choices before us, but those choices are also starkly different in some crucial respects. Of all the reasons given for not voting, the idea that Romney and Obama are basically touting the same platform is the most offensive and scurrilous. Further, it ignores a deeper question about how much the character of a candidate counts.
We know Obama can be a little cool, a little remote, even a little imperious in his affect. But Obama has convictions, and he states them publicly. Obama's convictions on a Monday remain his convictions on a Thursday, and when his positions "evolve" (as they did with regard to marriage equality), it's a process that takes place by degrees; moreover, it's a visible and open process.
Romney simply changes his stripes according to which way the wind is blowing. And talk about imperious! As governor of Massachusetts, Romney went out of his way not to have to deal with the unwashed masses. (Whenever I see a Romney-Ryan bumper sticker around Boston, I wonder if the driver of that vehicle actually lived here during Romney's tenure. Or maybe that cell phone clutched in the driver's hand has fried some key memory circuits?)
Romney was a lousy governor, and he's a lousy candidate for the Oval Office. The rare Romney assertion that carries a ring of genuine truth is his claim that as president he would be able to "reach across the aisle" and work with Democrats more effectively than Obama could work with Congressional Republicans. But that's not a mark of his national leadership ability, any more than it was his ability to lead the Commonwealth of Massachusetts; it's the result of Democrats being willing to compromise and work together with someone of a different ideology.
The GOP, on the other hand, has shown itself to be so entrenched and intransigent over the last four years that they would rather hold America's families as economic hostages in order to smear Obama than work for the common good. Indeed, you have to wonder if the "common" good is even on their minds. The common citizen certainly isn't, at least not once he's cast his Election Day vote.
Personally, I get the impression that the Republicans believe in a kind of "trickle-down freedom" that hands the greatest measure of autonomy and choice to the few wealthiest elect. A few weeks ago, The New Yorker profiled billionaires who are working against Obama because the president has come out in favor of allowing Bush-era special tax exemptions for the rich to expire. The billionaires in the article talked about their liberal views on social issues, but they are hard-liners when it comes to defending their stash from the greedy masses who want them to kick in and help pay for roads, bridges, and that expensive military.
Because they have the cash to attempt to hand-pick our political leaders, America's billionaires intend to replace Obama with a man who would repeal, rescind, and roll back years... make that decades... of hard-won social progress, a man whose agenda means punishing a large swath of Americans in order to curry favor with a narrow ribbon of the best-to-do. But! Here's the thing: America is one big party, and Romney's not gonna ask the super-rich to pick up the tab. No, he's got another idea altogether: Serve the wealthy a whole lot more champagne and oysters, and then ask every other table in the joint to shoulder the bill. Who cares what Romney does to the little guy as long as he refrains from the "class warfare" of asking the rich to pay their share?
The whining from the super-rich about Obama's "class warfare" went so far as to compare the president to the sort of bipolar, drunken sot who beats his wife, then apologizes for it, then beats her some more. Really? I mean... really? As the New Yorker noted, "The growing antagonism of the super-wealthy toward Obama can seem mystifying, since Obama has served the rich quite well."
Mystifying, indeed, except that "Hostility toward the President is particularly strident among the ultra-rich," the segment of society that sees itself as above the need to pay its taxes because it's just so very, very special. (They are the "job creators," don't you know, and even if they aren't creating any jobs, well... they're rich, and isn't that what America is all about?)
The billionaires in question attempt to defend their special right to not pay as much in taxes as the poor by saying that their charitable donations to pet projects amounts to a kind of "self-taxation." Gee, I would love to be able to dictate where my self-imposed taxes go: I have plenty of beloved causes that are disgracefully cash starved. Public schools, for example. Museums. Libraries. All the public institutions that disciples of Ayn Rand (VP candidate Paul Ryan, for instance) keep claiming are not available to those who most deserve them. (Note to self-entitled Ayn Rand clones: Get a library card. Anyone can do it, even the terminally selfish. You'll feel better if you participate in the public good rather than standing in the corner scowling and griping about it.)
Then there's the strange case of Sheldon Adelson, a billionaire twenty times over who runs gambling establishments that are now under investigation by Obama's Justice Dept. As reported at The Huffington Post, "Adelson's casino empire, the bulk of which is based in Asia, is being investigated for bribery and money laundering." Adelson has said he is willing to spend $100 million to unseat Obama and, presumably, buy himself some relief from criminal investigation under a Romney administration. The New York Times noted how little Adelson's motivations mattered to a pair of GOP candidates who, if elected, will have been chosen of the rich, by the rich, and for the benefit of the rich: The Times noted that Romney and Ryan seemingly went out of their way to avoid giving the press a chance to pose "uncomfortable questions about the multiple federal investigations into the company behind Mr. Adelson's wealth" following Adelson's huge contributions to their war chest.
