Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.     

Muju Virus,Harbored by Myodes regulus in Korea,Might Represent a Genetic Variant of Puumala Virus,the Prototype Arvicolid Rodent-Borne Hantavirus

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.     

Identification of FactorsInfluencing the Puumala Virus Seroprevalence within Its Reservoir in aMontane Forest Environment

Puumala virus (PUUV) is a major cause of mild to moderate haemorrhagic fever with renal syndrome and is transmitted by the bank vole (Myodes glareolus). There has been a high cumulative incidence of recorded human cases in South-eastern Germany since 2004 when the region was first recognized as being endemic for PUUV. As the area is well known for outdoor recreation and the Bavarian Forest National Park (BFNP) is located in the region, the increasing numbers of recorded cases are of concern. To understand the population and environmental effects on the seroprevalence of PUUV in bank voles we trapped small mammals at 23 sites along an elevation gradient from 317 to 1420m above sea level. Generalized linear mixed effects models(GLMEM) were used to explore associations between the seroprevalence of PUUV in bank voles and climate and biotic factors. We found that the seroprevalence of PUUV was low (6%–7%) in 2008 and 2009, and reached 29% in 2010. PUUV seroprevalence was positively associated with the local species diversity and deadwood layer, and negatively associated with mean annual temperature, mean annual solar radiation, and herb layer. Based on these findings, an illustrative risk map for PUUV seroprevalence prediction in bank voles was created for an area of the national park. The map will help when planning infrastructure in the national park (e.g., huts, shelters, and trails).     

Population, environmental, and community effects on local bank vole (Myodes glareolus) Puumala virus infection in an area with low human incidence

In this study, the distribution of Puumala hantavirus (PUUV) infection in local bank vole Myodes glareolus populations in an area with low human PUUV infection (nephropathia epidemica [NE]) incidence in northern Belgium was monitored for 2 consecutive years. Bank voles were trapped in preferred habitat and tested for anti-PUUV IgG. Infection data were related to individual bank vole features, population demography, and environmental variables. Rare occurrence of PUUV infection was found and PUUV prevalence was low compared with data from the high NE incidence area in southern Belgium. Small-scale climatic differences seemed to play a role in PUUV occurrence, vegetation index and deciduous forest patch size both influenced PUUV prevalence and number of infected voles in a positive way. The data suggested a density threshold in vole populations below which PUUV infection does not occur. This threshold may vary between years, but the abundance of bank voles does not seem to affect the degree of PUUV seroprevalence further. We found indications for a dilution effect on PUUV prevalence, dependent on the relative proportion of nonhost wood mice Apodemus sylvaticus in a study site. In conclusion, we regard the combination of a dilution effect, a possible threshold density that depends on local conditions, and a higher fragmentation of suitable bank vole habitat in our study area as plausible explanations for the sparse occurrence of PUUV infection and low prevalence detected. Thus, beside human activity patterns, local environmental conditions and rodent community structure are also likely to play a role in determining PUUV infection risk for humans.     

Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans

We reviewed the associations of immunity-related genes with susceptibility of humans and rodents to hantaviruses, and with severity of hantaviral diseases in humans. Several class I and class II HLA haplotypes were linked with severe or benign hantavirus infections, and these haplotypes varied among localities and hantaviruses. The polymorphism of other immunity-related genes including the C4A gene and a high-producing genotype of TNF gene associated with severe PUUV infection. Additional genes that may contribute to disease or to PUUV infection severity include non-carriage of the interleukin-1 receptor antagonist (IL-1RA) allele 2 and IL-1β (-511) allele 2, polymorphisms of plasminogen activator inhibitor (PAI-1) and platelet GP1a. In addition, immunogenetic studies have been conducted to identify mechanisms that could be linked with the persistence/clearance of hantaviruses in reservoirs. Persistence was associated during experimental infections with an upregulation of anti-inflammatory responses. Using natural rodent population samples, polymorphisms and/or expression levels of several genes have been analyzed. These genes were selected based on the literature of rodent or human/hantavirus interactions (some Mhc class II genes, Tnf promoter, and genes encoding the proteins TLR4, TLR7, Mx2 and β3 integrin). The comparison of genetic differentiation estimated between bank vole populations sampled over Europe, at neutral and candidate genes, has allowed to evidence signatures of selection for Tnf, Mx2 and the Drb Mhc class II genes. Altogether, these results corroborated the hypothesis of an evolution of tolerance strategies in rodents. We finally discuss the importance of these results from the medical and epidemiological perspectives.     

