A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates.

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

HuR binding to AU-rich elements present in the 3' untranslated region of Classical swine fever virus.

BACKGROUND: Classical swine fever virus (CSFV) is the member of the genus Pestivirus under the family Flaviviridae. The 5' untranslated region (UTR) of CSFV contains the IRES, which is a highly structured element that recruits the translation machinery. The 3' UTR is usually the recognition site of the viral replicase to initiate minus-strand RNA synthesis. Adenosine-uridine rich elements (ARE) are instability determinants present in the 3' UTR of short-lived mRNAs. However, the presence of AREs in the 3' UTR of CSFV conserved in all known strains has never been reported. This study inspects a possible role of the ARE in the 3' UTR of CSFV. RESULTS: Using RNA pull-down and LC/MS/MS assays, this study identified at least 32 possible host factors derived from the cytoplasmic extracts of PK-15 cells that bind to the CSFV 3' UTR, one of which is HuR. HuR is known to bind the AREs and protect the mRNA from degradation. Using recombinant GST-HuR, this study demonstrates that HuR binds to the ARE present in the 3' UTR of CSFV in vitro and that the binding ability is conserved in strains irrespective of virulence. CONCLUSIONS: This study identified one of the CSFV 3' UTR binding proteins HuR is specifically binding to in the ARE region.

Genetic analysis of fish iridoviruses isolated in Taiwan during 2001-2009.

To investigate the genetic relationships between field strains of iridoviruses gathered from various fish species in Taiwan, viruses that were collected from 2001 to 2009 were analyzed. Open reading frames encoding the viral major capsid protein (MCP) and adenosine triphosphatase (ATPase) were sequenced for phylogenetic analysis. Our results indicated that iridoviruses from Taiwan aquaculture fishes could be classified into two groups: prior to 2005, the viruses were closely related to members of the genus Ranavirus; and after 2005, they were similar to members of the genus Megalocytivirus. Based on the analysis of MCP amino acid sequences, virus isolates were divided into 4 major genotypes that were related to ISKNV, RSIV, FLIV, and GIV, respectively. Pairwise comparisons of MCP genes showed that the ranavirus was an epidemic pathogen for economically important species in the major production regions and cultured marine fish, while the megalocytivirus isolates were sensitive to host range. In addition, the distribution of synonymous and non-synonymous changes in the MCP gene revealed that the iridoviruses were evolving slowly, and most of the variations were synonymous mutations. The Ka/Ks values were lower than one, and hence, the viruses were under negative selection.

In Vivo Demonstration of the Superior Replication and Infectivity of Genotype 2.1 with Respect to Genotype 3.4 of Classical Swine Fever Virus by Dual Infections.

In Taiwan, the prevalent CSFV population has shifted from the historical genotype 3.4 (94.4 strain) to the newly invading genotype 2.1 (TD/96 strain) since 1996. This study analyzed the competition between these two virus genotypes in dual infection pigs with equal and different virus populations and with maternally derived neutralizing antibodies induced by a third genotype of modified live vaccine (MLV), to simulate that occurring in natural situations in the field. Experimentally, under various dual infection conditions, with or without the presence of maternal antibodies, with various specimens from blood, oral and fecal swabs, and internal organs at various time points, the TD/96 had consistently 1.51-3.08 log higher loads than those of 94.4. A second passage of competition in the same animals further widened the lead of TD/96 as indicated by viral loads. The maternally derived antibodies provided partial protection to both wild type CSFVs and was correlated with lower clinical scores, febrile reaction, and animal mortality. In the presence of maternal antibodies, pigs could be infected by both wild type CSFVs, with TD/96 dominating. These findings partially explain the CSFV shift observed, furthering our understanding of CSFV pathogenesis in the field, and are helpful for the control of CSF.

Impact of oxidative stress on treatment outcomes in adult patients with sepsis: A prospective study.

