Impact of rapid ascent to high altitude on sleep.

PURPOSE: Sleep disturbance at high altitude is common in climbers. In this study, we intended to evaluate the effect of rapid ascent on sleep architecture using polysomnography (PSG) and to compare the differences between subjects with and without acute mountain sickness (AMS). METHODS: The study included 40 non-acclimatized healthy subjects completing PSG at four time points, 3 days before the ascent (T0), two successive nights at 3150 m (T1 and T2), and 2 days after the descent (T3). All subjects were transported by bus from 555 to 3150 m within 3 h. AMS was diagnosed using self-reported questionnaire of Lake Louise score. RESULTS: Twenty of 40 (50%) subjects developed AMS. At high altitude, awakening percentages increased in AMS group but changed insignificantly in non-AMS group. Arousal index and apnea/hypopnea index (AHI) increased irrespective of AMS. The increases of AHI were more evident in non-AMS group than in AMS group. Compared to subjects without AMS, those with AMS had significantly lower sleep efficiency, lower central apnea index, and longer latencies to sleep and rapid eye movement (REM) sleep at T1 and lower REM sleep percentages at T1 and T2. Subjects with older age and lower minimum arterial oxygen saturation during sleep at sea level were prone to develop AMS. CONCLUSIONS: Higher AHI did not cause more frequent awakenings and arousals at high altitude. Central sleep apneas were observed in non-AMS but not in AMS group. Subjects unacclimatized to acute hypobaric hypoxia might have delayed and less REM sleep.

Mouth opening/breathing is common in sleep apnea and linked to more nocturnal water loss.

BACKGROUND: Mouth opening/breathing during sleep is common in patients with obstructive sleep apnea (OSA), which is probably associated with more water loss and higher risk for nocturnal ischemic heart attack. This study aimed to evaluate nocturnal changes in hematocrit/hemoglobin levels and estimated plasma volume loss in OSA patients and its relation to their OSA severity and mouth open/breathing. METHODS: Sixty OSA patients and fifteen healthy controls were enrolled and underwent overnight polysomnography. Mouth status was evaluated via an infrared camera and nasal/mouth airflow. Hematocrit and hemoglobin levels in peripheral venous blood were measured before and after sleep to estimate the change of plasma volume. RESULTS: Compared to controls, OSA patients had a greater nocturnal increase in hematocrit (1.35% vs. 1.0%, p = 0.013), hemoglobin (0.50% vs. 0.30%, p = 0.002) and more estimated water loss (5.5% vs 3.7% of plasma volume, p < 0.013). The extent of increase was correlated to apnea-hypopnea index (AHI)_the marker of OSA severity (Spearman's ρ = 0.332, p = 0.004; ρ = 0.367, p = 0.001 for hematocrit, hemoglobin, respectively), which remained significant after serial multivariate adjustment. OSA patients had more sleep time with mouth open (96.7% vs 26.7% of total sleep time, p < 0.001) and time with complete mouth breathing (14.1% vs 2.7%, p < 0.001). The extent of mouth breathing was correlated to AHI (ρ=0.487, p < 0.001), nocturnal increase in hematocrit/hemoglobin levels (ρ = 0.236, p = 0.042; ρ = 0.304, p = 0.008, respectively) and estimated plasma volume loss (ρ = 0.262, p = 0.023). CONCLUSION: OSA patients had a greater increase in hematocrit/hemoglobin levels after sleep, which is probably linked to more water loss and more sleep time with mouth open/breathing.

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.

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms.

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.

Development of a quantitative real-time RT-PCR assay for detecting Taiwan ferret badger rabies virus in ear tissue of ferret badgers and mice.

In 2013, the first case of Taiwan ferret badger rabies virus (RABV-TWFB) infection was reported in Formosan ferret badgers, and two genetic groups of the virus were distinguished through phylogenetic analysis. To detect RABV-TWFB using a sensitive nucleic acid-based method, a quantitative real-time reverse transcription polymerase chain reaction targeting the conserved region of both genetic groups of RABV-TWFB was developed. This method had a limit of detection (LOD) of 40 RNA copies/reaction and detected viral RNA in brain and ear tissue specimens of infected and dead Formosan ferret badgers and mice with 100% sensitivity and specificity. The mean viral RNA load detected in the ear tissue specimens of ferret badgers ranged from 3.89 × 108 to 9.73 × 108 RNA copies/g-organ, which was 111-fold to 2,220-fold lower than the concentration detected in the brain specimens, but 2,000-fold to 5,000-fold higher than the LOD of the assay. This highly sensitive technique does not require facilities or instruments complying with strict biosafety criteria. Furthermore, it is efficient, safe, and labor-saving as only ear specimens need be sampled. Therefore, it is a promising technique for epidemiological screening of Taiwan ferret badger rabies.