Habitat factors associated with bank voles (Clethrionomys glareolus) and concomitant hantavirus in northern Sweden

Puumala virus (PUUV), genus hantavirus, causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome in humans. In this study, bank voles, the natural reservoir of PUUV, were captured at locations of previous human PUUV exposure and paired controls within a region of high incidence in northern Sweden. The aim of the study was to evaluate the influence of environmental factors on the abundance of bank voles and the occurrence of PUUV. The total number of voles and the number of PUUV-infected voles did not differ between locations of previous human PUUV exposure and paired controls. The number of bank voles expressing antibodies to PUUV infection increased linearly with total bank vole abundance implying density independent transmission. Using principal component and partial correlation analysis, we found that particular environmental characteristics associated with old-growth moist forests (i.e., those dominated by Alectoria spp., Picea abies, fallen wood, and Vaccinium myrtillus) were also associated with increased abundance of bank vole and hence the number of PUUV-infected bank voles, whereas there were no correlations with factors associated with dry environments (i.e., Pinus sylvestris and V. vitis-idea). This suggests that circulation and persistence of PUUV within bank vole populations was influenced by habitat factors. Future modeling of risk of exposure to hantavirus and transmission of PUUV within vole populations should include the influence of these factors.     

First Report of Cowpea Mild Mottle Carlavirus on Yardlong Bean (Vigna unguiculata subsp. sesquipedalis) in Venezuela

Yardlong bean (Vigna unguiculata subsp. sesquipedalis) plants with virus-like systemic mottling and leaf distortion were observed in both experimental and commercial fields in Aragua State, Venezuela. Symptomatic leaves were shown to contain carlavirus-like particles. RT-PCR analysis with carlavirus-specific primers was positive in all tested samples. Nucleotide sequences of the obtained amplicons showed 84%–74% similarity to corresponding sequences of Cowpea mild mottle virus (CPMMV) isolates deposited in the GenBank database. This is the first report of CPMMV in Venezuela and is thought to be the first report of CPMMV infecting yardlong bean.     

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92–95% nucleotide or 99–100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.     

Temporal variation in individual factors associated with hantavirus infection in bank voles during an epizootic: implications for Puumala virus transmission dynamics

Puumala virus (PUUV), the causal agent of nephropathia epidemica in humans, is one of the many hantaviruses included in the list of emerging pathogens. Hantavirus infection is not distributed evenly among PUUV reservoir hosts (i.e., bank voles [Myodes glareolus]). Besides environmental factors and local population features, individual characteristics play an important role in vole PUUV infection risk. Identifying the relative importance of these individual characteristics can provide crucial information on PUUV transmission processes. In the present study, bank voles were monitored during the nephropathia epidemica outbreak of 2005 in Belgium. Vole sera were tested for presence of immunoglobulin G against PUUV, and a logistic mixed model was built to investigate the temporal variation in individual characteristics and their relative importance to PUUV infection risk in bank voles. Relative risk calculations for individual vole characteristics related to PUUV infection in the reservoir host show that reproductive activity dominates infection risk. The gender effect is only found in reproductively active voles, where reproductively active males have the highest infection risk. Results also revealed a clear seasonal variation in the importance of reproductive activity linked to PUUV infection. In contrast to the main effect found in other trapping sessions, no difference in infection risk ratio was found between reproductively active and nonactive voles in the spring period. Combined with increased infection risk for the reproductively nonactive group at that time, these results indicate a shift in the transmission process due to changes in bank vole behavior, physiology, or climate conditions. Hence, our results suggest that mathematical models should take into account seasonal shifts in transmission mechanisms. When these results are combined with the seasonal changes in population structure during the epizootic period, we identify vole reproductive activity and length of the breeding season as potential drivers of PUUV epizootics in west-central European regions.     