PURPOSE: Oxidative stress has been shown to reflect on the development of sepsis and disease severity. In the present study, we evaluated the effects of increased levels of oxidative stress and decreased antioxidant coactivity in patients with sepsis, and the importance of oxidative stress on treatment outcomes. METHODS: Biomarkers of oxidative stress (thiobarbituric acid-reactive substances [TBARS]) and antioxidant capacity (glutathione peroxidase [GPx] and glutathione content [thiol]) were prospectively evaluated along with biochemical and clinical data in 100 patients with sepsis on days 1, 4, and 7 after admission. RESULTS: The TBARS level of the non-survivor group was significantly higher than that of the survivor group on day 1 and day 4 and negatively correlated with thiol upon admission. However, thiol was positively correlated with lactate concentration. The TBARS and lactate levels upon admission were independent predictors of fatality. CONCLUSIONS: We conclude that a TBARS cut-off value of 18.30 µM can be used to predict fatality, and an increase in the TBARS concentration by 1 µM will increase the fatality rate by 0.94%. In the panel of biomarkers, the TBARS assay can be considered as a prognostic biomarker for the treatment of patients with sepsis.

Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus.

The neutralization titer of a hemagglutinin (HA)-specific neutralizing antibody against new isolates reflect both the antigenic drift and the conformation status of HA protein in these new influenza viruses. Since most antigenic sites are in the HA1 domain of HA, using HA1 domain of influenza virus as antigen is of great importance in vaccine development. In this study, we investigate different purification processes for optimizing the immunological properties of an Escherichia coli-expressed HA1 domain (rH5HA1) of influenza H5N1 virus. rH5HA1 was expressed as inclusion bodies and extracted with 6M guanidine hydrochloride (GnHCl)/PBS buffer. The best condition for generating HA1-specific neutralization determinants is on-column oxidative refolding procedures with GSH/GSSG and l-arginine buffer. Others refolding procedures such as using high-pH buffer and/or different detergent solubilizations were found to be ineffective producing neutralization epitope recognized by a HA1-specific neutralizing monoclonal antibody that was raised against H5N1 virus.

Rapid quantification of Salmonella typhimurium inoculated to meat products by real-time PCR.

The objective of this study was to use a 5'-nuclease (TaqMan) real-time PCR method with primers and probe specific to the spaQ gene as a rapid approach to quantitatively determine Salmonella Typhimurium. The result showed that the correlation coefficient between real-time PCR estimates and bovine serum albumin (BSA) plate counts of S. Typhimurium was 0.99, independently of 10(5)-fold numbers of bystander Escherichia coli O157:H7 or total viable counts. The sensitivity of the real-time quantitative PCR assay was 10 CFU/mL for pure S. Typhimurium culture without enrichment. A known number of S. Typhimurium target cells were inoculated to dumpling fillings and chicken nuggets and DNA was extracted for real-time PCR analysis. The sensitivity was 60 CFU/g for S. Typhimurium inoculated to the food samples without any preceding procedure of enrichment. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h. This study demonstrated that real-time PCR is a rapid and reliable technique for quantifying S. Typhimurium possessing the spaQ gene in pure culture and in meat products.

Impact of Intra-Abdominal Adhesion on Dialysis Outcome in Peritoneal Dialysis Patients.

BACKGROUND: Peritoneal dialysis (PD) is an increasingly popular therapeutic option for patients with advanced renal failure. However, intra-abdominal adhesions (IAA) represent a major unsolved problem in adequate PD performance. In this study, we investigated the role of previous abdominal surgery on the presence of subsequent IAA as well as outcomes in those patients with PD who had subsequent IAA. METHODS: Two hundred and two patients who received continuous ambulatory peritoneal dialysis were prospectively enrolled in this study. We compared the PD adequacy indices and outcomes for technical failure in patients with and without subsequent IAA at presentation and a minimum of 2 years of follow-up. RESULTS: Subsequent IAA accounted for 19% (38/202) of patients. Patients who had previous abdominal surgery had higher risks of subsequent IAA especially those patients who had higher mean ages (P=0.023). PD adequacy indices including both 24-hour dialysate volume and peritoneal WCcr L/week/1.73 m2 were significantly lower in patients who had, as compared to those who did not have subsequent IAA (P=0.003 and 0.018, respectively). Although patients who had subsequent IAA had decreased PD adequacy, the development of technical failures during PD maintenance did not show significant differences at the 2-year minimum follow-up study. CONCLUSIONS: Subsequent IAA is not rare, especially in high-risk patients including those with previous abdominal surgery and higher mean ages. Although decreased PD adequacy after IAA was found, the development of technical failures was not significantly different at the 2-year minimum follow-up study.

