All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals through K-Ras at the plasma membrane of acute myeloid leukemia cells.

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3 ITD) is an important oncoprotein in acute myeloid leukemia (AML). Owing to its constitutive kinase activity FLT3 ITD partially accumulates at endomembranes, a feature shared with other disease-associated, mutated receptor tyrosine kinases. Because Ras proteins also transit through endomembranes we have investigated the possible existence of an intracellular FLT3-ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-type FLT3. Ligand stimulation activated both K- and N-Ras in cells expressing wild-type FLT3. Live-cell Ras-GTP imaging revealed ligand-induced Ras activation at the plasma membrane (PM). FLT3-ITD-dependent constitutive activation of K-Ras and N-Ras was also observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells. Accordingly, specific interference with FLT3-ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potently inhibited cell proliferation and promoted apoptosis. In conclusion, Ras signaling is crucial for FLT3-ITD-dependent cell transformation and FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.

A highly conserved retinoic acid responsive element controls wt1a expression in the zebrafish pronephros.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression.

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Virus de influenza aviar del subtipo H5 altamente patógenos y de baja patogenicidad en aves silvestres en Ucrania. Ha habido tres oleadas de brotes de influenza aviar altamente patógena en aves comerciales, de traspatio y en aves silvestres en Ucrania. La primera (2005-2006) y la segunda (2008) fueron causadas por el virus de influenza aviar de alta patogenicidad H5N1, con 45 brotes en aves comerciales (pollos) y aves de traspatio (pollos, patos y gansos) en cuatro regiones de Ucrania (AR Crimea, Kherson, Odesa y Sumy Oblast). Los virus de alta patogenicidad H5N1se aislaron de aves silvestres muertas: cormoranes (Phalacrocorax carbo) y de somormujos lavanco (Podiceps cristatus) en 2006 y 2008. La tercera ola del virus de influenza aviar de alta patogenicidad consistió en nueve brotes del virus de alta patogenicidad subtipo H5N8 en aves silvestres y domésticas, a partir de noviembre de 2016 en las regiones central y sur (Kherson, Odesa, Chernivtsi, Ternopil y Mykolaiv Oblast). Se detectó el virus al patogenicidad H5N8 en cisnes blancos muertos (Cygnus olor), pavos reales (Pavo cristatus) (en zoológicos), tarros canelos (Tadorna ferruginea), gansos caretos (Anser albifrons) y en muestras ambientales en 2016 y 2017. Una vigilancia más amplia de aves silvestres para detectar el virus de la influenza aviar se realizó entre 2006 y 2016 en las regiones de Ucrania sospechosas de ser rutas migratorias intercontinentales (norte-sur y este-oeste). Se recolectaron un total de 21,511 muestras de 105 especies de aves silvestres que representan a 27 familias y 11 órdenes. Se aislaron ochenta y dos virus de influenza aviar de baja patogenicidad (incluido un virus H5N2 de baja patogenicidad del 2010) de aves silvestres con un total de 23 combinaciones antigénicas de hemaglutininas (HA) y neuraminidasas (NA). Se aislaron quince de los 16 subtipos de HA aviar conocidos. Dos virus de alta patogenicidad H5N8 y dos virus H5N2 de baja patogenicidad se aislaron de aves silvestres vivas y de muestras ambientales (heces de aves frescas) durante la vigilancia antes del brote en avicultura. Los virus ucranianos de alta patogenicidad H5N1, H5N8 y de baja patogenicidad H5N2 pertenecen a diferentes grupos filogenéticos de H5. Estos resultados demuestran la gran diversidad de virus de la influenza aviar en aves silvestres en Ucrania, así como la importancia de esta región para estudiar la ecología de la influenza aviar.