Hantavirus Research in Finland: Highlights and Perspectives

Finland has the highest incidence of hantavirus infections globally, with a significant impact on public health. The large coverage of boreal forests and the cyclic dynamics of the dominant forest rodent species, the bank vole Myodes glareolus, explain most of this. We review the relationships between Puumala hantavirus (PUUV), its host rodent, and the hantavirus disease, nephropathia epidemica (NE), in Finland. We describe the history of NE and its diagnostic research in Finland, the seasonal and multiannual cyclic dynamics of PUUV in bank voles impacting human epidemiology, and we compare our northern epidemiological patterns with those in temperate Europe. The long survival of PUUV outside the host and the life-long shedding of PUUV by the bank voles are highlighted. In humans, the infection has unique features in pathobiology but rarely long-term consequences. NE is affected by specific host genetics and risk behavior (smoking), and certain biomarkers can predict the outcome. Unlike many other hantaviruses, PUUV causes a relatively mild disease and is rarely fatal. Reinfections do not exist. Antiviral therapy is complicated by the fact that when symptoms appear, the patient already has a generalized infection. Blocking vascular leakage measures counteracting pathobiology, offer a real therapeutic approach.     

PRSS1 and SPINK1 mutations in idiopathic chronic and recurrent acute pancreatitis

AIM: To identify gene mutations in PRSS1 and SPINK1 in individuals with early onset idiopathic chronic or recurrent acute pancreatitis.METHODS: The cationic trypsinogen gene (PRSS1; exons 2 and 3) and the serine protease inhibitor Kazal 1 gene (SPINK1; exon 3) were selectively amplified and sequenced from blood samples of 19 patients admitted to the Pancreas Clinic at our institution with chronic pancreatitis and/or idiopathic recurrent acute pancreatitis that were diagnosed or with onset before age 35. Fifty healthy volunteers served as controls. Whole blood samples were collected and gene specific sequences were amplified by polymerase chain reaction (PCR). All PCR products were subsequently sequenced in order to identify the presence of any mutations.RESULTS: Nineteen patients with pancreatitis (14 males; median age 24 years, range 15-48 years) were included in this study, of which five showed the presence of gene mutations. Direct sequencing results indicated the presence of two previously unidentified mutations in exon 2 of PRSS1 (V39E and N42S) in two patients with recurrent acute pancreatitis. Two cases had the N34S SPINK1 mutation. Analysis of the relatives of one patient homozygous for this mutation showed that five of the six family members carried the N34S SPINK1 mutation. Of these members, three were healthy heterozygous carriers and two were homozygotes (one sibling had diabetes, the other was healthy). Another patient was heterozygous for a novel SPINK1 mutation located on exon 3 (V46D). All members from this patient’s family had normal genotypes, indicating that it was a de novo mutation. No mutations in either gene were present in the control subjects.CONCLUSION: Two novel PRSS1 mutations and one novel SPINK1 mutation were identified in Mexican patients with early onset idiopathic recurrent acute pancreatitis.     

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.     

The Bank Vole (Clethrionomys glareolus)—Small Animal Model for Hepacivirus Infection

Many people worldwide suffer from hepatitis C virus (HCV) infection, which is frequently persistent. The lack of efficient vaccines against HCV and the unavailability of or limited compliance with existing antiviral therapies is problematic for health care systems worldwide. Improved small animal models would support further hepacivirus research, including development of vaccines and novel antivirals. The recent discovery of several mammalian hepaciviruses may facilitate such research. In this study, we demonstrated that bank voles (Clethrionomys glareolus) were susceptible to bank vole-associated Hepacivirus F and Hepacivirus J strains, based on the detection of hepaciviral RNA in 52 of 55 experimentally inoculated voles. In contrast, interferon α/β receptor deficient C57/Bl6 mice were resistant to infection with both bank vole hepaciviruses (BvHVs). The highest viral genome loads in infected voles were detected in the liver, and viral RNA was visualized by in situ hybridization in hepatocytes, confirming a marked hepatotropism. Furthermore, liver lesions in infected voles resembled those of HCV infection in humans. In conclusion, infection with both BvHVs in their natural hosts shares striking similarities to HCV infection in humans and may represent promising small animal models for this important human disease.     