"Those questions, though, aren't going away, and neither are the ones about the judgment of Mr. Romney and Mr. Ryan in drawing ever closer to a man whose business background should lead them to back away -- fast," the Times noted. "By not repudiating Mr. Adelson's vow to spend as much as $100 million on their behalf, the two candidates seem more eager to keep the 'super PAC' dollars flowing than to preserve the integrity of their campaign."
Unless, of course, the "integrity" of the Romney-Ryan campaign really is all about money. And why shouldn't it be? It seems that at this moment in our national history, money isn't simply speech; it's the rule of law, as well. Justice for the wealthy is a different matter than justice for the increasingly not-so-wealthy.
This matters in other ways, as well. The rich get richer, just as the saying goes, and the poor get screwed. Poverty restricts lives, closes doors to opportunity, and harms individuals and their families.
In unvarnished talk, money buys freedom. The people I know who support Romney say it's because Obama's economic policies bleed their pocketbooks too much. They don't seem to get two essential facts: One, Obama reduced taxes on most ordinary Americans; Two, Romney will raise them again, while cutting taxes for the wealthy. Either that, or he'll do what Republican presidents before him have done: Spend big and put it all on account, which is just another way of raising taxes, albeit on the next generation rather than the current one.
What Obama knows that Romney doesn't is that "we all do better when we all do better." Romney clings to the belief, demonstrably untrue, that when the rich do better, so do the poor. The rich have been doing better and better for thirty years now, while the middle class has been shrinking and the poor slipping further into the morass of debt and need.
But it's not the numbers or even the realities that matter to Romney, whose campaign has proudly run on facts that turn out not to be facts at all. A government-bailout-enabled Jeep factory moving to China? I'd say "You can do better than that," but if the aim was to tell a lie so bizarre and fathomlessly fictitious as to confound all reason, well, you probably can't do better than that. Facts may not matter to politicians whose convictions shift with the clouds overhead, but they do matter to auto executives, and Chrysler called Romney on his bullshit.
"I feel obliged to unambiguously restate our position: Jeep production will not be moved from the United States to China," Chrysler CEO Sergio Marchionne reassured the company's workers in an email sent after the Romney campaign saturated the airwaves around Toledo, where thousands of auto workers live, work, and vote, with its misleading ad.
Lies in the air, facts on the ground: While Romney favored letting big carmakers file for bankruptcy, Obama took action -- and now the auto industry is thriving to the point of potentially expanding its operation into the Asian market. That's a hell of a lot different than suggesting that Obama is giving American car-building jobs to the Chinese. Coming from Mitt "Bain Capital" Romney -- Bain being a company that really did export American jobs -- it's painfully, grimly aggravating.
Money is not freedom, but America's super-wealthy and its lapdog GOP seem to think that the two things are one and the same, just as the GOP view of money (shared by our ideologically tilted Supreme Court) is that it is also "speech." (Actually, you get the impression that to the money-mad and politically punitive, money is pretty much everything... and that, too, is worrisome.)
This confusion has brought us to the aforementioned "trickle-down freedom," the already-observable state of our society in which the rich simply have more rights and more privileges than those of us who get by on less. A lot less. Several orders of magnitude less. Just like the money that doesn't, in fact, "trickle down," those liberties remain ensconced far out of reach of the ordinary American.
The result? A presidential candidate who preaches opportunity, but plans to curtail options. Romney has said he favors amendments to this nation's foundational legal document, the Constitution, that would restrict the rights women have over their own bodies, and forcibly divorce same-sex couples in the states where their marriages are valid.
Romney's willingness to place policy over people's bodies and their family lives is not a surprise, exactly, because freedom, for the Romney camp, is not a right to be enjoyed by all. In a system where money = speech = freedom, there really is only so much to go around -- and the rich don't get rich by sharing and sharing alike. Voter suppression, predatory financial practices, and laws that focus on punishing people instead of helping them are the order of the day under such regimes. (What: You don't remember eight years under W and his corporate puppet masters? Dude, that mess came to an end only, like, four years ago. Right after the economy tanked following the second financial sector meltdown under a Bush administration.)
But listen, don't take my word for it... If you really like the guy on the Republican ticket, go and vote for him. All I ask is this: Don't try to blame "the liberals" for the disaster that will result from a Romney presidency. In the event of a Romney-Ryan debacle, try this instead: Four years from now, man the hell up and take some real responsibility. I mean, take it -- right back from a government that's so negligent it lets our kids grow up dumb, but so big it thinks it can tell me who to marry.
Understand that we got into the mess degree by neglectful degree, and that to get out of it we'll have to go about it step by responsible, civic-minded step. The first step is to vote for the slightly less bad choice on the ballot, and work from there.
So would you please take the trouble to vote today -- or else hold your peace until 2016. Not that you'll have a choice, unless, of course, you have a billion dollars in the bank. In that case, you can speak up and be heard even if it's not Election Day.