Molecular characterization of a new serotype of duck hepatitis virus.

Duck hepatitis strains 90D and 04G were determined to be antigenically unrelated to type 1 duck hepatitis virus (DHV-1) by in vitro cross-neutralization assay. The genome sequences of 90D and 04G revealed that both strains of the new serotype DHV (N-DHV) possessed a typical picornavirus genome organization apart from the unique possession of three in-tandem 2A genes present in DHV-1. The 2A1, 2A2, and 2A3 proteins represented an aphthovirus-like 2A protein, AIG1-like protein, and human parechovirus-like 2A protein, respectively. The N-DHV genome displayed unique features, compared to the DHV-1 genome. The 366 nt 3'UTR of N-DHV, the largest determined thus far among picornaviruses, was 52 nt longer than DHV-1. The pairwise percent identity of the nucleic acid and amino acid sequences at 1D region of N-DHV and DHV-1 were only 69.1-69.7 and 70.1-70.5%, respectively. Finally, phylogenetic and evolutionary analysis of N-DHV revealed that the N-DHV and DHV-1 belong to two different clusters of a novel genus in the Picornaviridae family.

Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus.

In a 1990 outbreak, a virus isolated in Taiwan from the intestines of ducks showing signs of hepatitis was tentatively classified as a picornavirus on the basis of physical, chemical, and morphological characteristics. The virus was cloned and then found not to be type 1 duck hepatitis virus (DHV-1) or a new serotype of duck hepatitis virus (N-DHV) by serum neutralization. Complete genome sequencing indicated that the virus genome had 8351 nucleotides and the typical picornavirus genome organization (i.e., 5' untranslated region (UTR)-L-P1 (VP 4-2-3-1)-P2 (2A-B-C)-P3 (3A-B-C-D)-3' UTR-poly A). One open reading frame encoded 2521 amino acids, which makes this virus one of the largest picornaviruses, second only to equine rhinitis B virus of the genus Erbovirus. Its L protein was the largest within the family Picornaviridae (451 amino acids) and suspected to be a trypsin-like protease. The 235-nucleotide 3' UTR region was of intermediate size, quite long compared to other picornaviruses but shorter than other picornaviruses of duck-origin (DHV-1 and N-DHV) and had four regions of secondary structure. The 2A protein was composed of only 12 amino acids, which is the shortest of any member of the family Picornaviridae. Phylogenetic analysis of the polyprotein and 3D sequences indicated that this virus (named duck picornavirus [DPV]) together with porcine enterovirus type 8 virus and several simian picornaviruses form a distinct branch of the family Picornaviridae and should be assigned to a new picornavirus genus.

Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus.

The genome sequences of three duck hepatitis virus type 1 (DHV-1) strains were determined. Comparative sequence analyses showed that they possessed a typical picornavirus genome organization apart from the unique possession of three in-tandem 2A genes. The 2A1 protein of DHV-1 is an aphthovirus-like 2A protein; the 2A2 protein is not related to any known picornavirus protein; the 2A3 protein is a human parechovirus-like 2A protein. Several other features were found to be unique to the DHV-1 genome when compared with other picornaviruses: (i) the 3' UTR of DHV-1 was composed of 314 nt, the largest among the picornaviruses; (ii) pair-wise amino acid sequence identities between polyprotein of DHV-1 and other picornaviruses are all less than 30%. The pair-wise amino acid sequence identities in the 3D region of DHV-1 with LV and HPeV-1 is only 38.6 and 36.6%, respectively, and less than 30% with all other picornaviruses; (iii) the DHV-1 capsid polypeptide VP0 is not proteolytically cleaved into VP4 and VP2; and (iv) phylogenetic and evolutionary analysis of DHV-1 reveals a new picornavirus clade. It is therefore proposed that DHV-1 should be assigned to a new genus in the Picornaviridae.