Interspecific introgressive origin of genomic diversity in the house mouse

We report on a genome-wide scan for introgression between the house mouse (Mus musculus domesticus) and the Algerian mouse (Mus spretus), using samples from the ranges of sympatry and allopatry in Africa and Europe. Our analysis reveals wide variability in introgression signatures along the genomes, as well as across the samples. We find that fewer than half of the autosomes in each genome harbor all detectable introgression, whereas the X chromosome has none. Further, European mice carry more M. spretus alleles than the sympatric African ones. Using the length distribution and sharing patterns of introgressed genomic tracts across the samples, we infer, first, that at least three distinct hybridization events involving M. spretus have occurred, one of which is ancient, and the other two are recent (one presumably due to warfarin rodenticide selection). Second, several of the inferred introgressed tracts contain genes that are likely to confer adaptive advantage. Third, introgressed tracts might contain driver genes that determine the evolutionary fate of those tracts. Further, functional analysis revealed introgressed genes that are essential to fitness, including the Vkorc1 gene, which is implicated in rodenticide resistance, and olfactory receptor genes. Our findings highlight the extent and role of introgression in nature and call for careful analysis and interpretation of house mouse data in evolutionary and genetic studies.Classical laboratory mouse strains, as well as newly established wild-derived ones, are widely used by geneticists for answering a diverse array of questions (1). Understanding the genome contents and architecture of these strains is important for studies of natural variation and complex traits, as well as evolutionary studies in general (2). Mus spretus, a sister species of Mus musculus, impacts the findings in M. musculus investigations for at least two reasons. First, it was deliberately interbred with laboratory M. musculus strains to introduce genetic variation (3). Second, Mus musculus domesticus is partially sympatric (naturally cooccurring) with M. spretus (Fig. 1).Open in a separate windowFig. 1.Species ranges and samples used in our study. The species range of M. spretus is shown in green (4), and the species range of M. m. domesticus includes the blue regions, the range of M. spretus, and beyond (1). M. m. domesticus and M. spretus samples were obtained from locations marked with red circles and purple diamonds, respectively. The samples originated from within and outside the area of sympatry between the two species. (SI Appendix, Table S1, provides additional details about the samples used in our study.)Recent studies have examined admixture between subspecies of house mice (5–8), but have not studied introgression with M. spretus. In at least one case (5), the introgressive descent of the mouse genome was hidden due to data postprocessing that masked introgressed genomic regions as missing data. In another study reporting whole-genome sequencing of 17 classical laboratory strains (6), M. spretus was used as an outgroup for phylogenetic analysis. The authors were surprised to find that 12.1% of loci failed to place M. spretus as an outgroup to the M. musculus clade. The authors concluded that M. spretus was not a reliable outgroup but did not pursue their observation further. On the other hand, in a 2002 study (9), Orth et al. compiled data on allozyme, microsatellite, and mitochondrial variation in house mice from Spain (sympatry) and nearby countries in western and central Europe. Interestingly, allele sharing between the species was observed in the range of sympatry but not outside in the range of allopatry. The studies demonstrated the possibility of natural hybridization between these two sister species. Further, the study of Song et al. (10) demonstrated a recent adaptive introgression from M. spretus into some M. m. domesticus populations in the wild, involving the vitamin K epoxide reductase subcomponent 1 (Vkorc1) gene, which was later shown to be more widespread in Europe, albeit geographically restricted to parts of southwestern and central Europe (11).Major, unanswered questions arise from these studies. First, is the vicinity around the Vkorc1 gene an isolated case of adaptive introgression in the house mouse genome, or do many other such regions exist? Second, is introgression between M. spretus and M. m. domesticus common outside the range of sympatry? Third, have there been other hybridization events, and, in particular, more ancient ones? Fourth, what role do introgressed genes, and, more generally, genomic regions, play?To investigate these open questions, we used genome-wide variation data from 20 M. m. domesticus samples (wild and wild-derived) from the ranges of sympatry and allopatry, as well as two M. spretus samples. For detecting introgression, we used PhyloNet-HMM (12), a newly developed method for statistical inference of introgression in genomes while accounting for other evolutionary processes, most notably incomplete lineage sorting (ILS).Our analysis provides answers to the questions posed above. First, we find signatures of introgression between M. spretus and each of the M. m. domesticus samples. The amount of introgression varies across the autosomes of each genome, with a few chromosomes harboring all detectable introgression, and most of the chromosomes have none. We detected no introgression on the X chromosome. Further, the amount of introgression varied widely across the samples. Our analyses demonstrate introgression outside the range of sympatry. In fact, our results show more signatures of introgression in the genomes of allopatric samples from Europe than in sympatric samples from Africa. For the third question, we used the length distribution and sharing patterns of introgressed regions across the samples to show support for at least three hybridization events: an ancient hybridization event that predates the colonization of Europe by M. m. domesticus and two more recent events, one of which presumably occurred about 50 y ago and is related to warfarin resistance selection (10). For the fourth question, our functional analysis of the introgressed genes shows enrichment for certain categories, most notably olfaction—an essential trait for the fitness of rodents. Understanding the genomic architecture and evolutionary history of the house mouse has broad implications on various aspects of evolutionary, genetic, and biomedical research endeavors that use this model organism. The PhyloNet-HMM method (12) can be used to detect introgression in other eukaryotic species, further broadening the impact of this work.     