Manifestations and Outcomes of Patients with Parkinson's Disease and Serious Infection in the Emergency Department.

BACKGROUND: Several comorbidities contribute to an increased risk of infections in Parkinson's disease (PD) as the disease progresses. However, few studies have examined the correlation between sepsis and PD. AIM: The aim of this study is to disclose the presentation and outcome of serious infection in patients with PD in the emergency department. METHODS: This retrospective cohort study enrolled patients with PD who had serious infection and were admitted to the emergency department between January 2007 and December 2013. For clinical comparison, we compared the clinical features, laboratory data, and outcomes with those of age- and sex-matched patients who had serious infection but not PD. RESULTS: There were a total of 1,200 episodes of infected PD patients and 2,400 age- and sex-matched infected patients without PD as disease controls. PD patients had fewer comorbidities and lower severity of infectious disease but longer hospital stays than control group patients. The incidences of respiratory tract and urinary tract infections were higher in PD patients. The levels of inflammatory and organ dysfunction biomarkers in PD were lower and compatible with the severity of infectious disease. A total of 86 (7.2%) infected PD patients died during the 28-day admission compared to 339 (14.1%) in non-PD patients. Serum C-reactive protein, bandemia, and lactate could be used to predict mortality in infected PD patients. CONCLUSIONS: In infected patients with PD, respiratory and urinary tract infections were the two most common infectious sources. Empiric therapy based on experience could treat both respiratory and urinary tract infections. Early diagnosis and treatment are essential for survival.

Concentration and value of endocan on outcome in adult patients after severe sepsis.

BACKGROUND: Endothelial dysfunction plays a central role in severe sepsis. Endocan is constitutively expressed in human endothelial cells when sepsis occurs. We tested the hypothesis that endocan concentrations are substantially increased in severe sepsis and decrease after antimicrobial therapy, and that endocan concentrations can predict treatment outcomes. METHODS: Biomarkers of the endothelium including endocan and cell adhesion molecules were prospectively evaluated in 153 patients with severe sepsis on days 1, 4, and 7 after admission along with biochemical and clinical data. RESULTS: Sepsis non-survivors had significantly higher endocan, ICAM-1, and VCAM-1 concentrations and lower platelet concentrations upon admission than the survivors. Non-survivors had significantly higher endocan and VCAM-1 concentrations than the survivors on serial analysis (days 1, 4, and 7). After stepwise logistic regression model and AUC analysis, endocan was revealed as a good predictor of outcome in severe sepsis, and the cut-off value for predicting fatality was 6.28 ng/ml. An increase in the endocan concentration by one ng/ml indicated an increase in fatality rate by 11.1%. CONCLUSIONS: Based on our results, serial endocan concentration meets the major requirements for predicting outcome in patients with severe sepsis. An assay of endocan concentration may be a good prognostic biomarker in the clinic for severe sepsis.

Competitive replication kinetics and pathogenicity in pigs co-infected with historical and newly invading classical swine fever viruses.

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field.

Isolation and characterization of a Sagiyama virus from domestic pigs.

In 2002, a strain of Sagiyama virus (SAGV) designated ML/Taiwan/02 was isolated from farmed pigs in Taiwan. The nsP1 and E1 gene sequences of the ML/Taiwan/02 strain shared 98.6 and 96.7% homology, respectively, with corresponding genes of a Japanese strain of SAGV. Nucleotide and amino acid sequence comparison revealed this strain of SAGV to be most closely related to Getah virus, as opposed to its current classification as a subtype of Ross River virus. To investigate the seroprevalence of SAGV infection in Taiwan, a total of 586 pig sera collected from 11 of 17 Taiwanese districts were tested for serum neutralizing antibodies (SNA) against SAGV. Results indicated that 51% of the samples had SNA titer > or = 4, and 40% had SNA titer > or = 48, indicative of repeated exposure to SAGV in the field. To study the pathogenicity of the ML/Taiwan/02 strain, this strain was experimentally inoculated into 4-week-old specific-pathogen-free pigs that were seronegative for SAGV. Viremia was detected during postinoculation days (PID) 2-4, when the SNA titer was < or = 16. By PID 7, viremia was no longer detectable, coinciding with the increase of SNA titer to > or = 48. Clinical illnesses or remarkable lesions were not observed. To the authors' knowledge, this is the first reported isolation of a strain of SAGV from pigs in the field. The virus is experimentally nonpathogenic to pigs but is moderately widespread, most likely via repeated exposure to virus-carrying mosquitoes.