Safety, efficacy and immunogenicity evaluation of the SAG2 oral rabies vaccine in Formosan ferret badgers.

Since 2013, rabies cases have been reported among Formosan ferret badgers in Taiwan, and they have been shown to be the major reservoirs for Taiwanese enzootics. To control and eradicate rabies, the authorities plan to implement a vaccination programme. Before distributing live vaccines in the field, this study assessed the safety, efficacy, and immunogenicity of SAG2 vaccine on ferret badgers by direct oral instillation. After application of 109 TCID50/dose, no virus was excreted into the oral cavity 1-7 days post-application, and safety was also satisfactorily verified over a 266-day period. Moreover, despite the low level of rabies virus neutralising antibodies induced after vaccination of a 108 TCID50/dose, the efficacy assessment revealed a 100% survival rate (15/15) of vaccinees and an 87.5% fatality rate (7/8) in control animals after a challenge on the 198th day post-vaccination. The immunisation and protection rates obtained more than 6 months after a single vaccination dose demonstrated that SAG2 is an ideal vaccine candidate to protect Formosan ferret badgers against rabies in Taiwan.

Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan.

BACKGROUND/PURPOSE(S): Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). METHODS: After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. RESULTS: To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. CONCLUSION: The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus.

Antigenic and genotypical characterization of Newcastle disease viruses isolated in Taiwan between 1969 and 1996.

Three major epidemics of Newcastle disease (ND) occurred in Taiwan over the past three decades (in 1969, 1984, and 1995). In order to gain a better understanding of the relationships between past ND epizootics in Taiwan, 36 ND viruses (NDVs) isolated between 1969 and 1996 were characterized antigenically and genotypically. The antigenicity of these viruses was analysed by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Using a panel of 22 mAbs to divide NDVs into subgroups, a total of 18 binding patterns were revealed. The sequences covering the cleavage site of the fusion protein gene of these isolates were also determined. The results of the phylogenetic analysis placed 36 NDVs into I, II, VIb, VIIa, VIII and two novel genotypes (provisionally termed X and VIh). The 1969 velogenic isolates were of genotypes X and VIh; the 1984-1985 velogenic isolates were genotyped VIb, VIh, VIIa, and X; while the 1995-1996 velogenic isolates were genotyped VIIa or VIII. Some 1969 and 1984 velogenic isolates were of the same mAbs binding pattern and genotype, and the mAbs binding patterns of the 1995-1996 isolates have not been seen before. It is concluded that velogenic NDVs of different genotype and antigenic type have co-circulated in Taiwan at least since 1969. Also there were epizootiological links between strains isolated in 1969 and 1984, whereas the 1995-1996 epidemic was caused by new antigenic variants.

Genetic variation of viral protein 1 genes of field strains of waterfowl parvoviruses and their attenuated derivatives.

To understand the genetic variations between the field strains of waterfowl parvoviruses and their attenuated derivatives, we analyzed the complete nucleotide sequences of the viral protein 1 (VP1) genes of nine field strains and two vaccine strains of waterfowl parvoviruses. Sequence comparison of the VP1 proteins showed that these viruses could be divided into goose parvovirus (GPV) related and Muscovy duck parvovirus (MDPV) related groups. The amino acid difference between GPV- and MDPV-related groups ranged from 13.1% to 15.8%, and the most variable region resided in the N terminus of VP2. The vaccine strains of GPV and MDPV exhibited only 1.2% and 0.3% difference in amino acid when compared with their parental field strains, and most of these differences resided in residues 497-575 of VP1, suggesting that these residues might be important for the attenuation of GPV and MDPV. When the GPV strains isolated in 1982 (the strain 82-0308) and in 2001 (the strain 01-1001) were compared, only 0.3% difference in amino acid was found, while MDPV strains isolated in 1990 (the strain 90-0219) and 1997 (the strain 97-0104) showed only 0.4% difference in amino acid. The result indicates that the genome of waterfowl parvovirus had remained highly stable in the field.

A complete molecular diagnostic procedure for applications in surveillance and subtyping of avian influenza virus.

INTRODUCTION: The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. METHOD: This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. RESULT AND CONCLUSION: This diagnostic procedure may prove exceedingly useful for virological and ecological advancements in global avian influenza research.

Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer.

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.