Discovery of a Novel Coronavirus in Swedish Bank Voles (Myodes glareolus)

The unprecedented pandemic COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with bats as original reservoirs, has once again highlighted the importance of exploring the interface of wildlife diseases and human health. In this study, we identified a novel Betacoronavirus from bank voles (Myodes glareolus) in Grimsö, Sweden, and this virus is designated as Grimso virus. Repeated detection over three years and an overall prevalence of 3.4% suggest that the virus commonly occurs in bank voles. Furthermore, phylogenetic analyses indicate that the Grimso virus belongs to a highly divergent Embecovirus lineage predominantly associated with bank voles. Given that bank voles are one of the most common rodent species in Sweden and Europe, our findings indicate that Grimso virus might be circulating widely in bank voles and further point out the importance of sentinel surveillance of coronaviruses in wild small mammalian animals, especially in wild rodents.     

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron–molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum–iron–sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.     

Degenerate evolution of the hedgehog gene in a hemichordate lineage

The discovery of a set of highly conserved genes implicated in patterning during animal development represents one of the most striking findings from the field of evolutionary developmental biology. Existence of these “developmental toolkit” genes in diverse taxa, however, does not necessarily imply that they always perform the same functions. Here, we demonstrate functional evolution in a major toolkit gene. hedgehog (hh) encodes a protein that undergoes autocatalytic cleavage, releasing a signaling molecule involved in major developmental processes, notably neural patterning. We find that the hh gene of a colonial pterobranch hemichordate, Rhabdopleura compacta, is expressed in a dramatically different pattern to its ortholog in a harrimaniid enteropneust hemichordate, Saccoglossus kowalevskii. These represent two of the three major hemichordate lineages, the third being the indirect developing ptychoderid enteropneusts. We also show that the normally well-conserved amino acid sequence of the autoproteolytic cleavage site has a derived change in S. kowalevskii. Using ectopic expression in Drosophila, we find that this amino acid substitution reduces the efficiency of Hh autocatalytic cleavage and its signaling function. We conclude that the Hh sequence and expression in S. kowalevskii represent the derived state for deuterostomes, and we argue that functional evolution accompanied secondary reduction of the central nervous system in harrimaniids.     

The Prognostic Relevance of BAALC and ERG Expression Levels in Cytogenetically Normal Pediatric Acute Myeloid Leukemia

Cytogenetic aberrations are important prognostic factors in acute myeloid leukemia (AML). About 45 % of de novo adult AML and 20 % of pediatric AML lack cytogenetic abnormalities, so identification of predictive molecular markers might improve therapy. Mutation status of FLT3, NPM1 genes and gene expression levels of ERG, BAALC have been postulated as possible prognostic markers in pediatric AML with normal karyotype. Pretreatment blood samples from 47 cytogenetically normal AML patients were analysed for BAALC and ERG expression using real time RT-PCR. The patients were dichotomized at BAALC and ERG mean expression into low and high expression based on the median expression as cutoff. BAALC showed high expression in (24/47; 51.1 %) of patients and ERG high expression was detected in (22/47; 46.6 %). With follow-up for 1 year, patients with high BAALC and high ERG had inferior EFS (P = 0.001, P = 0.017 respectively), overall survival (P = 0.001, 0.08 respectively), and low rates of induction remission (P = 0.001, P = 0.0017 respectively) as compared to those with low expression. Also there was significant positive association between high expression of BAALC; ERG and FLT-ITD mutations (P = 0.016; P = 0.007 respectively). Multivariable analysis confirmed that high BAALC expression is an independent risk factor for EFS [HR for EFS 1.9(1.04–3.46) P = 0.037]; and OS [HR OS 1.55(1.7–3.36) P = 0.03]. In conclusion: Over expression of BAALC could predict adverse clinical outcome and may define important risk factor in cytogenetically normal pediatric AML.