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Lean Cuisine Steam Pouch Is the Trendiest Bag in Town
This season's hottest new bag comes from Nestle USA. Eight new varieties of Lean Cuisine Market Creations, from Nestle's Solon, Ohio-based Prepared Foods div., use a cooking pouch to steam the ingredients, locking in nutritious ingredients and unlocking delicious flavor. With the authentic taste and texture of a freshly prepared meal, these new entrées contain 300 calories or less, can be beautifully plated and are ready to eat in minutes.
Varieties include: Asiago Cheese Tortelloni (tomatoes, yellow and green zucchini and yellow carrots in a tomato vodka sauce); Chicken Alfredo (white meat chicken with penne pasta, broccoli, yellow carrots and orange carrots in a creamy Alfredo sauce); Chicken Margherita (white meat chicken, penne pasta, tomatoes, red bell peppers and spinach, with mozzarella cheese in a savory balsamic sauce); Garlic Chicken (white meat chicken with bowtie pasta, asparagus, broccoli and carrots in a creamy garlic Parmesan sauce); Mushroom Tortelloni (Portobello mushroom and cheese stuffed tortelloni with snap peas, sun-dried tomatoes, peas and red peppers in a creamy mushroom sauce); Shanghai-Style Shrimp (shrimp with Udon noodles, snap peas, red bell peppers and carrots in a sweet and spicy Asian-style sauce); Shrimp Scampi (shrimp with pasta, broccoli, yellow carrots and red peppers in a lemon garlic sauce); and Sweet & Spicy Ginger Chicken (chicken tenderloins with Udon noodles, broccoli, carrots, red peppers and water chestnuts in a sweet and spicy ginger sauce).
"Lean Cuisine Market Creations offer high quality steamed entrées that can be plated for a premium dining experience," says Christine Dahm, vice president of Lean Cuisine marketing. "Women can treat themselves to great-tasting meals without the hassle while staying on track with real ingredients they can feel good about – living a life full of flavor without a bite of compromise. That's what we call living the delicious life."
Suggested retail price for a 10-oz. to 10.5-oz. package is $3.99.


text: Cardiff - Proteas one-day captain AB de Villiers has revealed his side's plan to take advantage of England's lightweight batting line-up in the forthcoming one-day series.De Villiers feels England's recent one-day success has masked a lack of depth in their batting that could be exposed by a bowling attack of South Africa's quality.England have risen to the top of the one-day world rankings on the back of 10 wins in 2012, including a 4-0 series rout of Australia.But they will be without star batsman Kevin Pietersen after the text message controversy that led to his omission from the squad.And while De Villiers believes England are a "wonderful" team, he is confident the South Africans have enough quality bowlers to knock their hosts from pole position for the second time in quick succession.South Africa ended England's reign as the top-ranked Test team thanks to dominant displays from fast bowlers Dale Steyn and Morné Morkel and they will be unleashed again in the five-match one-day series that starts in Cardiff on Friday."I think they are a little bit light in the batting department, that might be something that we will be attacking. We'll see," De Villiers said on Thursday."They've got Bresnan that I think they rely on quite a bit coming in at seven."The reason why they're playing conservatively up front is because they're a batter light. They don't go with the wicketkeeper at seven like most teams do."But I don't think they've got a set blueprint. They can play around with options and they've got a wonderful ODI side."While De Villiers is in confident mood, he knows England have not lost a home series since 2009.The wicketkeeper-batsman is also wary that Alastair Cook and Ian Bell set up the crushing series win over Australia with some impressive displays and he admits the Proteas will have to be at their best to win the series."You've got to play the situation and the conditions that you're dealt. In England it moves around a little bit with the two new balls," he said."You can't just go out there and hit every ball for six. You will be three or four down very quickly."You have to try to get through the new ball with as a little damage as possible."After knocking England off their coveted Test perch De Villiers denied that taking over at the top of the 50-over format was the main objective of the five-match series.South Africa will go top with a series win but De Villiers is more concerned with developing a new-look side."It's definitely not our main goal. We're not here focused on world rankings, certainly not as much as the Test series," he said."It would be nice to get that as a bonus but we feel we've got other stuff to focus on. We're trying to build a brand of cricket that we want to play."We want to play attacking, good cricket. I'd like to think of us as a team that has done fairly well under pressure, so when we are put under pressure this series we will be looking to attack that's for sure."England have had just four days to get over their Test series defeat before they return to action in Cardiff.But with captain Cook welcoming a number of new names into the squad, De Villiers doesn't think England will suffer a Test hangover."That won't be huge, maybe a tiny little bit of a factor. It's the same situation we find ourselves in with a new-look team under Cook," he said."They'll feel refreshed and re-energised. Their ODI team is a totally different unit and I can't see them carrying a lot of scars from the Test series. "There is just a tiny little mental factor but if we don't start well tomorrow we lose that momentum right away."