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Virus de influenza aviar del subtipo H5 altamente patógenos y de baja patogenicidad en aves silvestres en Ucrania. Ha habido tres oleadas de brotes de influenza aviar altamente patógena en aves comerciales, de traspatio y en aves silvestres en Ucrania. La primera (2005-2006) y la segunda (2008) fueron causadas por el virus de influenza aviar de alta patogenicidad H5N1, con 45 brotes en aves comerciales (pollos) y aves de traspatio (pollos, patos y gansos) en cuatro regiones de Ucrania (AR Crimea, Kherson, Odesa y Sumy Oblast). Los virus de alta patogenicidad H5N1se aislaron de aves silvestres muertas: cormoranes (Phalacrocorax carbo) y de somormujos lavanco (Podiceps cristatus) en 2006 y 2008. La tercera ola del virus de influenza aviar de alta patogenicidad consistió en nueve brotes del virus de alta patogenicidad subtipo H5N8 en aves silvestres y domésticas, a partir de noviembre de 2016 en las regiones central y sur (Kherson, Odesa, Chernivtsi, Ternopil y Mykolaiv Oblast). Se detectó el virus al patogenicidad H5N8 en cisnes blancos muertos (Cygnus olor), pavos reales (Pavo cristatus) (en zoológicos), tarros canelos (Tadorna ferruginea), gansos caretos (Anser albifrons) y en muestras ambientales en 2016 y 2017. Una vigilancia más amplia de aves silvestres para detectar el virus de la influenza aviar se realizó entre 2006 y 2016 en las regiones de Ucrania sospechosas de ser rutas migratorias intercontinentales (norte-sur y este-oeste). Se recolectaron un total de 21,511 muestras de 105 especies de aves silvestres que representan a 27 familias y 11 órdenes. Se aislaron ochenta y dos virus de influenza aviar de baja patogenicidad (incluido un virus H5N2 de baja patogenicidad del 2010) de aves silvestres con un total de 23 combinaciones antigénicas de hemaglutininas (HA) y neuraminidasas (NA). Se aislaron quince de los 16 subtipos de HA aviar conocidos. Dos virus de alta patogenicidad H5N8 y dos virus H5N2 de baja patogenicidad se aislaron de aves silvestres vivas y de muestras ambientales (heces de aves frescas) durante la vigilancia antes del brote en avicultura. Los virus ucranianos de alta patogenicidad H5N1, H5N8 y de baja patogenicidad H5N2 pertenecen a diferentes grupos filogenéticos de H5. Estos resultados demuestran la gran diversidad de virus de la influenza aviar en aves silvestres en Ucrania, así como la importancia de esta región para estudiar la ecología de la influenza aviar.

Impact of antibiotic treatment intensity on long-term sepsis-associated kidney injury in a polymicrobial peritoneal contamination and infection model.

BACKGROUND/AIMS: Long-term kidney affections after sepsis are poorly understood. Animal models for investigating kidney damage in the late phase of disease progression are limited. The aim of this study was to investigate the impact of two antibiotic regimes on persistence of kidney injury after peritonitis. METHODS: Kidney damage was investigated 65 days after polymicrobial peritoneal contamination and infection (PCI) sepsis induction in C57BL/6 mice. Short-term antibiotic therapy (STA, 4 days) was compared to long-term (LTA, 10 days) treatment using plasma creatinine, plasma and urine neutrophil gelatinase-associated lipocalin (NGAL), urine albumin/creatinine ratio and renal histology. RESULTS: Sepsis resulted in mortality rates of 68.2% (STA) and 61.0% (LTA). Surviving STA animals showed the most pronounced kidney damage indicated by significantly elevated levels of creatinine and acute tubular damage (ATD), whereas NGAL was significantly increased in LTA survivors only. A creatinine level above 0.3 mg/dl was used to define kidney injury, found in 21.4% of STA animals and 7.8% of LTA animals. While animals with kidney injury demonstrated significantly higher ATD scores and persistent tubular damage, no significant differences were found for plasma or urine NGAL levels or urine albumin/creatinine ratios. CONCLUSION: Prolonged antibiotic treatment reduced the rate of ongoing peritonitis-induced kidney injury in a C57BL/6 mouse model. Plasma or urine NGAL levels were not able to identify animals with or without persistent kidney injury. The kidney injury after the PCI mouse model represents prototypic clinical findings and should be used for further studies investigating disease mechanisms.