Circulating endothelial progenitor cells may predict outcomes in adult patients with severe sepsis in the emergency department.

BACKGROUND: Dysfunctional and decreased numbers of endothelial progenitor cells (EPCs) may play an essential role in the initiation of organ dysfunction caused by severe sepsis. We evaluated the role of serial circulating EPCs in outcomes of patients with severe sepsis. METHODS: In total, 101 adult patients with severe sepsis and septic shock were evaluated. Circulating levels of EPCs (CD133(+)/CD34(+) and KDR(+)/CD34(+) cells) were determined at different time points. RESULTS: The levels of CD133(+)/CD34(+) and KDR(+)/CD34(+) EPCs were significantly higher in the severe sepsis group than in the healthy controls. Levels of CD133(+)/CD34(+) EPCs were significantly higher in the mortality group than in the survival group on day 1 of admission (p<0.05), but decreased significantly with time among non-survivors (p<0.05), and were lowest on day 4 at the emergency department. The Sequential Organ Failure Assessment score and number of CD133(+)/CD34(+) EPCs on admission were independently associated with in-hospital mortality. CONCLUSION: The level of CD133(+)/CD34(+) EPCs on admission is independently associated with in-hospital mortality, and the trend of a sharp decrease in the number of EPCs is related to outcomes in patients with severe sepsis.

Phylogenetic analysis of classical swine fever virus isolated from Taiwan.

By analyzing the E2 sequences of classical swine fever virus from field outbreaks in Taiwan during 1993-2001, three virus populations with distinct genotypes were determined including one historical (subgroup 3.4) and two exotic (subgroup 2.1) strains. The first subgroup 2.1 virus was isolated in 1994 and further sporadic outbreaks occurred after 1996. Phylogenetic analysis using the E2 region has segregated the Taiwanese strains of 2.1 virus into two different genotypes (termed 2.1a and 2.1b). The 2.1b viruses were only isolated in 2001 and shared approximately 94.8% nucleotide identities to the 2.1a viruses in the total genomic sequences. The results suggest that the 2.1a and 2.1b viruses may be introduced from different origins.

Natural infections of pigs with akabane virus.

Akabane (AKA) virus is considered a pathogen of herbivores in nature. However, we found that pig populations in fields were infected in Taiwan. An isolate (NT-14) of AKA virus was obtained from pigs. The NT-14 virus was able to infect pigs by the oronasal route. Subsequently, low levels of infectious virus particles were excreted into the oronasal discharge during the stage of viremia but they were not sufficient to infect new porcine hosts via contact transmission. The prevalence of serum neutralizing antibodies to AKA virus in pig populations was investigated, indicating that approximately 75% of pigs in Taiwan were seropositive. Sows and newborn piglets have the highest titers of neutralizing antibodies. Contrarily, fattening pigs aged at approximately 20 weeks old contained the lowest titers of specific antibodies. Our results suggest that pigs in natural situations are part of the AKA virus transmission cycle.

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from naïve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.

Characterization of porcine circovirus type 2 in Taiwan.

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.

Co-firing of paper mill sludge and coal in an industrial circulating fluidized bed boiler.

Co-firing of coal and paper mill sludge was conducted in a 103 MWth circulating fluidized bed boiler to investigate the effect of the sludge feeding rate on emissions of SOx, NOx, and CO. The preliminary results show that emissions of SOx and Nx decrease with increasing sludge feeding rate, but CO shows the reverse tendency due to the decrease in combustion temperature caused by a large amount of moisture in the sludge. All emissions met the local environmental requirements. The combustion ashes could be recycled as feed materials in the cement manufacturing process.

The challenges of classical swine fever control: modified live and E2 subunit vaccines.

Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA.

Long-term immunogenicity studies of formalin-inactivated enterovirus 71 whole-virion vaccine in macaques.

Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.